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https://openalex.org/W3203798575
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https://www.jhsci.ba/ojs/index.php/jhsci/article/download/1387/784
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Evaluation of serum levels of malondialdehyde and endogenous non-enzymatic antioxidants in relation to colorectal cancer stage and intestinal wall infiltration
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Journal of Health Sciences
| 2,021
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| 5,784
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RESEARCH ARTICLE Open Access © 2020 Rašić, et al.; licensee University of Sarajevo - Faculty of Health Studies. This is an Open Access article
distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/
by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work
is properly cited. ABSTRACT Introduction: Oxidative stress and lipid peroxidation are pointed as possible factors in the development of colorectal
cancer (CRC). The aim of this study was to assess the serum malondialdehyde (MDA) and non-enzymatic antioxidants
concentration (albumin, bilirubin, uric acid, and ferritin) and their relation with the stage and histopathologic size (pT)
of CRC. Methods: One hundred and twenty patients with clinically and histopathologically confirmed CRC and the need for
surgical treatment were included in a cross-sectional study. All patients were divided into groups according to the disease
stage and depth of tumor invasion. The control group included 30 subjects with no signs of malignant and inflammatory
bowel disease. The patients and controls did not receive vitamin supplementation. Peripheral venous blood was sampled
before the surgical treatment of CRC patients and on the day of the examination of control subjects for determination
of serum MDA and the concentration of the non-enzymatic antioxidants. Results: The serum levels of MDA were progressively increased in CRC patients with the highest level in the fourth
stage of disease and pT4 group. Ferritin levels increased significantly with the CRC stage and decreased with the depth
of bowel wall invasion. Serum albumin concentration significantly decreased with increasing stage and increasing depth
of tumor invasion of the intestinal wall, while serum bilirubin level showed no change compared to the control group. Serum uric acid concentration was significantly higher in CRC patients, but no difference was observed with CRC pro
gression. It was confirmed that serum albumin significantly negatively correlated with the CRC stage (rho = −0.649,
p < 0.001), while serum MDA significantly positively correlated with the CRC stage (rho = 0.750, p < 0.001). Conclusion: These results indicate that serum MDA concentrations are related to the progression of CRC,
imbalance in non-enzymatic antioxidants also contributes. Conclusion: These results indicate that serum MDA concentrations are related to the progression of CRC, to which the
imbalance in non-enzymatic antioxidants also contributes. Keywords: Colon cancer; malondialdehyde; non-enzymatic antioxidants Evaluation of serum levels of malondialdehyde and
endogenous non-enzymatic antioxidants in relation to
colorectal cancer stage and intestinal wall infiltration Ismar Rašić1*, Sandin Holjan2, Vedad Papović3, Sanjin Glavaš3, Adi Mulabdić2, Azra Rašić4 1Department of Surgery, General Hospital “Prim. Dr. Abdulah Nakaš,” Sarajevo, Bosnia and Herzegovina, 2Clinic for General and Abdominal
Surgery, Clinical Center University of Sarajevo, Sarajevo, Bosnia and Herzegovina, 3Clinic for Gastroenterohepatology, Clinical Center
University of Sarajevo, Sarajevo, Bosnia and Herzegovina 4The Oncology Clinic, Clinical Center University of Sarajevo, Sarajevo, Bosnia and
Herzegovina Journal of Health Sciences *Corresponding author: Ismar Rašić, Department of Surgery, General
Hospital “Prim. dr. Abdulah Nakaš”, Bosnia and Herzegovina.
E-mail: rasicismar@gmail.com *Corresponding author: Ismar Rašić, Department of Surgery, General
Hospital “Prim. dr. Abdulah Nakaš”, Bosnia and Herzegovina.
E-mail: rasicismar@gmail.com
Submitted: 25 June 2021/Accepted: 10 September 2021
DOI: https://doi.org/10.17532/jhsci.2021.1387 Submitted: 25 June 2021/Accepted: 10 September 2021 METHODS One hundred and twenty patients both genders, 68 (56.7%)
male and 52 (43.3%) female, with radiologically, colonos
copically, and histopathologically confirmed CRC and with
a need for surgical treatment of that cancer were included
in a cross-sectional study lasting 4 years at the Clinic
for General and Abdominal Surgery, Clinical Center of
the University of Sarajevo. The ethical committee of the
Clinical Centre of the University of Sarajevo approved this
study. All patients who participated in this study gave their
written informed consent. The mean age of CRC patients
was 67.1 (range 49–79) years without a significant differ
ence according to the sex of the patients (67.4-years-old in
male vs. 64.8-years-old in female). CRC surgery was performed according to the princi
ple of en bloc resection of colon cancer with associated
lymph-vascular arcade under general anesthesia. After
resection and macroscopic examination of surgically
obtained tissue material, samples of tumor were fixed in
10% phosphate-buffered formalin for further histopatho
logical analysis. Stage of CRC was determined according
to the TNM classification of the American Association
of Cancer (American Joint Committee on Cancer, AJCC
guidelines) from 2010 (10), in which “T” marks the
depth of tumor invasion (pT), “N” lymph node metasta
sis (pN), and “M” distal metastasis. Staging of the CRC
was marked with numbers I–IV. All patients were divided
into groups according to the disease stage and depth of
tumor invasion. Exclusion criteria included evidence of neoplasm on an
organ unrelated to colon cancer, patients undergoing onco
logical treatment (radiotherapy or chemotherapy) before
surgery, presence of inflammatory bowel disease, history of
familial adenomatous polyposis, and coexistence of other
systemic or autoimmune diseases. The control group included 30 healthy volunteers (53.3%
males and 46.7% females), mean age of 59.1 (45–78) years,
who underwent preventive examination at the Counseling
Centre for Gastroenterohepatology, Clinical Center of the
University of Sarajevo. They had no family history of cancer
or clinical signs of malignant or inflammatory bowel dis
ease, comorbid conditions such as diabetes, hypertension,
coronary heart disease or autoimmune diseases, lung, thy
roid, liver, kidney, and infectious diseases (HCV and HIV
infection). In addition, patients and the control group did
not receive vitamin supplementation for the past 3 months
before inclusion in the study and they did not smoke or use
medications such as antibiotics, nonsteroidal anti-inflam
matory, and steroid medications. Statistical analyses were performed using the MedCalc
Software for Windows, version 12.6.1.0. INTRODUCTION past 10 years, there is growing support for the concept that
overproduction of reactive oxygen species (ROS) could
result in mutations and promote oncogenic phenotypes
involved in carcinogenesis, implicated in a range of diseases,
including CRC (2,3). ROS oxidize structural proteins and
inhibit the proteolytic system (4). ROS also have the ability
to oxidize polyunsaturated fatty acids, which take part in
cell membrane constitution. This reaction initiates a chain
reaction of lipid peroxidation, that produces other free rad
icals and substances such as malondialdehyde (MDA), to
which a significant role in the development of cancer has
been attributed (5). Colorectal cancer (CRC) is classified as the third most com
mon malignancy worldwide, accounting for approximately
9.4% of all causes of cancer death (1). Due to the ever-in
creasing incidence, CRC is becoming an increasingly
important diagnostic and therapeutic problem. Several risk factors are related to the onset and progression
of CRC, such as physical inactivity, environmental factors,
alcohol consumption, smoking, diet, and obesity. For the To prevent damage from ROS, the body has an antiox
idant protection system, which includes enzymatic and DOI: https://doi.org/10.17532/jhsci.2021.1387 81,9(56,7<2)6$5$-(92
)$&8/7<2)+($/7+678',(6 www.jhsci.ba Five milliliters of peripheral venous blood were sampled
from fasting patients before the surgical treatment of CRC
and from controls on the day of physical examination. The
blood was collected in BD Vacutainer test tube and centri
fuged at 5.000 rpm/min at room temperature for 10 min,
with separation of serum into aliquots. All serum samples
for determination of MDA concentration were stored at
−80°C until analysis, while serum levels of albumin, uric
acid, and bilirubin were determined on the day of the blood
sampling as well as the level of ferritin in plasma sample. non-enzymatic antioxidants. Enzymatic antioxidants belong
to cellular antioxidants and are found in the cells of the
arterial walls. Non-enzymatic antioxidants are present both
extracellularly and intracellularly and represent the first line
of defense of the body against the action of oxidizing sub
strates. Non-enzymatic antioxidants include exogenous anti
oxidants such as Vitamin E, Vitamin C and carotene, and
endogenous antioxidants such as albumin, ferritin, uric acid,
bilirubin, and ceruloplasmin. More than 70% of the plasma’s
antioxidant capacity is albumin and uric acid. Bilirubin is
also a strong antioxidant, which prevents lipid peroxidation. Serum concentration of MDA was estimated using a
commercial kit for the overall level of MDA (USCN Life
Science Inc., Houston, USA). INTRODUCTION Reading of the results was
carried out spectrophotometrically at 450 nm on a plate
reader STAT FAX 2100 (Awareness Technology, Palm City,
Florida, USA). The measured MDA concentration was
expressed in nanograms per milliliter (ng/mL). Several studies have documented the importance of antiox
idants in slowing down and reducing adverse effects of oxi
dative stress and preventing colorectal carcinogenesis (6,7). However, data were performed mostly in vitro on cell cul
tures, or in vivo in experimental animal models (8,9). The aim of the study was to parallel monitoring and analyz
ing the levels of serum MDA concentration and non-enzy
matic antioxidants in patients with CRC and to determine
the relationship of monitored biomarkers with the histo
pathologic size and stage of CRC. Serum albumin level was determined by electrophore
sis, after spectrophotometric total protein concentration
measurement on Dimension X Pand Plus system analyzer
(Siemens AG, Germany), with reference value for albumin
35.0–50.0 g/L. Plasma ferritin levels were quantified on
Cobas 6000 analyzer (Roche Diagnostics International Ltd,
Switzerland) using an electrochemiluminescent immunoas
say (reference range: 21.81–274.66 ng/mL). Quantitative
determination of serum uric acid concentration was per
formed spectrophotometrically using a modified Kalckar
method (URCA method) on Dimension RxL Max (Siemens
AG, Germany; reference rank: 155–428 µmol/L). The total
serum bilirubin concentration was also measured on the
same analyzer by spectrophotometric method (reference
range: 1.7–20.5 μmol/L). RESULTSh The most common location of CRC was the rectum, in
47 (39.2%) patients, while the rarest location was cecum, in
three cases (2.5%). All the CRC patients had histologically
adenocarcinoma type of cancer, in two-thirds of cases mod
erately differentiated (G2 grade). According to the depth
of the intestinal wall infiltration, 23 (19.2%) patients had
tumor with invasion of muscularis propria (pT2). Tumor
that spread through the muscularis propria into non-peri
tonealized pericolorectal tissues without involvement the
other organs (pT3) was confirmed in 67 (55.8%) patients,
while 30 (25.0%) patients had a tumor that penetrated the
visceral peritoneum or directly affected the other organs or
structures (pT4). The percentage of patients without lymph
node (N0) and distant metastasis (M0) was 37.5% and
75.0%, respectively. FIGURE 1. Serum malondialdehyde concentration in patients with different
stages of CRC. The data are presented as median, with minimum–max
imum values. *p<0.05 - difference between control and stage II of CRC,
**p<0.001- difference between II and III stage of CRC, ***p<0.001 - differ
ence between II and IV stage of CRC. NS: No significance between III and
IV stage of CRC. CRC: colorectal cancer, MDA: Malondialdehyde. FIGURE 2. Serum malondialdehyde concentration in CRC patients with
different depth of tumor invasion. The data are presented as median, with
minimum–maximum values. p<0.001 - significance difference between
pT3 and pT4, *p<0.05 - significance difference between pT2 and pT4, NS:
No significance between pT2 and pT3. CRC: Colorectal cancer, MDA:
Malondialdehyde, pT: Depth of tumor invasion. According to the laboratory examination, 75 (62.5%)
patients had decreased serum albumin concentration,
while 45 (37.5%) cases had normal serum albumin values. Normal plasma ferritin levels were confirmed in about half
of the patients (63; 52.5%), decreased in 21 (17.5%) cases
and increased in 36 (30.0%) cases. Most patients had nor
mal serum uric acid concentration (95; 72.2%) and serum
bilirubin concentration (117; 97.5%). The basic informa
tion about the study patients are summarized in Table 1. Serum MDA concentration was significantly higher
in patients with second stage of colorectal malignancy
compared to controls (47.5 [16.5–58.3] vs. 26.9 [17.4-
31.2] ng/mL; p < 0.05). The results of the study indicate
that serum MDA concentration shows a progressive increase
through the stages (II–IV stage) of CRC (47.5 [16.5–
58.3] vs. 61.35 [54.6–69.5] vs. 66.3 [60.8–69.5] ng/mL;
p < 0.001) (Figure 1). FIGURE 2. RESULTSh Serum malondialdehyde concentration in CRC patients with
different depth of tumor invasion. The data are presented as median, with
minimum–maximum values. p<0.001 - significance difference between
pT3 and pT4, *p<0.05 - significance difference between pT2 and pT4, NS:
No significance between pT2 and pT3. CRC: Colorectal cancer, MDA:
Malondialdehyde, pT: Depth of tumor invasion. According to the depth of tumor invasion (pT), there was
no difference between the pT2 and pT3 groups in terms
of serum MDA level (p = 0.590). The concentration of
serum MDA was significantly higher in the pT4 group
(66.3 [59.2–71.0] ng/mL) compared to the pT3 group of
patients (58.3 [46.8–66.8] ng/mL, p = 0.002) and the pT2
group of patients (57.6 [46.0–60.4] ng/mL, p = 0.012)
(Figure 2). malignancy (p < 0.05). While plasma ferritin levels increased
with the increasing stage of CRC, it also showed a tendency
to decline with depth of tumor invasion, with the lowest
level observed in the pT4 group (Table 2). While plasma
ferritin levels increased with the increasing stage of CRC,
it also showed a tendency to decline with depth of tumor
invasion, with the lowest level observed in the pT4 group
(Table 2). Serum uric acid concentration did not change
significantly through the CRC stages and different depth of
malignant infiltration of the intestinal wall, but was signifi
cantly higher in CRC patients compared to controls. Serum
bilirubin values were indistinguishable from the control
group and had no specific oscillations according to the stage
or depth of tumor invasion. Parallel monitoring of non-enzymatic parameters of the
antioxidant system indicated a gradual decrease of serum
albumin concentrations in patients with progression of
CRC (Table 1). The highest reduction in serum albumin
level was observed in the fourth stage of CRC (p < 0.001). The serum albumin level also decreased significantly with
the depth of tumor invasion. A significant difference in
the serum albumin concentration was detected in the pT4
group of patients with CRC compared to pT2 and pT3
group (p = 0.014). It was found that serum albumin had significant negative
correlation with CRC stage (r = −0.649, p < 0.001), while
the negative insignificant correlation of this biomarker
with the depth of tumor invasion was confirmed (r =
−0.189, p = 0.062). METHODS Kolmogorov–
Smirnov test or Shapiro–Wilk test was used to examine
the normal distribution of data. Variables with normal
distribution were presented as mean ± standard deviation
and compared using the t-test for independent samples. Variables not displaying normal distribution were pre
sented as median and interquartile range and compared
by Mann–Whitney U-test. ANOVA and Kruskal–Wallis
test was used for statistical evaluation of more than three
groups. The correlation between the monitored biomark
ers of oxidative and antioxidant system and the stage and
histopathological depth of intestinal wall invasion was
determined by Spearman correlation coefficient. Multiple Informed consent was obtained from all respondents
included in this study. The study protocol was approved by
the local Ethics Committee and was performed in accor
dance with the Helsinki Declaration. 143 www.jhsci.ba
Ismar Rašić, et al.: Serum levels of MDA and endogenous non-enzymatic antioxidants in relation to CRC stage and intestinal wall infiltration
Journal of Health Sciences 2021;11(3):142-148 FIGURE 1. Serum malondialdehyde concentration in patients with different
stages of CRC. The data are presented as median, with minimum–max
imum values. *p<0.05 - difference between control and stage II of CRC,
**p<0.001- difference between II and III stage of CRC, ***p<0.001 - differ
ence between II and IV stage of CRC. NS: No significance between III and
IV stage of CRC. CRC: colorectal cancer, MDA: Malondialdehyde. regression analysis was used to examine the impact of
MDA and non-enzymatic antioxidants (albumin, biliru
bin, uric acid, and ferritin) on the stage of CRC and the
depth of tumor invasion. Statistical significance was estab
lished at p < 0.05. DISCUSSION The oxidative stress usually results either due to excessive
ROS production or impaired antioxidant system, or a
combination of these factors. The pro-oxidative/antioxi
dative imbalance between the ROS formation and ability
of the several antioxidant defense mechanisms (including
enzymes and non-enzymatic antioxidants) to eliminate
that disturbances leads to various pathophysiological con
ditions. ROS overproduction associated with insufficient
antioxidant defense lead to protein, DNA and lipid oxi
dation and oxidative cell damage, which can trigger cancer
initiation and progression, including CRC (2). Several published studies suggest that ROS overproduction,
including also MDA as a product of lipid peroxidation, may
play a significant role in all stages of carcinogenesis (11,12). Increased concentrations of MDA in plasma or urine have
been observed in patients with various neoplasms, includ
ing gastric cancer (13), breast cancer (14), and bladder
cancer (15). The study by Zinczuk et al. also indicated sig
nificantly higher plasma concentrations of MDA in CRC
patients compared to healthy controls (16). In addition,
the research of Janion et al. demonstrated differentiation
in the intensity of lipid peroxidation process in relation to
the location of the primary tumor on the right side of the
colon (17). In this study, serum MDA levels reached the
highest values in patients with stage IV CRC, but these
results were not statistically significant. Branković et al. proved the significant presence of oxidative
stress in tumor tissue samples from resected colon prepa
ration with a highly significant increase of MDA concen
tration in both tumor and adjacent tissue compared to the
values in healthy tissue specimens (18). In the study by Leung et al., it was shown that the patients
with advanced inoperable CRC had a much higher con
centration of MDA serum compared to those with primary
localized CRC (19). Our research demonstrated statistically
significant differences in serum MDA levels in the third
and the fourth stage of the CRC compared to the second
stage of CRC. Similar results were reported by Surinenaite
et al. (20), who also found that MDA levels decreased sig
nificantly after surgical treatment compared to preoperative
status. RESULTSh Serum MDA had a significant positive The decline in serum albumin level was accompanied by a
significant increase in plasma ferritin concentration, with
the highest level of ferritin in the fourth stage of colorectal 144 www.jhsci.ba TABLE 1. Basic characteristics of the study group
Parameter
Patient number (%)
Age (years)
>60
31 (25.8%)
<60
89 (74.2%)
Gender
Male
68 (56.7%)
Female
52 (43.3%)
Tumor location
Cecum
3 (2.5%)
Ascending colon
18 (15.0%)
Transverse colon
3 (2.5%)
Descending colon
9 (7.5%)
Sigmoid colon
40 (33.3%)
Rectum
47 (39.2%)
Histological type
Adenocarcinoma
120 (100.0%)
Grade I
14 (11.6%)
Grade II
80 (66.7%)
Grade III
20 (16.7%)
Grade IV
6 (5.0%)
Depth of tumor invasion (pT)
pT1
0 (0.0%)
pT2
23 (19.2%)
pT3
67 (55.8%)
pT4
30 (25.0%)
Lymph node metastasis (pN)
N0
45 (37.5%)
N1
39 (32.5%)
N2
36 (30.0%)
Distant metastasis (pM)
M0
90 (75.0%)
M1
30 (25.0%)
Stage of CRC
I
0 (0.0%)
II
23 (19.2%)
III
67 (55.8%)
IV
30 (25.0%)
Serum concentration of albumin
Normal
45 (37.5%)
Decreased
75 (62.5%)
Increased
0 (0.0%)
Plasma ferritin concentration
Normal
63 (52.5%)
Decreased
21 (17.5%)
Increased
36 (30.0%)
Serum uric acid concentration
Normal
95 (72.2%)
Decreased
5 (4.2%)
Increased
20 (16.6%
Serum bilirubin concentration
Normal
117 (97.5%)
Decreased
0 (0.0%)
Increased
3 (2.5%)
CRC: Colorectal cancer
correlation with CRC stage (r = 0.750, p < 0.001) and
intestinal wall infiltration (r = 0.380, p < 0.001) (Table 3). while albumin was an independent negative predictor of
CRC stage (Table 3). In addition, an independent positive
predictor of tumor size or depth of tumor invasion (pT)
was serum MDA concentration (Table 4). DISCUSSION Correlation of monitored biomarkers with stage of CRC and
depth of tumor invasion
Biomarkers
Stage
pT
Albumin
rho
−0.649**
−0.189
p
0.000
0.062
Bilirubin
rho
0.106
−0.121
p
0.247
0.236
Uric acid
rho
0.062
−0.011
p
0.500
0.916
Ferritin
rho
0.137
0.022
p
0.136
0.830
MDA (ng/mL)
rho
0.750**
0.380**
p
0.000
0.000
pT: Depth of tumor invasion, rho: Correlation coefficient, *p<0.05,
**p<0.01. MDA: Malondialdehyde, CRC: Colorectal cancer TABLE 3. Correlation of monitored biomarkers with stage of CRC and
depth of tumor invasion hypoalbuminemia is a systemic inflammatory response
to malignancy (23,24). However, the results of our study
indicated that CRC progression was associated with a pro
gressive decrease in serum albumin as a reflection of dis
orders of the antioxidant system. In a systematic review
of 927 participants from seven case–control studies, Feng
et al. found that patients with CRC had lower serum fer
ritin levels than healthy controls (25). Ferritin is a protein
that binds to iron and belongs to the antioxidant system. Based on in vitro evidence, it is assumed that iron facil
itates DNA mutation through augmentation of oxygen
radical synthesis through the Haber-Weiss reaction, as well
as suppresses tumoricidal activity of macrophages. The results of our study suggest that the antioxidant imbal
ance present in patients with CRC contributes to the
increase in MDA concentration and progression of colorec
tal carcinoma. Furthermore, serum MDA was found to be
in significant positive correlation with CRC stage, whereas
serum albumin was in significantly negative correlation
with CRC stage, indicating the association of these param
eters with CRC. between oxidative stress index, MDA and colon tumor
budding, which suggest the participation of oxidative stress
in the remodeling of tumor (21). In addition, this study
observed significantly higher MDA level in patients with
lymph node metastasis in comparison to those without
metastasis. The research of Gopcevic et al. confirmed that lipid peroxi
dation is higher in all stages of CRC compared to the con
trol group, but with no significant differences among the
stages of the disease (26). At the same time, these authors
found a significantly lower activity of superoxide dismutase
and glutathione reductase as enzymatic indicators of anti
oxidant status in all stages of CRC compared to the control
group, but with a significant increase in stage IV disease,
concluding that CRC was associated with an increase in
oxidative stress followed by increase in antioxidant imbal
ance. DISCUSSION Our study also confirmed significantly higher serum MDA
concentrations in patients in whom the tumor perforated
the visceral peritoneum or involved adjacent organs (pT4)
compared to the group in which the tumor spread through
the muscularis propria to non-peritonealized pericolorectal
tissues without involvement of other organs (pT3) and to
the group in which the tumor invaded muscularis propria
(pT2). Serum MDA concentration significantly positively
correlated with CRC stage and depth of intestinal wall infil
tration. These findings indicate the carcinogenic potential
of MDA and its association with the progression of CRC. In recently published study, Zinczuk et al. observed a link CRC: Colorectal cancer correlation with CRC stage (r = 0.750, p < 0.001) and
intestinal wall infiltration (r = 0.380, p < 0.001) (Table 3). In the linear regression analysis model, an independent pos
itive predictor of CRC stage was serum MDA concentration, correlation with CRC stage (r = 0.750, p < 0.001) and
intestinal wall infiltration (r = 0.380, p < 0.001) (Table 3). In the linear regression analysis model, an independent pos
itive predictor of CRC stage was serum MDA concentration, 145 TABLE 2. The serum concentration of non‑enzymatic antioxidants in relation to the stage and histopathological features of CRC
Variabe
Control
Stage of CRC
II
III
IV
p
Albumin (g/L)
42.0 (39.0–44.0) a
38.0 (32.0–40.0) b
37.0 (32.0–39.0) b
29.5 (25.0–33.0) c
<0.001
Bilirubin (µmol/L)
8.7 (6.6–10.2)
8.1 (6.9–11.9)
10.7 (7.6–13.1)
9.5 (5.8–12.1)
NS
Uric acid (µmol/L)
272.0 (220.0–288.0) a
288.5 (237.0–362) b
281.5 (204.0–363.0) b
263.0 (215.0–340.0) b
<0.05
Ferritin (ng/mL)
49.54 (38.0–115.0) a
84.7 (39.7–189.3) b
80.3 (32.2–150.1) b
95.2 (50.9–226.6) c
<0.05
pT
pT2
pT3
pT4
p
Albumin (g/L)
33.8±6.5a
34.5±6.2a
29.8±7.3b
<0.05
Bilirubin (µmol/L)
11.6 (10.5–12.8) a
9.2 (6.9–12.9) a
9.4 (6.7–11.9) a
NS
Uric acid (µmol/L)
281.8±105.1
309.3±111.5
275.5±88.8
NS
Ferritin (ng/mL)
99.2 (54.7–116.2) a
71.0 (28.6–150.9) b
66.1 (38.8–136.6) b
<0.05
Data are presented as mean±standard deviation (SD) or median and interquartile range q1‑q3.abcValues in the same row that do not contain a same letter
differ significantly. NS: Insignificantly, CRC: Colorectal cancer, pT: Depth of tumor invasion ABLE 2. The serum concentration of non‑enzymatic antioxidants in relation to the stage and histopathological features of CRC TABLE 2. The serum concentration of non‑enzymatic antioxidants in relation to the stage and histopathologic TABLE 3. https://doi.org/10.1245/s10434-010-0985-4. 11. Mena S, Ortega A, Estrela JM. Oxidative stress in environmental-induced carcinogen
esis. Mutat Res 2009;674(1-2):36-44. 11. Mena S, Ortega A, Estrela JM. Oxidative stress in environmental-induced carcinogen
esis. Mutat Res 2009;674(1-2):36-44. Although our study has certain limitations regarding the
lack of enzyme parameters of the antioxidant system and
the relatively small sample of patients with CRC, the results
of this study can be the starting point for further clinical
research in a large population of colon cancer patients to
assess diagnostic values of these biomarkers in CRC and for
potential strengthening of the antioxidant protection sys
tem as a therapeutic approach in this disease. 12. Federico A, Morgillo F, Tuccillo C, Ciardiello F, Loguercio C. Chronic inflammation and
oxidative stress in human carcinogenesis. Int J Cancer 2007;121(11):2381-6. https://doi.org/10.1002/ijc.23192. 12. Federico A, Morgillo F, Tuccillo C, Ciardiello F, Loguercio C. Chronic inflammation and
oxidative stress in human carcinogenesis. Int J Cancer 2007;121(11):2381-6. https://doi.org/10.1002/ijc.23192. 13. Borrego S, Vazquez A, Dasi F, Cerda C, Iradi A, Tormos C, et al. Oxidative stress
and DNA damage in human gastric carcinoma: 8-oxo-7 ‘8-dihydro-2’-deoxyguanosine
(8-oxo-dG) as a possible tumor marker. Int J Mol Sci 2013;14(2):3467-86. https://doi.org/10.3390/ijms14023467. 14. Sawczuk B, Maciejczyk M, Sawczuk-Siemieniuk M, Posmyk R, Zalewska A, Car H. Salivary gland function, antioxidant defence and oxidative damage in the saliva of
patients with breast cancer: Does the BRCA1 mutation disturb the salivary redox pro
file? Cancers (Basel) 2019;11(10):1501. DISCUSSION Dolfi SC, Yang Z, Lee MJ, Guan F, Hong J, Y
forms of tocopherols, tocopherol phosphates a
colon cancer cells. J Agric Food Chem 2013;61
htt
//d i
/10 1021/jf401076 presence of complex disharmony in the balance of oxidative
stress and the antioxidant barrier. 8. Dolfi SC, Yang Z, Lee MJ, Guan F, Hong J, Yang CS. Inhibitory effects of different
forms of tocopherols, tocopherol phosphates and tocopherol quinones on growth of
colon cancer cells. J Agric Food Chem 2013;61(36):8533-40. The authors declare no conflict of interest. https://doi.org/10.2478/rjim-2020-0008. CONFLICT OF INTEREST https://doi.org/10.3390/cancers11101501. 15. Lepara Z, Lepara O, Fajkić A, Rebić D, Alić J, Spahović H. Serum malondialdehyde
(MDA) level as a potential biomarker of cancer progression for patients with bladder
cancer. Rom J Intern Med 2020;58(3):146-52. https://doi.org/10.2478/rjim-2020-0008. REFERENCES 16. Zinczuk J, Maciejczyk M, Zareba K, Romaniuk W, Markowski A, Kedra B, et al. Antioxidant barrier, redox status, and oxidative damage to biomolecules in patients
with colorectal cancer. Can malondialdehyde and catalase be markers of colorectal
cancer advancement? Biomolecules 2019;9(10):637. https://doi.org/10.3390/biom9100637. 1. Sung H. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence
and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin
2020;71(3):209-49. https://doi.org/10.3322/caac.21609. https://doi.org/10.3322/caac.21609. https://doi.org/10.3390/biom9100637. 2. Perše M. Oxidative stress in the pathogenesis of colorectal cancer: Cause or conse
quence? Biomed Res Int 2013;2013:725710. https://doi.org/10.1155/2013/725710. 17. Janion K, Szczepanska E, Nowakowska-Zajdel E, Strzelczyk J, Copija A. Selected
oxidative stress markers in colorectal cancer patients in relation to primary tumor loca
tion-a preliminary research. Medicina (Kaunas) 2020;56(2):47. https://doi.org/10.3390/medicina56020047. https://doi.org/10.3390/medicina56020047. 3. Kruk J, Aboul-Enein HY. Reactive oxygen and nitrogen species in carcinogenesis:
Implications of oxidative stress on the progression and development of several cancer
types. Mini Rev Med Chem 2017;17(11):904-19. https://doi.org/10.2174/1389557517666170228115324. 18. Brankovic B, Stanojevic G, Nestorovic M, Veljkovic A, Stojanovic I, Petrovic D, et al. Trosative stress parameters in colon cancer tumor, adjacent and healthy tissue. Acta
Med Med 2016;55(1):44-50. https://doi.org/10.5633/amm.2016.0107. https://doi.org/10.5633/amm.2016.0107. 4. Shringarpure R, Davies KJ. Protein turnover by the proteasome in aging and disease. Free Radic Biol Med 2002;32(11):1084-9. 19. Leung EY, Crozier JE, Talwar D, O‘Reilly DS, McKee RF, Horgan PG, et al. Vitamin
antioxidants, lipid peroxidation, tumour stage, the systemic inflammatory response
and survival in patients with colorectal cancer. Int J Cancer 2008;123(10):2460-4. https://doi.org/10.1002/ijc.23811. 5. Cejas P, Casado E, Belda-Iniesta C, de Castro J, Espinosa E, Redondo A, et al. Implications of oxidative stress and cell membrane lipid peroxidation in human cancer. Cancer Causes Control 2004;15(7):707-19. https://doi.org/10.1023/b: caco.0000036189.61607.52. 5. Cejas P, Casado E, Belda-Iniesta C, de Castro J, Espinosa E, Redondo A, et al. Implications of oxidative stress and cell membrane lipid peroxidation in human cancer. Cancer Causes Control 2004;15(7):707-19. https://doi.org/10.1023/b: caco.0000036189.61607.52. 20. Surinenaite B, Prasmickiene G, Milasiene V, Stratilatovas E, Didziapetriene J. Influence of surgical treatment and red blood cell transfusion on changes in antioxida
tive and immune system parameters in colorectal cancer patients. Medicina (Kaunas)
2009;45(10):785-91. htt
//d i
/10 3390/
di i
45100102 CONCLUSION A significant increase in serum MDA concentration rela
tive to the CRC stage and depth of tumor invasion suggests
that MDA plays a significant role in the carcinogenesis and
progression of CRC. The changes found in the selected
non-enzymatic parameters of the antioxidant system indi
cate an imbalance in the network of non-enzymatic activity
of system, especially emphasizing the association of hypo
albuminemia with the progression of colorectal carcinoma. 9. Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J. Free radicals and anti
oxidants in normal physiological functions and human disease. Int J Biochem Cell Biol
2007;39(1):44-84. https://doi.org/10.1016/j.biocel.2006.07.001. 10. Edge SB, Compton CC. The American joint committee on cancer: The 7th edi
tion of the AJCC cancer staging manual and the future of TNM. Ann Surg Oncol
2010;17(6):1471-4. DISCUSSION The results by Stone et al. point out that the oxidative
status expression is caused by a reduction of the potential
antioxidant defense (27), which is also consistent with our
results. Oxidative stress may arise from an imbalance between the
production of ROS and the mechanisms of cellular anti
oxidant defense (7). Ozgonul et al. found that patients
with CRC have lower levels of total antioxidative capacity
compared to healthy controls (22). However, information
on biochemical alterations in tissue and blood, especially
antioxidant status, and their correlation with the clinical
stage of the disease are lacking. Due to the increase in
oxidative stress in these patients, the classification of oxi
dative-antioxidant specificities of different stages of CRC
is of particular importance. In our study, serum albumin
concentration showed a gradual decrease in higher stages
of CRC, while plasma ferritin concentrations showed
irregular oscillations in different stages of the disease. A significant decrease in serum albumin in the fourth stage
of CRC compared to the second and third stages of this
disease was accompanied by a significant increase in serum
ferritin concentration. Some authors have suggested that Several studies have found some genetic factors that may
affect susceptibility to CRC, which relate to several sin
gle-nucleotide polymorphisms in genes implicated in
antioxidative protective system, such as eosinophil perox
idase, myeloperoxidase, and selenoprotein (28,29). It is
considered that genetic variation of antioxidative protec
tive proteins can be associated with CRC risk and survival
after diagnosis. All these studies and their results highlight
the complexity of the pathogenetic basis of CRC and the 146 TABLE 4. Independent predictors of the CRC progression
B
Standarderror
Beta
p
95% Confidence interval
Albumin
−0.043
0.011
−0.207
0.000
−0.065–0.021
Bilirubin
0.005
0.015
0.014
0.746
−0.025–0.035
Uric acid
0.002
0.001
0.113
0.909
0.000–0.003
Ferritin
−3.329E–05
0.001
−0.002
0.958
−0.001–0.001
MDA
0.021
0.004
0.335
0.000
0.013–0.031
Dependent variable: CRC stage
Albumin
−0.010
0.010
−0.118
0.307
–0.030–0.010
Bilirubin
−0.025
0.014
−0.188
0.081
–0.053–0.003
Uric acid
0.000
0.001
0.027
0.790
–0.001–0.001
Ferritin
0.000
0.001
0.123
0.241
0.000–0.002
MDA
0.010
0.002
0.367
0.001
0.005–0.017
Dependent variable: Depth of tumor invasion
B: Regression coefficient, Beta: Ratio probability, p: level of significance, MDA: Malondialdehyde, CRC: Colorectal cancer TABLE 4. Independent predictors of the CRC progression presence of complex disharmony in the balance of oxidative
stress and the antioxidant barrier. CONCLUSION
2017;8(6):469-73. https://doi.org/10.4328/jcam.5210. 8. https://doi.org/10.1023/b: caco.0000036189.61607.52. 6. Carini F, Mazzola M, Rappa F, Jurjus A, Geagea AG, Al Kattar S, et al. Colorectal
carcinogenesis: Role of oxidative stress and antioxidants. Anticancer Res
2017;37(9):4759-66. https://doi.org/10.3390/medicina45100102. 7. Dusak A, Atasoy N, Demir H, Dogan E, Gursoy T, Sarikaya E. Investigation of lev
els of oxidative stress and antioxidant enzymes in colon cancers. J Clin Anal Med 7. Dusak A, Atasoy N, Demir H, Dogan E, Gursoy T, Sarikaya E. Investigation of lev
els of oxidative stress and antioxidant enzymes in colon cancers. J Clin Anal Med 21. Zinczuk J, Maciejczyk M, Zareba K, Pryczynicz A, Dymicka-Piekarska V, Kaminska J, 147 serum ferritin with colorectal cancer. Int J Clin Exp Med 2015;8(12):22293-9. et al. Pro-oxidant enzymes, redox balance and oxidative damage to proteins, lipids
and DNA in colorectal cancer tissue. Is oxidative stress dependent on tumour budding
and inflammatory infiltration? Cancers (Basel) 2020;12(6):1636. serum ferritin with colorectal cancer. Int J Clin Exp Med 2015;8(12):22293-9. et al. Pro-oxidant enzymes, redox balance and oxidative damage to proteins, lipids
and DNA in colorectal cancer tissue. Is oxidative stress dependent on tumour budding
and inflammatory infiltration? Cancers (Basel) 2020;12(6):1636. et al. Pro-oxidant enzymes, redox balance and oxidative damage to proteins, lipids
and DNA in colorectal cancer tissue. Is oxidative stress dependent on tumour budding
and inflammatory infiltration? Cancers (Basel) 2020;12(6):1636. 26. Gopcevic KR, Rovcanin BR, Tatic SB, Krivokapic ZV, Gajic MM, Dragutinovic VV. Activity of superoxide dismutase, catalase, glutathione peroxidase, and glutathione
reductase in different stages of colorectal carcinoma. Dig Dis Sci 2013;58(9):2646-52. https://doi.org/10.1007/s10620-013-2681-2. https://doi.org/10.3390/cancers12061636. 22. Ozgonul A, Aksoy N, Dilmec F, Uzunkoy A, Aksoy S. Measurement of total antioxi
dant response in colorectal cancer using a novel automated method. Turk J Med Sci
2009;39(4):503-6. 27. Stone WL, Krishnan K, Campbell SE, Palau VE. The role of antioxidants and pro-oxi
dants in color cancer. World J Gastrointest Oncol 2014;6(3):55-66. 23. Nazha B, Moussaly E, Zaarour M, Weerasinghe C, Azab B. Hypoalbuminemia in
colorectal cancer prognosis: Nutritional marker or inflammatory surrogate? World J
Gastrointest Surg 2015;7(12):370-7. https://doi.org/10.4240/wjgs.v7.i12.370.l 28. Hong Y, Wu G, Li W, Liu D, He K. A comprehensive meta-analysis of genetic
associations between five key SNPs and colorectal cancer risk. Oncotarget
2016;7(45):73945-59. https://doi.org/10.18632/oncotarget.12154. 28. Hong Y, Wu G, Li W, Liu D, He K. A comprehensive meta-analysis of genetic
associations between five key SNPs and colorectal cancer risk. Oncotarget
2016;7(45):73945-59. https://doi.org/10.18632/oncotarget.12154. 24. Almasaudi AS, Dolan RD, Edwards CA, McMillan DC. https://doi.org/10.1023/b: caco.0000036189.61607.52. Hypoalbuminemia reflects nutri
tional risk, body composition and systemic inflammation and is independently associ
ated with survival in patients with colorectal cancer. Cancers (Basel) 2020;12(7):1986. https://doi.org/10.3390/cancers12071986. 29. Fedirko V, Jenab M, Meplan C, Jones JS, Zhu W, Schomburg L, et al. Association
of selenoprotein and selenium pathway genotypes with risk of colorectal cancer and
interaction with selenium status. Nutrients 2019;11(4):935. 25. Feng Z, Chen JW, Feng JH, Shen F, Cai WS, Cao J, et al. The association between https://doi.org/10.1201/9780429423482-77. RELATED ARTICLES PUBLISHED IN JHSCI 1. Gubaljevic J, Srabović N, Jevrić-Čaušević A, Softić A, Rifatbegović A, Mujanović-Mustedanagić J, Dautović E,
Smajlović A, Mujagić Z. Serum levels of oxidative stress marker malondialdehyde in breast cancer patients in relation
to pathohistological factors, estrogen receptors, menopausal status, and age. JHSCI. 2018;8(3):154-61. 1. Gubaljevic J, Srabović N, Jevrić-Čaušević A, Softić A, Rifatbegović A, Mujanović-Mustedanagić J, Dautović E,
Smajlović A, Mujagić Z. Serum levels of oxidative stress marker malondialdehyde in breast cancer patients in relation
to pathohistological factors, estrogen receptors, menopausal status, and age. JHSCI. 2018;8(3):154-61. 148
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Indonesian
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Analisis Pesan Dakwah dalam Novel Religi
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Muttaqien
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cc-by-sa
| 9,734
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Keywords:
Bil-Qalam's Da'wah,
Novels, Messages of
Da'wah, Women,
Violence Against
Women. ABSTRAK ABSTRAK
Dakwah merupakan kegiatan menyeru manusia ke jalan Allah SWT. Dakwah
bisa dilakukan dengan berbagai cara salah satunya yaitu menggunakan media
tulisan. Karena sifatnya yang terdokumentasi, dakwah dengan tulisan
memiliki umur yang panjang sehingga dapat menyentuh audiens dalam
jumlah banyak bahkan lintas generasi. Dakwah dengan tulisan ini disebut
dengan dakwah bil qalam. Dakwah bil qalam, bisa dilakukan dengan menulis
sebuah karya sastra seperti novel. Sastra novel menjadi salah satu jenis tulisan
yang dijadikan media dakwah yang efektif, salah satu novel yang menarik
untuk dianalisis yakni Novel “Hilda” karya Muyassarotul Hafidzoh. Novel ini
merupakan salah satu novel yang mengangkat isu kekerasan terhadap
perempuan dengan berlatar pesantren. Novel ini menceritakan seorang gadis
bernama Hilda yang menjadi korban kekerasan seksual (baca: perkosaan)
yang harus berjuang menghadapi berbagai diskriminasi dan stereotip yang
menuju kepadanya yang tidak lain adalah korban, belum lagi kejadian itu
membuat Hilda mengalami trauma yang sulit disembuhkan. Penelitian ini
bertujuan untuk menemukan pesan anti kekerasan terhadap perempuan dan
pesan dakwah yang terkandung dalam novel dengan menggunakan
pendekatan kualitatif dengan jenis penelitian studi pustaka atau library
research, yakni sumber utama berupa buku novel “Hilda” karya Muyassarotul
Hafidzoh. Selain dari pada itu, dalam penelitian ini juga menggunakan
hermeneutika Gadamer sebagai pisau analisis dan sebagai prosedur
penafsiran. Muttaqien: E-ISSN : 2723-5963 | 15
Copyright © 2022 Rina dkk. DOI: https://doi.org/10.52593/mtq.03.1.02 Muttaqien, Vol. 3. No. 1 Januari 2022, 15-41 Muttaqien, Vol. 3. No. 1 Januari 2022, 15-41 ANALISIS PESAN DAKWAH DALAM NOVEL RELIGI
(Pesan Anti Kekerasan terhadap Perempuan dalam Novel “Hilda” Karya Muyassarotul
Hafidzoh) Rina1, Erfian Syah2, AD Kusumaningtyas3 Program Studi Komunikasi dan Penyiaran Islam STAI DR.KH.EZ.Muttaqien Indonesia
l i
16@
i
1
i
h
@
il
2
i i
d @
h
3 Program Studi Komunikasi dan Penyiaran Islam STAI DR.KH.EZ.Muttaqien Indonesia
nurmalarina16@gmai.com1 , erviansyahprayoga@gmail.com2, nining_ade@yahoo.com3 Informasi artikel
Kata kunci:
Dakwah Bil-Qalam,
Novel, Pesan Dakwah,
Perempuan, Kekerasan
terhadap Perempuan. ABSTRACT Da'wah is an activity that calls people to the path of Allah SWT. Da'wah can be done in
various ways, one of which is using written media. Because of its documented nature,
da'wah with writing has a long life so that it can touch large audiences, even across
generations. Da'wah with this writing is called da'wah bil qalam. Da'wah bil qalam, can
be done by writing a literary work such as a novel. Novel literature is one type of writing
that is used as an effective propaganda medium, one of the interesting novels to analyze is
the novel "Hilda" by Muyassarotul Hafidzoh. This novel is one of the novels that raises
the issue of violence against women with a pesantren background. This novel tells of a girl
named Hilda who is a victim of sexual violence (read: rape) who has to struggle with
various discriminations and stereotypes that lead to her who is none other than the victim,
not to mention that the incident left Hilda traumatized that is difficult to heal. This study
aims to find the message of anti-violence against women and the message of da'wah
contained in the novel by using a qualitative approach with the type of library research,
namely the main source in the form of the novel "Hilda" by Muyassarotul Hafidzoh. Apart from that, this study also uses Gadamer's hermeneutics as an analytical tool and as
an interpretive procedure. Keywords:
Bil-Qalam's Da'wah,
Novels, Messages of
Da'wah, Women,
Violence Against
Women. Muttaqien: E-ISSN : 2723-5963 | 15
p
Copyright © 2022 Rina dkk. DOI: https://doi.org/10.52593/mtq.03.1.02 Muttaqien: E-ISSN : 2723-5963 | 15
p
Copyright © 2022 Rina dkk. DOI: https://doi.org/10.52593/mtq.03.1.02 Muttaqien: E-ISSN : 2723-5963 | 15
DOI: https://doi.o g/10.52593/mtq.03.1.02 Muttaqien, Vol. 3. No. 1 Januari 2022, 15-41 Naskah diterima: 28 Desember 2021, direvisi: 19 Januari 2022, disetujui 29 Januari 2022 Naskah diterima: 28 Desember 2021, direvisi: 19 Januari 2022, disetujui 29 Januari 2022 A. Pendahuluan Dakwah merupakan suatu upaya yang dilakukan mukmin untuk mengubah keadaan
individu, masyarakat dan kondisi yang kurang islami dalam berbagai aspek agar menjadi
lebih islami , atau dengan kata lain dakwah merupakan kegiatan mengajak manusia
kepada jalan Allah SWT sebagaimana firman Allah dalam QS. An-Nahl ayat 125 sebagai
berikut: Dakwah merupakan suatu upaya yang dilakukan mukmin untuk mengubah keadaan
individu, masyarakat dan kondisi yang kurang islami dalam berbagai aspek agar menjadi
lebih islami , atau dengan kata lain dakwah merupakan kegiatan mengajak manusia
kepada jalan Allah SWT sebagaimana firman Allah dalam QS. An-Nahl ayat 125 sebagai
berikut: Dakwah merupakan suatu upaya yang dilakukan mukmin untuk mengubah keadaan
individu, masyarakat dan kondisi yang kurang islami dalam berbagai aspek agar menjadi lebih islami , atau dengan kata lain dakwah merupakan kegiatan mengajak manusia
kepada jalan Allah SWT sebagaimana firman Allah dalam QS. An-Nahl ayat 125 sebagai
berikut: َسَبِيلِ رَ ب ِكَ بِالْحِ كْمَةِ وَ الْمَوْ عِظَةِ الْحَسَنَةِ ۖ وَ جَادِلْهُمْ بِالَّتِي هِيَ أَحْ سَنُ ۚ إِنَّ رَ بَّك ادْعُ إِلَى
َهُوَ أَعْلَمُ بِمَنْ ضَلَّ عَنْ سَبِيلِهِ ۖ وَ هُوَ أَعْلَمُ بِالْمُهْتَدِين Artinya : “Serulah manusia ke jalan Tuhanmu dengan hikmah dan pelajaran yang baik dan
bantahlah mereka dengan cara yang baik. Sesungguhnya Tuhanmu Dialah yang lebih mengetahui
tentang siapa yang tersesat dari jalan-Nya dan Dialah yang lebih mengetahui orang-orang yang
mendapat petunjuk. Hikmah: ialah perkataan yang tegas dan benar yang dapat membedakan antara
yang hak dengan yang bathil.” Dakwah adalah setiap usaha ataupun aktivitas dengan lisan maupun sebuah tulisan dan
lainnya dengan sifat menyeru, mengajak, serta memanggil manusia untuk senantiasa
beriman dan mematuhi Allah SWT sesuai dengan garis-garis aqidah dan syariat serta
akhlak Islamiyah. Jika dakwah secara sederhana memiliki maksud sebagai setiap usaha seseorang (Muslim)
guna memengaruhi orang lain agar melakukan suatu perubahan, berupa perubahan
pikiran, perasaan, sikap dan perilaku ke arah yang lebih baik, apa pun bentuk dari
kegiatannya, termasuk juga menulis, maka seorang penulis bisa disebut seorang da’i. Melalui karyanya, penulis berusaha untuk memengaruhi para pembacanya dan mampu
menyentuh audiens dalam jumlah banyak yang mungkin melebihi pendengar ceramah
akbar sekalipun. Bahkan, karena sifatnya terdokumentasi, dakwah dengan tulisan
memiliki umur yang panjang dibandingkan dakwah lisan.(Muhtadi 2012). Dakwah dengan tulisan disebut dakwah dakwah bil qalam. Dakwah bil qalam adalah
metode atau cara untuk menyampaikan pesan kebaikan yang memiliki nilai dakwah
kepada mad’u. Dakwah bil qalam adalah metode dakwah melalui pena. Dakwah bil
qalam disebut juga dengan istilah “Dakwah Bil Kitabah”. | Muttaqien: E-ISSN : 2723-5963 A. Pendahuluan Dakwah ini dilaksanakan 16 | Muttaqien: E-ISSN : 2723-5963 Analisis Pesan Dakwah … (Rina, dkk) melalui aktivitas yang melibatkan kepenulisan seperti risalah, majalah, artikel, cerpen,
sajak, poster, buku, internet, Koran, dan tulisan-tulisan yang mengandung pesan
dakwah.(Tata Sukayat 2009). Salah satu jenis tulisan yang cukup populer yang hari ini banyak diminati oleh
masyarakat adalah novel. Novel merupakan salah satu karya sastra yang banyak
dimanfaatkan oleh penulis muslim dan muslimah sebagai sarana menyampaikan pesan-
pesan Islam. Karya sastra berupa novel menawarkan sebuah dunia kepada para
pembacanya, model yang ditawarkan adalah model kehidupan yang diidealkan oleh
penulis, dunia yang bersifat imajinatif, serta dibangun melalui berbagai unsur. Dari sisi tertentu sebuah novel dapat dipandang sebagai sebuah karya yang
mengandung
keinginan pengarang
untuk
menawarkan,
menyampaikan
atau
mendialogkan suatu pesan tertentu. Pesan tersebut dapat berupa pandangan hidup,
moral, gagasan, atau sebuah amanat yang ingin disampaikan kepada para pembaca. Pesan moral berupa nilai-nilai religius, banyak ditemukan dalam sebuah karya novel. Hal tersebut merupakan “lahan” yang memberikan banyak inspirasi kepada para
penulis. Sebab selama ini banyak realita kehidupan yang tidak sesuai dengan harapan,
lalu kemudian para penulis mencoba untuk menawarkan suatu hal yang diidealkan
dalam karyanya.(Tata Sukayat 2009). Dakwah bil qalam menjadi salah satu metode dakwah yang banyak digunakan para
penulis untuk menyampaikan ide, gagasan maupun kritik sosial kepada para pembaca. Dakwah bil qalam juga memiliki kelebihan dibandingkan dengan metode dakwah
lainnya, sifatnya yang terdokumentasi membuat dakwah bil qalam memiki umur yang
panjang dan menjadi investasi masa bagi masa depan, boleh jadi penulisnya sudah wafat
namun tulisannya masih bisa terus dibaca oleh lintas generasi. Saat ini kerinduan masyarakat terhadap nilai-nilai Islam sangat tinggi, ini dibuktikan
dengan banyaknya kemunculan berbagai novel Islam yang menjadi best seller dan
munculnya film-film religi yang digemari oleh masyarakat. Seorang da’i kini dituntut Muttaqien: E-ISSN : 2723-5963 | 17 Muttaqien, Vol. 3. No. 1 Januari 2022, 15-41 untuk berperan aktif dalam berbagai hal, salah satunya, da’i kini harus memiliki
keterampilan dalam menulis untuk dapat menyampaikan dakwah dengan metode bil
qalam. Hal ini dikarenakan keadaan masyarakat saat ini khususnya kesibukan
masyarakat metropolis, sulit meluangkan waktu untuk sekedar menghadiri kajian
keagamaan ataupun majelis taklim karena berbagai latar belakang pekerjaannya. Salah satu penulis muda Muslimah yang memilih menggunakan metode bil qalam
sebagai media penyampai pesan-pesan Islam adalah Muyassarotul Hafidzoh. Muyassarotul Hafidzoh telah berhasil menerbitkan sebuah novel yang dapat memicu
turning point (titik balik) dalam kehidupan pembacanya yang berjudul “Hilda”. 18 | Muttaqien: E-ISSN : 2723-5963 1. Dakwah Ditinjau dari segi bahasa, dakwah berasal dari bahasa Arab “da’wah” (الدعوة). Da’wah mempunyai tiga huruf asal, yaitu د, ع, dan ؤ. Dari ketiga huruf asal ini,
terbentuk beberapa kata dan ragam makna. Makna tersebut adalah memanggil,
mengundang, minta tolong, meminta, memohon, menamakan, menyuruh datang,
mendorong, menyebabkan, mendatangkan, mendoakan, menangisi, dan meratapi
(Ahmad Warson Munawwir, 1997: 406). Dalam Al-Qur’an, kata da’wah dan
berbagai bentuk katanya ditemukan sebanyak 198 kali menurut hitungan
Muhammad Sulthon (2003:4), 299 kali versi Muhammad Fu’ad ‘Abd al-Baqi’ (dalam
A. Ilyas Isma’il, 2006: 144-145), atau 212 kali menurut Asep Muhiddin (2002: 40). Ini berarti, Al-Qur’an mengembangkan makna dari kata da’wah untuk berbagai
penggunaan.(Aziz 2004). A. Pendahuluan Keberanian Muyas dalam membidik tema sensitif dalam novel “Hilda” ini perlu
diapresiasi. “Hilda” merupakan novel yang penuh dengan nuansa spiritualitas untuk
menguatkan perempuan yang terluka oleh laki-laki, tepatnya “Hilda” adalah novel yang
menceritakan tentang seorang perempuan yang mengalami kekerasan seksual, sebuah
peristiwa yang perempuan di belahan dunia mana pun tidak ingin mengalaminya. Salah satu isu penting yang menarik perhatian penulis adalah soal isu kekerasan seksual. Hilda adalah seorang santri yang pernah menjadi korban kekerasan seksual oleh salah
satu temannya saat Hilda masih Sekolah Menengah Atas. Peristiwa tersebut membuat
Hilda dikeluarkan dari bangku sekolah karena dianggap sebagai pembawa aib. Hilda
harus menanggung caci maki dari teman-temannya dan para tetangga di lingkungan
tempat ia tinggal oleh suatu kejahatan yang tidak ia kehendaki dan sangat ia benci
namun sayangnya harus menimpa dirinya, inilah kenyataan pahit yang dialami oleh
Hilda sang tokoh utama. 8 | Muttaqien: E-ISSN : 2723-5963
Hilda adalah korban kekerasan seksual yang harusnya dilindungi karena beban berat
yang
harus
ditanggungnya, namun sebaliknya, stereotip dalam masyarakat
menyimpulkan bahwa perempuan dianggap sebagai penyebab terjadinya perbuatan keji
tersebut. Hilda sekuat tenaga berusaha untuk sembuh dari luka masa lalunya, berusaha
melupakan tragedi yang mengerikan dan kelam itu, melupakan kejadian yang hampir
mengubur impiannya juga pengalaman yang membuatnya selalu dalam bayang-bayang 18 | Muttaqien: E-ISSN : 2723-5963 18 Analisis Pesan Dakwah … (Rina, dkk) ketakutan. Novel ini mengambil tema konteks sosial berupa isu kekerasan terhadap
perempuan, yang dikemas dengan nuansa pesantren yang tidak biasa atau anti
mainstream. 2. Pesan Dakwah Dalam ilmu komunikasi pesan dakwah adalah message, yaitu simbol-simbol. Dalam
literatur bahasa Arab, pesan dakwah disebut maudlu’ al-da’wah (مؤضؤع الدعؤة). Istilah ini lebih tepat dibanding dengan istilah “materi dakwah” yang diterjemahkan
dalam bahasa Arab menjadi maaddah al-da’wah (مادة
الدعؤة). Sebutan terakhir ini bisa
menimbulkan kesalahpahaman sebagai logistik dakwah. Istilah pesan dakwah
dipandang lebih tepat untuk menjelaskan “isi dakwah berupa kata, gambar, lukisan
dan sebagainya yang diharapkan dapat memberikan pemahaman bahkan perubahan
sikap dan perilaku mitra dakwah”. Jika dakwah melalui tulisan umpamanya, maka
yang ditulis itulah pesan dakwah. Jika dakwah melalui lisan, maka yang diucapkan
pembicara itulah pesan dakwah. Jika melalui tindakan, maka perbuatan baik yang
dilakukan itulah pesan dakwah.(Aziz 2004). Muttaqien: E-ISSN : 2723-5963 | 19 Muttaqien, Vol. 3. No. 1 Januari 2022, 15-41 3. Materi Dakwah Materi dakwah disebut juga dengan maddah atau isi pesan yang disampaikan da’i
kepada mad’u. Dalam hal ini sudah jelas bahwa yang menjadi maddah dakwah adalah
ajaran Islam itu sendiri. Materi dakwah disebut juga dengan maddah atau isi pesan yang disampaikan da’i
kepada mad’u. Dalam hal ini sudah jelas bahwa yang menjadi maddah dakwah adalah
ajaran Islam itu sendiri. Secara umum materi dakwah dapat diklarifikasikan menjadi empat masalah pokok,
yaitu:(munir muhammad wahyu ilaihi 2009). Secara umum materi dakwah dapat diklarifikasikan menjadi empat masalah pokok,
yaitu:(munir muhammad wahyu ilaihi 2009). a. Masalah Akidah [keimanan] Masalah pokok yang menjadi materi dakwah adalah akidah islamiyah. Aspek
akidah ini yang membentuk moral [akhlak] manusia. Oleh karena itu, yang
pertama kali dijadikan materi dalam dakwah Islam adalah masalah akidah atau
keimanan. Akidah yang menjadi materi dakwah ini memiliki ciri-ciri yang
membedakannya dengan kepercayaan agama lain, yaitu: Masalah pokok yang menjadi materi dakwah adalah akidah islamiyah. Aspek
akidah ini yang membentuk moral [akhlak] manusia. Oleh karena itu, yang
pertama kali dijadikan materi dalam dakwah Islam adalah masalah akidah atau
keimanan. Akidah yang menjadi materi dakwah ini memiliki ciri-ciri yang
membedakannya dengan kepercayaan agama lain, yaitu: • Keterbukaan melalui persaksian [syahadat]. Dengan demikian seorang
Muslim harus jelas dengan identitasnya dan mengakui identitas keagamaan
orang lain. • Cakrawala pandangan yang luas dengan memperkenalkan bahwa Allah
adalah Tuhan seluruh alam, bukan Tuhan kelompok atau bangsa tertentu. • Ketahanan antara iman dan Islam atau antara iman dan amal perbuatan. Dalam ibadah pokok yang merupakan manifestasi dari iman dipadukan
dengan segi-segi pengembangan diri dan kepribadian seseorang dengan
kemaslahatan masyarakat yang menuju pada kesejahteraannya. Karena
akidah memiliki keterlibatan dengan soal-soal kemasyarakatan. \ b. Masalah Syari’ah
Hukum atau syari’ah sering disebut sebagai cermin peradaban dalam pengertian
bahwa ketika ia tumbuh matang dan sempurna, maka peradaban mencerminkan
dirinya dalam hukum-hukumnya. Pelaksanaan syari’ah merupakan sumber yang
melahirkan peradaban Islam, yang melestarikan dan melindunginya dalam
sejarah. Syari’ah inilah yang akan selalu menjadi kekuatan peradaban di kalangan
kaum muslim. 20 | Muttaqien: E-ISSN : 2723-5963 20 | Muttaqien: E-ISSN : 2723-5963 Analisis Pesan Dakwah … (Rina, dkk) Melestarikan dakwah yang bersifat syari’ah ini sangat luas dan mengikat seluruh
umat Islam. Ia merupakan jantung yang tidak terpisahkan dari kehidupan umat
Islam di berbagai penjuru dunia, dan merupakan hal yang patut dibanggakan. Kelebihan dari materi syari’ah ini antara lain adalah, ia tidak dimiliki oleh umat-
umat lain. 3. Materi Dakwah Berdasarkan pengertian ini, maka ajaran akhlak dalam Islam pada dasarnya
meliputi kualitas perbuatan manusia yang merupakan ekspresi dari kondisi
kejiwaannya. Akhlak dalam Islam bukanlah norma ideal yang tidak dapat
diimplementasikan, dan bukan pula sekumpulan etika yang terlepas dari kebaikan
norma sejati. Dengan demikian yang menjadi materi akhlak dalam Islam adalah
mengenai sifat dan kriteria perbuatan manusia serta kewajiban yang harus
dipenuhinya. Karena
semua
manusia
harus
mempertanggungjawabkan
perbuatannya, maka Islam mengajarkan kriteria perbuatan dan kewajiban yang
mendatangkan kebahagiaan, bukan siksaan. 4. Media Dakwah Media berasal dari dari bahasa Latin medius yang secara harfiah berarti perantara,
tengah atau pengantar. Dalam bahasa Inggris media merupakan bentuk jamak dari
medium yang berarti tengah, antara, rata-rata. Dari pengertian ini ahli komunikasi
mengartikan media sebagai alat yang menghubungkan pesan komunikasi yang
disampaikan oleh komunikator kepada komunikan (penerima pesan). Dalam bahasa
Arab media sama dengan wasilah atau bentuk jamak, wasail yang berarti alat atau
perantara.(munir muhammad wahyu ilaihi 2009) . Dalam ilmu komunikasi, media dapat juga diklasifikasi menjadi tiga yaitu: Dalam ilmu komunikasi, media dapat juga diklasifikasi menjadi tiga yaitu: • Media terucap (the spoken word) alat yang bisa mengeluarkan bunyi seperti
radio, telepon dan sejenisnya. • Media tertulis (the printed writing) yaitu media berupa tulisan atau cetakan
seperti majalah, surat kabar, buku, pamflet, lukisan, gambar dan sejenisnya. • Media dengar pandang (the audio visual) yaitu media yang berisi gambar hidup
yang bisa dilihat dan didengar, yaitu film, video, televisi dan sejenisnya. 3. Materi Dakwah Syari’ah ini bersifat universal, yang menjelaskan hak-hak umat muslim
dan nomuslim, bahkan hak seluruh umat manusia. Dengan adanya materi syari’ah
ini, maka tatanan sistem dunia akan teratur dan sempurna. Masalah Mu’amalah Islam merupakan agama yang menekankan urusan mu’amalah lebih besar
porsinya daripada urusan ibadah. Islam lebih banyak memerhatikan aspek
kehidupan sosial daripada aspek kehidupan ritual. Islam adalah agama yang
menjadikan seluruh bumi ini masjid, tempat mengabdi kepada Allah SWT. Cakupan aspek mu’amalah jauh lebih luas dari pada ibadah Statement ini dapat Cakupan aspek mu’amalah jauh lebih luas dari pada ibadah. Statement ini dapat
dipahami dengan alasan: • Dalam Al-Qur’an dan Hadis mencakup proporsi terbesar sumber hukum
yang berkaitan dengan urusan mu’amalah. • Ibadah yang mengandung segi kemasyarakatan diberi ganjaran lebih besar
daripada ibadah yang bersifat perorangan. Jika urusan ibadah dilakukan tidak
sempurna atau batal, karena melanggar pantangan tertentu, maka tebusannya
adalah melakukan sesuatu yang berhubungan dengan mu’amalah. Sebaliknya,
jika orang tidak baik dalam urusan mu’amalah, maka urusan ibadah tidak dapat
menutupinya. d. Masalah Akhlak
Secara etimologis, kata akhlaq berasal dari bahasa Arab yang artinya budi pekerti,
perangai, dan tingkah laku atau tabiat. Sedangkan secara terminologi, pembahasan
akhlak berkaitan dengan masalah tabiat atau kondisi temperatur batin yang
memengaruhi perilaku manusia. Ilmu akhlak bagi Al-Farabi, tidak lain dari
bahasan tentang keutamaan-keutamaan yang dapat menyampaikan manusia
kepada tujuan hidupnya yang tertinggi, yaitu kebahagiaan, dan tentang berbagai
kejahatan atau kekurangan yang dapat merintangi usaha pencapaian tujuan
tersebut. d. Masalah Akhlak
Secara etimologis, kata akhlaq berasal dari bahasa Arab yang artinya budi pekerti,
perangai, dan tingkah laku atau tabiat. Sedangkan secara terminologi, pembahasan
akhlak berkaitan dengan masalah tabiat atau kondisi temperatur batin yang
memengaruhi perilaku manusia. Ilmu akhlak bagi Al-Farabi, tidak lain dari
bahasan tentang keutamaan-keutamaan yang dapat menyampaikan manusia
kepada tujuan hidupnya yang tertinggi, yaitu kebahagiaan, dan tentang berbagai
kejahatan atau kekurangan yang dapat merintangi usaha pencapaian tujuan
tersebut. Muttaqien: E-ISSN : 2723-5963 | 21 Muttaqien, Vol. 3. No. 1 Januari 2022, 15-41 Berdasarkan pengertian ini, maka ajaran akhlak dalam Islam pada dasarnya
meliputi kualitas perbuatan manusia yang merupakan ekspresi dari kondisi
kejiwaannya. Akhlak dalam Islam bukanlah norma ideal yang tidak dapat
diimplementasikan, dan bukan pula sekumpulan etika yang terlepas dari kebaikan
norma sejati. Dengan demikian yang menjadi materi akhlak dalam Islam adalah
mengenai sifat dan kriteria perbuatan manusia serta kewajiban yang harus
dipenuhinya. Karena
semua
manusia
harus
mempertanggungjawabkan
perbuatannya, maka Islam mengajarkan kriteria perbuatan dan kewajiban yang
mendatangkan kebahagiaan, bukan siksaan. Muttaqien: E-ISSN : 2723-5963 | 23 ن ؤلقلم ؤما يسطرون ن ؤلقلم ؤما يسطرون 5. Metode Dakwah Dakwah dalam pelaksanaannya memerlukan sebuah metode dan juga teknik. Metode dan teknik ini harus terkonsep agar tujuan dakwah terimplementasi. Dakwah bertujuan untuk merubah sikap mental dan perilaku manusia yang kurang
baik menjadi lebih baik atau meningkatkan kualitas iman dan Islam seseorang secara 22 | Muttaqien: E-ISSN : 2723-5963 Analisis Pesan Dakwah … (Rina, dkk) sadar dan timbul dari kemauannya sendiri tanpa merasa terpaksa. Dakwah juga
bertujuan agar menjadikan manusia yang dapat menciptakan “Hablum Minallah dan
Hablum Minannas”. Secara garis besar dakwah memiliki tiga metode, di antaranya: Dakwah Lisan
(da’wah bil lisan), Dakwah Tulisan (da’wah bil qalam), dan Dakwah Tindakan (da’wah
bil hal). •
Dakwah Lisan (bil lisan) (
)
Dakwah dengan metode lisan merupakan metode yang digunakan oleh para
rosul untuk menyampaikan ajaran Allah SWT kepada umatnya, bahkan
hingga saat ini dakwah lisan masih banyak digunakan oleh para da’i. Salah
satu contoh dakwah lisan ini misalnya khotbah jum’at, ceramah-ceramah
yang disampaikan di masjid dan juga majelis taklim. Umumnya pesan-pesan
dakwah yang disampaikan dalam dakwah lisan memiliki sifat ringan,
informatif, dan tidak mengundang perdebatan. Dalam dakwah lisan seorang
da’i memegang otoritas untuk menyampaikan informasi keagamaan kepada
mad’u (audiens).(munir muhammad wahyu ilaihi 2009). Dakwah Tindakan (da’wah bil hal)
Secara arti kata dakwah bil hal berarti menyampaikan ajaran Islam dengan
amaliyah nyata. Berdasarkan pengertian di atas bisa dikatakan bahwa
dakwah bil hal mempunyai prospek, peran penting dalam dakwah. Da’i
sebagai agen perubahan dapat melakukan perubahan dan pembangunan
umat dengan cara dakwah bil hal. Secara arti kata dakwah bil hal berarti menyampaikan ajaran Islam dengan
amaliyah nyata. Berdasarkan pengertian di atas bisa dikatakan bahwa
dakwah bil hal mempunyai prospek, peran penting dalam dakwah. Da’i
sebagai agen perubahan dapat melakukan perubahan dan pembangunan
umat dengan cara dakwah bil hal. Dakwah
bil
hal
bisa
dikatakan
sebagai
upaya
yang
bersifat
menumbuhkembangkan kesadaran dan kemampuan mad’u untuk mengatasi
berbagai masalah kehidupan dengan cara turut serta dalam mengatasi
persoalan yang terjadi. Dakwah bil hal juga merupakan suatu upaya dakwah
dengan melakukan perbuatan nyata, dengan wujud yang beraneka ragam,
dapat berupa bantuan materi ataupun nonmateri seperti pembuatan
sekolah, rumah sakit, rumah yatim piatu dan lain sebagainya.(Sagir Akhmad
n.d.) Muttaqien: E-ISSN : 2723-5963 | 23 Muttaqien, Vol. 3. No. 1 Januari 2022, 15-41 •
Dakwah Tulisan (da’wah bil qalam)
Da’wah bil qalam merupakan suatu upaya mengajak atau menyeru manusia
dengan cara bijaksana kepada jalan yang benar sesuai dengan perintah Allah
SWT melalui karya tulis. 5. Metode Dakwah Ali bin Abi Thalib pernah berkata “Ikatlah ilmu
dengan tulisan” dan “Tulisan ialah taman para ulama”. Melalui tulisanlah
para ulama mengabadikan dan menyebarluaskan pandangan dan pemikiran
keislamannya. Kitab kuning yang saat ini menjadi pegangan para pelajar,
santri dan yang lainnya merupakan salah satu produk dari dakwah bil qalam
oleh para da’i terdahulu. Da’wah bil qalam memiliki efisiensi dalam kegiatan penyampaiannya kepada
khalayak luas. Para ulama maupun pemimpin menggunakan ilmu jurnalistik
untuk mendesain dengan sedemikian rupa sampai akhirnya pembaca suatu
buku, surat kabar, majalah, maupun karya tulis lainnya mampu disisipkan
unsur Islam maupun dakwah dalam tulisannya.(Fitria, Rini n.d.). •
Dakwah Tulisan (da’wah bil qalam)
Da’wah bil qalam merupakan suatu upaya mengajak atau menyeru manusia
dengan cara bijaksana kepada jalan yang benar sesuai dengan perintah Allah
SWT melalui karya tulis. Ali bin Abi Thalib pernah berkata “Ikatlah ilmu
dengan tulisan” dan “Tulisan ialah taman para ulama”. Melalui tulisanlah
para ulama mengabadikan dan menyebarluaskan pandangan dan pemikiran
keislamannya. Kitab kuning yang saat ini menjadi pegangan para pelajar,
santri dan yang lainnya merupakan salah satu produk dari dakwah bil qalam
oleh para da’i terdahulu. Da’wah bil qalam memiliki efisiensi dalam kegiatan penyampaiannya kepada
khalayak luas. Para ulama maupun pemimpin menggunakan ilmu jurnalistik
untuk mendesain dengan sedemikian rupa sampai akhirnya pembaca suatu
buku, surat kabar, majalah, maupun karya tulis lainnya mampu disisipkan
unsur Islam maupun dakwah dalam tulisannya.(Fitria, Rini n.d.). 6. Pengertian Dakwah Bil Qalam 6. Pengertian Dakwah Bil Qalam
Istilah “Dakwah Bil qalam” mungkin masih asing di telingan banyak orang, tidak
seperti istilah “Dakwah Bil-Lisan” dan “Dakwah Bil hal”. Penggunaan nama
“Qolam” merujuk kepada firman Allah SWT dalam (QS Al-Qalam: 1):(Asep
Syamsul M. Romli 2003)
ن
ط
ن ؤلقل ؤ ا 6. Pengertian Dakwah Bil Qalam
Istilah “Dakwah Bil qalam” mungkin masih asing di telingan banyak orang, tidak
seperti istilah “Dakwah Bil-Lisan” dan “Dakwah Bil hal”. Penggunaan nama
“Qolam” merujuk kepada firman Allah SWT dalam (QS Al-Qalam: 1):(Asep
Syamsul M. Romli 2003) Artinya :“Nun, demi kalam dan apa yang mereka tulis” Dakwah bil qalam adalah metode dakwah melalui pena. Dakwah bil qalam disebut
juga dengan istilah “Dakwah Bil Kitabah”. Dakwah ini dilaksanakan melalui aktivitas
yang melibatkan kepenulisan seperti risalah, majalah, artikel, cerpen, sajak, poster,
buku, internet, koran, dan tulisan-tulisan yang mengandung pesan dakwah.(Tata
Sukayat 2009) Sebuah peradaban akan lenyap tanpa sebuah tulisan. Al-Qur’an,
hadis, dan juga ilmu fiqih dari berbagai mazhab yang dibaca hari ini merupakan
tulisan yang telah dipublikasikan. Metode dakwah bil qalam ini merupakan buah
dari keterampilan menulis seorang da’i. Keterampilan tangan ini tidak hanya 24 | Muttaqien: E-ISSN : 2723-5963 Analisis Pesan Dakwah … (Rina, dkk) berbentuk tulisan, namun juga berbentuk gambar yang mengandung misi
dakwah.(Aziz 2004) . Dakwah bil qalam merupakan sebuah investasi bagi masa mendatang. Buku yang
terdokumentasi memiliki kelebihan yaitu umur yang panjang, boleh jadi penulisnya
sudah meninggal dunia, tetapi ide dan pemikirannya masih bisa dibaca oleh lintas
generasi. Hadis Rasulullah SAW tidak akan bisa diketahui jika saja tidak ada
pendakwah yang menulis hadis tersebut pada masa sebelumnya. Inilah yang menjadi
motivasi dakwah bil qalam. Keunggulan dakwah bil qalam dibandingkan dengan format dakwah bentuk lain
adalah sifat objeknya yang massif dan cakupannya yang luas. Pesan dakwah bil
qalam dapat diterima oleh ratusan, ribuan, ratusan ribu, bahkan jutaan orang
pembaca.(Asep Syamsul M. Romli 2003) Rasulullah SAW bersabda “Sesungguhnya
tinta para ulama adalah lebih baik dari darahnya para syuhada.”(Aziz 2004)
Demikian beberapa kelebihan dari model dakwah bil qalam. Seorang da’i yang melakukan dakwah dengan tulisan tidak memiliki beban
fsikologis yang berat, sebagaimana dakwah dengan lisan. Biasanya banyak para
mad’u (audiens) yang berekspektasi tinggi terhadap pendakwah, misalnya seorang
da’i harus berperilaku seperti nabi, baik dari segi pakaian, sikap dan bertindak,
apabila ada hal dari diri da’i yang tidak sejalan dengan harapan mad’u, maka akan
berpengaruh terhadap kredibilitas da’i itu sendiri. Lain halnya dengan tulisan,
seorang pembaca hanya akan membaca tulisan saja tanpa melihat siapa dan seperti
apa penulisnya.(Asep Syamsul M. Romli 2003). “Tulisan adalah tamannya para ulama,” kata Ali bin Abi Thalib. Lewat tulisan-
tulisanlah para ulama “mengabadikan” dan menyebar-luaskan pandangan-
pandangan keislamannya. Dakwah bil qalam yang telah dilakukan para ulama salaf
atau cendekiawan muslim terdahulu, telah melahirkan sejumlah “Kitab Kuning”
(buku teks para santri di pesantren-pesantren). Mungkin, jika tidak dituangkan Muttaqien: E-ISSN : 2723-5963 | 25 Muttaqien, Vol. 3. No. 1 Januari 2022, 15-41 dalam tulisan, maka pendapat para ulama dan mujtahid sulit dipelajari dan diketahui
dewasa ini.(Asep Syamsul M. Romli 2003). Artinya :“Nun, demi kalam dan apa yang mereka tulis” Tulisan dan bacaan adalah media dakwah yang tidak kurang vitalnya dari angkatan
mujahidin dan mubalighin yang bergerak setiap masa ke segala pelosok dunia;
membuka hati masyarakat dari genggaman dan belenggu paham dan aliran luar
Islam. Masyarakat Islam dalam segala tingkatan, keluarga dan rumah tangga kaum
muslimin, harus kita masuki dengan bacaan-bacaan Islam, mengembalikan mereka
kepada kehidupan Islam.(Asep Syamsul M. Romli 2003). 7. Pengertian Novel Istilah novel berasal dari bahasa Italia novella yang berarti ‘sebuah barang baru yang
kecil’. Dewasa ini istilah novella dan novelle mengandung pengertian yang sama
dengan istilah Indonesia ‘novelet’ (Inggris novelette), yang berarti sebuah karya
prosa fiksi yang panjangnya cukupan, tidak terlalu panjang, namun juga tidak terlalu
pendek. Novel menceritakan berbagai masalah kehidupan manusia dalam interaksinya
dengan sesama dan lingkungannya, juga interaksinya dengan diri sendiri dan Tuhan. Novel merupakan hasil dialog, kontempelasi, dan reaksi pengarang terhadap
kehidupan dan lingkungannya, setelah melalui penghayatan dan perenungan secara
intens. Pendek kata, novel merupakan karya imajinatif yang dilandasi kesadaran dan
tanggung jawab kreatif sebagai karya seni yang berunsur estetik dengan
menawarkan model-model kehidupan yang diidealkan pengarang.(Pradotokusumo
2005) C. Metode Penelitian 26 | Muttaqien: E-ISSN : 2723-5963
C. Metode Penelitian
Penelitian ini menggunakan pendekatan kualitatif dengan jenis penelitian studi
kepustakaan atau library research, yakni memiliki sumber utama berupa buku novel
“Hilda” karya Muyassarotul Hafidzoh. Penelitian kualitatif adalah penelitian yang digunakan untuk meneliti pada kondisi objek
alamiah, dimana peneliti merupakan instrumen kunci.(Harahap 2020) penelitian Penelitian ini menggunakan pendekatan kualitatif dengan jenis penelitian studi
kepustakaan atau library research, yakni memiliki sumber utama berupa buku novel
“Hilda” karya Muyassarotul Hafidzoh. Penelitian ini menggunakan pendekatan kualitatif dengan jenis penelitian studi
kepustakaan atau library research, yakni memiliki sumber utama berupa buku novel
“Hilda” karya Muyassarotul Hafidzoh. 26 | Muttaqien: E-ISSN : 2723-5963
Penelitian kualitatif adalah penelitian yang digunakan untuk meneliti pada kondisi objek
alamiah, dimana peneliti merupakan instrumen kunci.(Harahap 2020) penelitian 26 | Muttaqien: E-ISSN : 2723-5963
Penelitian kualitatif adalah penelitian yang digunakan untuk meneliti pada kondisi objek
alamiah, dimana peneliti merupakan instrumen kunci.(Harahap 2020) penelitian Analisis Pesan Dakwah … (Rina, dkk) kualitatif merupakan pendekatan yang bergantung pada pengamatan manusia yang
memiliki kawasan sendiri serta memiliki hubungan dengan orang lain tersebut dalam
bahasa dan peristilahannya.(Lexy J. Moleong 2018). Selain dari pada itu, dalam penelitian ini juga menggunakan hermeneutik sebagai pisau
analisis dan sebagai prosedur penafsiran. Peneliti menggunakan analisis hermeneutika
Effective History (Sejarah-Efektif) Gadamer dalam proses penggalian pesan dakwah
(pesan anti kekerasan terhadap perempuan) dan pesan dakwah dalam novel “Hilda”. Effective History dalam penjelasannya disebutkan bahwa terdapat empat tahap yang harus
dilakukan agar dapat memahami teks, yaitu: Pertama, kesadaran keterpengaruhan oleh
sejarah. Situasi hermeneutis seperti tradisi, kultur maupun pengalaman hidup tertentu
mempengaruhi pemahaman hermeneutis penafsir. Seorang penafsir harus mampu
mengatasi subyektifitasnya ketika menafsirkan sebuah teks. Kedua, keterpengaruhan
oleh situasi hermeneutik tertentu membentuk pra pemahaman (prejudice) pada diri
penafsir terhadap teks yang ditafsir. Pra pemahaman sebagai posisi awal atau prior
knowledge penafsir untuk membantu dalam memahami teks. Pra pemahaman harus
bersifat terbuka, dapat dikritisi dan direhabilitasi. Ketiga, penggabungan cakrawala atau
asimilasi horizon. Dalam proses penafsiran seorang interpretator harus sadar bahwa ada
dua cakrawala pengetahuan, atau horizon, yaitu horizon di dalam teks, dan horizon
pemahaman horizon pembaca. Kedua horizon ini selalu hadir dalam proses
pemahaman dan penafsiran. Kedua horizon tersebut dikomunikasikan, sehingga
“ketegangan antara keduanya dapat diatasi”. Keempat, penerapan atau aplikasi. Artinya
penafsir harus menangkap makna objektif dan menemukan “meaningful sense” (makna
yang berarti) sebagai pesan dari teks, disamping makna objektifnya.(Hanif 2018). C. Metode Penelitian Muttaqien: E ISSN : 2723 5963 | 27
Teori Effective History dalam hermeneutika Gadamer menjadikan teks sebagai sesuatu
yang tidak terpisahkan dengan sejarah. Sebagaimana novel “Hilda” yang tidak bisa
dipisahkan dari historis pengarangnya (Muyassarotul Hafidzoh). Oleh karena itu, untuk
mendapatkan pemahaman yang sempurna terhadap teks, peneliti melakukan Asimilasi
(peleburan) cakrawala pemahaman antara peneliti (sebagai pembaca), teks dalam novel Muttaqien: E-ISSN : 2723-5963 | 27 Muttaqien, Vol. 3. No. 1 Januari 2022, 15-41 “Hilda” dan Muyassarotul Hafidzoh (sebagai pengarang dan pemilik historis). Proses
Asimilasi Horizon dilakukan dengan cara relasi-dialogis atau melakukan dialog dengan
teks dan pengarang novel untuk kemudian menemukan pemahaman dan interpretasi
yang kaya dan bersifat produktif dengan tidak mengabaikan horizon pengarang, hal ini
sebagaimana potongan dari gagasan Gadamer Asimilasi Horizon “Peleburan
Cakrawala”. D. Hasil Penelitian dan Pembahasan 1. Pesan Anti Kekerasan Terhadap Perempuan dalam Novel “Hilda” 1. Pesan Anti Kekerasan Terhadap Perempuan dalam Novel “Hilda”
Peneliti menemukan pesan anti kekerasan dalam novel “Hilda” menggunakan
asimilasi horizon (peleburan cakrawala) yaitu, cakrawala peneliti (sebagai pembaca),
teks novel “Hilda” dan juga penulis novel (sebagai pemiliki histori). Narasi-narasi
tentang pesan anti kekerasan terhadap perempuan baik yang tersirat maupun
tersurat, yang peneliti temukan adalah sebagai berikut: a. Jangan Menyamakan Perkosaan dengan Perzinahan
Banyak yang berpendapat bahwa, perkosaan memiliki persamaan dengan
perzinahan. Dan tidak jarang perempuan juga dianggap sebagai penyebab
utama perkosaan itu terjadi dengan alasan tidak menutup aurat, bersolek
berlebihan dan terlalu cantik. Walaupun perkosaan dan perzinahan sama-sama
termasuk pada kategori seksual, tetapi keduanya adalah dua hal yang berbeda. Jika perzinahan adalah hubungan seksual di luar pernikahan yang dilakukan
dengan ‘suka sama suka’, maka perkosaan adalah hubungan seksual yang
dilakukan dengan ‘terpaksa’ karena ada pihak pemaksa (tersangka), dan yang
dipaksa (korban). Hal ini sebagaimana narasi kutipan dalam novel sebagai
berikut, “Memang, perkosaan dan perzinahan sama-sama termasuk kategori seksual yang dilarang
oleh agama. Tetapi keduanya berbeda. Perzinahan adalah hubungan seksual yang
dilakukan di luar ikatan pernikahan dan biasanya dilakukan secara sukarela antara
kedua pihak. Sementara dalam perkosaan ada unsur paksaan, sehingga ada pihak
pemaksa yakni pemerkosa, dan ada pihak yang dipaksa yakni korban. Dalam kasus ini
para laki-laki sebagai pelaku telah melakukan dua kejahatan sekaligus; dia melakukan “Memang, perkosaan dan perzinahan sama-sama termasuk kategori seksual yang dilarang
oleh agama. Tetapi keduanya berbeda. Perzinahan adalah hubungan seksual yang
dilakukan di luar ikatan pernikahan dan biasanya dilakukan secara sukarela antara
kedua pihak. Sementara dalam perkosaan ada unsur paksaan, sehingga ada pihak
pemaksa yakni pemerkosa, dan ada pihak yang dipaksa yakni korban. Dalam kasus ini
para laki-laki sebagai pelaku telah melakukan dua kejahatan sekaligus; dia melakukan 28 | Muttaqien: E-ISSN : 2723-5963 Analisis Pesan Dakwah … (Rina, dkk) perzinahan dan tindakan pemaksaan atas perempuan sehingga dengan terpaksa mau
melakukannya. Karena itu, perempuan yang jadi korban perkosaan, tidak boleh
disamakan dengan mereka yang melakukan zina, sebab mereka dipaksa, disakiti, dinodai,
dan ini berbeda dengan zina. Perempuan korban perkosaan tidak boleh dikenai hukuman
karena mereka tidak melakukan pelanggaran. Mereka adalah korban. Sebaliknya, laki-
laki pelaku perkosaan itulah yang seharusnya dihukum karena dialah yang melakukan
pelanggaran.” (Kutipan di atas merupakan kalimat yang disampaikan oleh tokoh Hilda dalam
sebuah seminar tentang dialog keagamaan). Dari kutipan di atas, menjelaskan bahwa menyamakan perkosaan dengan
perzinahan adalah hal yang fatal. D. Hasil Penelitian dan Pembahasan Karena hal itu sangat menyakiti korban. Muyas dalam narasi ini ingin lebih menekankan mengenai perbedaan antara
perkosaan dan perzinahan. Dalam kasus perkosaan, perempuan telah dipaksa,
disakiti dan dinodai, hal ini jelas berbeda dengan perzinahan. Seharusnya
perempuan yang menjadi korban perkosaan diberi perlindungan dan keadilan,
dan pelaku perkosaan harus dihukum karena tindakan kejahatannya. Menyamakan perkosaan dengan perzinahan akan membuat korban terluka
kembali. Pesan utama dalam kutipan narasi tersebut sebagaimana pernyataan Muyas
adalah pesan Ghadldlul Bashar (mengontrol cara pandang) dan hifdhul farji
(menjaga kehormatan, bukan kemaluan). Menurut Dr. Nur Rofiah, Kata bashar
tidaklah bermakna mata fisik seperti kata ‘ainun, melainkan kondisi mental saat
memandang sesuatu. Ghadldlul Bashar dengan demikian bukan penundukkan
mata, melainkan kontrol atas cara pandang. Ketika cara pandang kita pada
lawan jenis hanya sebatas makhluk seksual, interaksi pun menjadi sebatas
pejantan dan betina. Karenanya, farji menjadi sulit dijaga. Muttaqien: E-ISSN : 2723-5963 | 29
Dalam cara pandang seperti ini, interaksi dengan lawan jenis bisa menjadi arena
bersama untuk mengasah intelektualitas dan spiritualitas. Inilah perbedaan Muttaqien, Vol. 3. No. 1 Januari 2022, 15-41 mendasar antara manusia yang dikaruniai akal budi dengan makhluk lain,
seperti hewan misalnya, yang tidak dikaruniai. Libasut Taqwa dan Ghadldlul
Bashar dalam makna ini sama-sama penting untuk membangun relasi
kemitraan laki-laki dan perempuan yang bisa menjaga farji (dan aneka
pelecehan seksual), sekaligus produktif melahirkan aneka kemaslahatan di muka
bumi. b. Derajat Perempuan dan Laki-laki Sama b. Derajat Perempuan dan Laki-laki Sama
Manusia dihadapan Allah SWT adalah sama, yang membedakan keduanya adalah
ketakwaannya. Allah tidak pernah membeda-bedakan manusia berdasarkan jenis
kelaminnya baik itu laki-laki maupun perempuan, semua sama yaitu diciptakan sebagai
hamba. Maka dari itu, segala bentuk perbudakan berdasarkan perbedaan jenis kelamin
sangat tidak dianjurkan. Laki-laki dilarang untuk menggauli secara paksa (memperkosa)
dan melacurkan perempuan apabila ia menghendaki kesucian bahkan jika perempuan itu
sendiri adalah seorang budak. Hal ini sebagaimana kutipan dalam novel sebagai berikut :
“Apakah kalian tahu, dalam Al-Qur’an Allah melarang manusia menggauli atau
melacurkan seorang budak jika mereka menginginkan kesucian. Anda bisa baca firman
Allah dalam Surat An-Nur ayat 33, Wala tukrihu fatayatiku ‘ala al-bighai in aradna
tahashanan fainna allaha min ba’di ikrahihinna ghofururohim.’ Dan janganlah kamu
paksa budak-budak perempuanmu untuk melakukan pelacuran –sedang mereka sendiri
menginginkan kesucian– karena kamu hendak mencari keuntungan duniawi. Dan barang
siapa yang memaksa mereka, sesungguhnya Alah adalah Maha Pengampun dan
Penyayang,” Hilda menarik ujung jilbabnya dan menyeka air matanya. “Mari perhatikan status perempuan dalam ayat tersebut. Dia budak, yang oleh
masyarakat Arab dianggap wajar kalau diperkosa dan dilacurkan. Tapi dalam ayat itu,
Allah dengan tegas melindungi perempuan meskipun ia budak dari tindakan keji berupa
perkosaan dan pelacuran. Lalu bagaimana bisa anda dengan mudah mengatakan bahwa
tindakan perkosaan itu penyebabnya semata-mata karena perempuan?” Berikut ungkapan Hilda masih dalam sebuah dialog keagamaan. Kutipan di atas
berisi tentang larangan untuk memperkosa dan melacurkan seorang budak
perempuan ketika ia menginginkan kesucian, Pernyataan tersebut berdasarkan
dalil Qur’an Surat an-Nur ayat 33. 30 | Muttaqien: E-ISSN : 2723-5963 Analisis Pesan Dakwah … (Rina, dkk) Hal ini sebagaimana pernyataan Muyassarotul Hafidzoh sebagai berikut,
“Perempuan bahkan seorang budak saja dilindungi oleh Allah SWT ketika dia
menginginkan kesucian, di mana kita tahu, budak bisa diperlakukan semau tuannya, tapi
Al-Qur’an dengan jelas menyebutkan hal ini. Seandainya budak ini tidak kuasa menjaga
kesuciannya (tuannya tetap ingin menggauli) maka Allah Maha Pengampun dan
Penyayang. b. Derajat Perempuan dan Laki-laki Sama Artinya budak itu tidak perlu merasa kotor, begitu pula orang yang tidak
berdaya seperti Hilda, yang jelas sebagai korban, maka dia tidak menanggung dosa, karena
ampunan Allah dan Maha penyayang Allah kepada perempuan yang tidak berdaya,
bukan dikucilkan, dikeluarkan dari sekolah, dibuat bahan gunjingan, dipinggirkan,
Sebaliknya korban seperti Hilda harusnya ditolong, dilindungi, dibimbing, dan tetap diberi
haknya dalam pendidikan, hak aman dalam bersosial dll.” Allah menciptakan manusia dengan berbagai perbedaannya, yaitu perbedaan
jenis kelamin, bangsa dan suku semuanya bertujuan agar manusia saling
mengenal satu-sama lain, bukan untuk saling mendominasi dan saling
menyakiti. Hal ini telah Allah Firmankan dalam Surat Al-Hujurat ayat 13
sebagai berikut: ي ِاَ يُّهَا النَّا سُ ا
نَّا خَلَقْن كُمْ م ِنْ ذَكَرٍ وَّاُنْث ى وَ جَعَلْن كُمْ شُعُوْ بًا وَّقَبَآئِلَ لِتَعَا رَفُوْ ا
ۗ
ْاِنَّ اَكْرَمَكُمْ عِنْدَ ّٰللاه ِ اَ ت ْق ٮكُم
ۗ
اِن
ٌّٰللاه َ عَلِيْمٌ خَبِيْر ي ِاَ يُّهَا النَّا سُ ا
نَّا خَلَقْن كُمْ م ِنْ ذَكَرٍ وَّاُنْث ى وَ جَعَلْن كُمْ شُعُوْ بًا وَّقَبَآئِلَ لِتَعَا رَفُوْ ا
ۗ
ْاِنَّ اَكْرَمَكُمْ عِنْدَ ّٰللاه ِ اَ ت ْق ٮكُم
ۗ
َّاِن
ٌّٰللاه َ عَلِيْمٌ خَبِيْر Artinya : "Wahai manusia! Sungguh, Kami telah menciptakan kamu dari seorang laki-
laki dan seorang perempuan, kemudian Kami jadikan kamu berbangsa-bangsa dan
bersuku-suku agar kamu saling mengenal. Sungguh, yang paling mulia di antara kamu di
sisi Allah ialah orang yang paling bertakwa. Sungguh, Allah Maha Mengetahui, Maha
Teliti." Ayat di atas menjelaskan bahwa, sesungguhnya derajat manusia di hadapan
Allah itu adalah sama dan setara, baik laki-laki maupun perempuan. Adapun
yang membedakan keduanya adalah tingkat ketakwaannya. Maka dari itu,
tindakan saling mendominasi dan menyakiti salah satu pihak adalah tindakan
yang tidak dibenarkan oleh agama terlebih jika itu adalah sebuah tindakan
perkosaan yang sangat menyakiti kaum perempuan. Muttaqien: E-ISSN : 2723-5963 | 31 Muttaqien, Vol. 3. No. 1 Januari 2022, 15-41 c. Penghapusan Segala Bentuk Kekerasan Terhadap Perempuan Segala bentuk kekerasan haruslah diberantas, karena semua tindak kejahatan
itu sangat membahayakan kehidupan kemanusiaan. Kekerasan terhadap
perempuan ini memperlihatkan tindakan yang tidak memiliki adab dan tidak
manusiawi. Oleh karena itu, dalam hal ini pemerintah harus hadir dan memberi
perlindungan hukum kepada perempuan dari segala bentuk kekerasan. Hal ini
juga dibahas dalam novel “Hilda” berikut kutipannya: “Malam harinya Rindang tidak bisa tidur. Dia mencoba mempelajari kasus ini dan
mencari dasar-dasar hukum untuk dijadikan penguat ketika Hilda dan ibunya
menginginkan masalah yang dihadapinya diproses secara hukum. b. Derajat Perempuan dan Laki-laki Sama Dia memahami betul
bahwa peran negara harus hadir dalam kasus seperti itu, karena sudah sejak tahun 1984
pemerintah Indonesia sudah meratifikasi konvensi CEDAW (Convention on The
Elimination of All Forms of Discrimination Against Women- Konvensi Penghapusan
Segala Bentuk Diskriminasi Terhadap Perempuan) melalui undang-undang (UU) untuk
menghapuskan segala bentuk kekerasan terhadap perempuan.” Muyas dalam narasi ini ingin memberi pemahaman mengenai landasan hukum
tentang penghapusan segala bentuk kekerasan terhadap perempuan. Muyas
berharap
pemerintah
memiliki
perhatian
lebih
mengenai
pentingya
perlindungan terhadap perempuan. Sebagaimana pernyataan Muyas sebagai
berikut, “Seandainya ada pembaca dari golongan legislatif maupun eksekutif
semoga tersadar dengan hal ini, sehingga berusaha maksimal untuk
menghentikan praktik kekerasan seksual dan melindungi perempuan dengan
kebijakan-kebijakan yang adil”. Penghapusan terhadap segala bentuk kekerasan haruslah diupayakan dan
dilakukan secara serius. Perempuan sebagai warga negara harus mendapat
perlindungan hukum, agar perempuan merasa aman dan terlindungi. Sehingga
kejahatan berbasis gender seperti, KDRT, perkosaan, diksriminasi dan lainnya
tidak lagi terjadi di Indonesia, setidaknya apabila pemerintah bersikap tegas
maka kejahatan seperti ini bisa berkurang. 32 | Muttaqien: E-ISSN : 2723-5963 Analisis Pesan Dakwah … (Rina, dkk) d. Keadilan Bagi Perempuan d. Keadilan Bagi Perempuan d. Keadilan Bagi Perempuan Dalam sebuah masyarakat patriarki, yang memiliki cara pikir dan juga sikap
yang sangat didominasi oleh laki-laki. Perempuan yang menjadi korban
perkosaan cenderung disalahkan oleh berbagai pihak, tidak terkecuali oleh
pihak media. Semua terlihat dari sudut pandang penulisan sebuah judul berita
yang sarat dengan maskulinitas dan seolah kekerasan seksual itu terjadi karena
kesalahan perempuan itu sendiri. Hal ini sebagaimana narasi kutipan berikut:
“Perhatikan judul berita pertama ‘Karena Sering Berpakaian seksi, Seorang Remaja
Diperkosa Ayah Tirinya’, kemudian judul berita kedua ‘Hamil di Luar Nikah Pelajar
Ini Dilarang Melanjutkan sekolah’. Coba kalian perhatikan kesamaan dalam kedua judul
tersebut.” “Iya memang ini hanya sebuah berita, tetapi efeknya akan jauh berbeda bagi
orang yang jadi korban. Berita pun bisa menjadi sesuatu yang mengerikan bagi
korban. Kesalahan mengambil sudut pandang dalam berita, akan lebih
menyakitkan bagi korban. Jadi, menurut saya, seorang penulis atau jurnalis
harus memiliki sudut pandang yang adil dan berimbang.”
* “Yang disampaikan Hilda memang betul, ketika penulis atau jurnalis belum memiliki
pemahaman gender dengan baik, maka berita seperti contoh tersebut akan selalu disalahpahami. Efeknya adalah perempuanlah yang selalu dianggap menyebabkan kejahatan itu, perempuanlah
yang harus menanggung beban kejahatan orang lain, perempuanlah yang disudutkan, dikucilkan,
didiskriminasi, sehingga perempuan dipandang lemah.” Jelas Khotim “Oleh karena itu, kalian sebagai calon penulis harus memiliki perspektif gender yang bagus. Supaya bisa menyuarakan kebenaran dan keadilan.” “Oleh karena itu, kalian sebagai calon penulis harus memiliki perspektif gender yang bagus. Supaya bisa menyuarakan kebenaran dan keadilan.” Muttaqien: E-ISSN : 2723-5963 | 33
Dalam kutipan di atas menjelaskan bahwa pihak media juga ikut terlibat dalam
budaya patriarki. Padahal sejatinya media harus memiliki sudut pandang yang
berimbang. Karena apabila media tidak berimbang dalam menulis sebuah judul
berita, tentu ini akan menentukan persepsi para pembacanya. Disini pihak Muttaqien, Vol. 3. No. 1 Januari 2022, 15-41 perempuan kembali disakiti oleh media dengan penulisan judul yang seolah
menyalahkan perempuan, dan pelaku kejahatan bebas dari pembahasan publik. Seperti pernyataan Muyas, “Terkadang media pun menyajikan hal yang tidak ramah
terhadap pemberitaan tentang kekerasan seksual. Bahkan banyak judul yang
terkesan perempuannya yang disalahkan. Jika ada jurnalis atau penulis yang
membaca ini, harapannya menjadi pertimbangan untuk kembali belajar tentang
keadilan gender”. perempuan kembali disakiti oleh media dengan penulisan judul yang seolah
menyalahkan perempuan, dan pelaku kejahatan bebas dari pembahasan publik. Seperti pernyataan Muyas, “Terkadang media pun menyajikan hal yang tidak ramah
terhadap pemberitaan tentang kekerasan seksual. Bahkan banyak judul yang
terkesan perempuannya yang disalahkan. e. Memuliakan Perempuan Perempuan haruslah dipandang sebagai manusia seutuhnya yang patut dilindungi
dan dimuliakan. Bukan sebaliknya, memandang perempuan sebagai objek seksual. Apabila perempuan dipandang sebagai objek seksual, maka yang terjadi adalah
tindak pelecehan bahkan kekerasan seksual. Hal ini tergambar dari kutipan novel
sebagai berikut: “Jadi, tolong sampaikan kepada para laki-laki tundukkan pandanganmu, tundukkan cara
pandangmu jika ingin melihat perempuan, jangan jadikan mereka objek seksual. Tapi lihatlah
mereka sebagai manusia yang patut dimuliakan, bukan dilecehkan, patut dicintai dan dikasihi
bukan dinodai dan disakiti.” Hilda semakin tidak kuasa menahan tekanan perasaannya
ketika menyampaikan kalimat-kalimat terakhirnya ini.” Seperti yang dikatakan Muyas bahwa, “Narasi ini hampir sama ya dengan sebelumnya yaitu
tentang konsep ghodlul basyar atau cara pandang” Seperti yang dikatakan Muyas bahwa, “Narasi ini hampir sama ya dengan sebelumnya yaitu
tentang konsep ghodlul basyar atau cara pandang” Kutipan di atas mengisyaratkan tentang bagaimana seharusnya laki-laki memandang
perempuan. Cara pandang ini akan menentukan perlakuan terhadap perempuan itu
sendiri. Oleh karena itu, pandanglah perempuan sebagai manusia seutuhnya yang
harus dijaga, dilindungi dan dimuliakan bukan sebaliknya disakiti dan dizalimi. d. Keadilan Bagi Perempuan Jika ada jurnalis atau penulis yang
membaca ini, harapannya menjadi pertimbangan untuk kembali belajar tentang
keadilan gender”. Oleh karena itu, sangat penting membangun kesadaran adil gender bagi sebuah
media. Dalam paragraf kutipan terakhir di atas menjelaskan bahwa, seorang jurnalis
haruslah memiliki perspektif gender yang bagus agar bisa menyuarakan sebuah
kebenaran dan keadilan. Bukan malah sebaliknya ikut menjadi pihak yang menyakiti
perempuan yang menjadi korban. Selanjutnya dalam novel juga menjelaskan tentang keadilan yang hakiki bagi
perempuan yaitu sebagai berikut: “Nah, konsep keadilan hakiki bagi perempuan adalah dengan memastikan apa yang
disebut maslahah, itu tidak membuat perempuan tambah sakit ketika mengalami
kondisi biologis perempuan seperti saat menstruasi, saat hamil, melahirkan, nifas,
dan menyusui. Kemudian memastikan perempuan tidak mengalami stigmatisasi,
marginalisasi, subordinasi, kekerasan dan beban ganda hanya karena menjadi
perempuan. Inilah yang disebut kondisi sosial.” Secara biologis perempuan mengalami hal-hal seperti menstruasi, hamil,
melahirkan, nifas dan menyusui. Durasinya bermacam-macam, bisa menit, jam,
hari, minggu dan bulan sampai tahun. Semua hal biologis yang perempuan rasakan
memberi sensasi rasa sakit di samping rasa bahagia secara psikis. Belum lagi apabila
perempuan hamil, melahirkan, nifas dan menyusui itu akibat dari sebuah perkosaan. Tentu hal ini akan memberi rasa sakit secara lahir dan batin (fisik dan psikis). Muyas
dalam narasi ini ingin memberi pemahaman tentang keadilan hakiki bagi perempuan
dan kadar kemaslahatan bersama. 34 | Muttaqien: E-ISSN : 2723-5963 Analisis Pesan Dakwah … (Rina, dkk) e. Memuliakan Perempuan Narasi 1 : “Apakah kalian semua tahu, dalam Al-Qur’an Allah melarang manusia menggauli atau melacurkan
seorang budak jika mereka menginginkan kesucian. Anda bisa baca firman Allah dalam Surat An-Nur
ayat 33, ‘Wala tukrihu fatayatikum ‘ala al bighai in aradna tahashanan litabtaghu ‘aradha al hayati al
dunya wa man yukrihhunna fainna Allaha min ba’di ikrahihinna ghofururohiim.’ Dan janganlah kamu
paksa budak-budak perempuanmu untuk melakukan pelacuran sedang mereka sendiri menginginkan
kesucian. Karena kamu hendak mencari keuntungan duniawi. Dan barang siapa yang memaksa mereka
sesungguhnya Allah adalah Maha Pengampun dan Penyayang”, Hilda menarik ujung jilbabnya dan
menyeka air matanya. “Apakah kalian semua tahu, dalam Al-Qur’an Allah melarang manusia menggauli atau melacurkan
seorang budak jika mereka menginginkan kesucian. Anda bisa baca firman Allah dalam Surat An-Nur
ayat 33, ‘Wala tukrihu fatayatikum ‘ala al bighai in aradna tahashanan litabtaghu ‘aradha al hayati al
dunya wa man yukrihhunna fainna Allaha min ba’di ikrahihinna ghofururohiim.’ Dan janganlah kamu
paksa budak-budak perempuanmu untuk melakukan pelacuran sedang mereka sendiri menginginkan
kesucian. Karena kamu hendak mencari keuntungan duniawi. Dan barang siapa yang memaksa mereka
sesungguhnya Allah adalah Maha Pengampun dan Penyayang”, Hilda menarik ujung jilbabnya dan
menyeka air matanya. “Apakah kalian semua tahu, dalam Al-Qur’an Allah melarang manusia menggauli atau melacurkan
seorang budak jika mereka menginginkan kesucian. Anda bisa baca firman Allah dalam Surat An-Nur
ayat 33, ‘Wala tukrihu fatayatikum ‘ala al bighai in aradna tahashanan litabtaghu ‘aradha al hayati al
dunya wa man yukrihhunna fainna Allaha min ba’di ikrahihinna ghofururohiim.’ Dan janganlah kamu
paksa budak-budak perempuanmu untuk melakukan pelacuran sedang mereka sendiri menginginkan
kesucian. Karena kamu hendak mencari keuntungan duniawi. Dan barang siapa yang memaksa mereka
sesungguhnya Allah adalah Maha Pengampun dan Penyayang”, Hilda menarik ujung jilbabnya dan
menyeka air matanya. Pesan dakwah dalam kutipan di atas yakni perlakukanlah perempuan sebagai
manusia seutuhnya, ia tidak boleh dipaksa melakukan hal-hal yang dapat
mendatangkan penderitaan bagi dirinya hanya untuk mencari keuntungan pribadi. Dan apabila perempuan tidak berdaya untuk menolak dan terus dipaksa, maka
Allah Maha Pengampun dan Penyayang. Jadi perempuan tidak perlu merasa jijik
dengan dirinya sendiri, tidak perlu merasa tidak pantas hidup, dan tidak punya masa
depan. Selagi perempuan terus berusaha memperbaiki diri dan mendekatkan diri
kepada Allah, Maka Allah akan mengampuni segala dosanya. Pesan dakwah dalam kutipan di atas yakni perlakukanlah perempuan sebagai
manusia seutuhnya, ia tidak boleh dipaksa melakukan hal-hal yang dapat
mendatangkan penderitaan bagi dirinya hanya untuk mencari keuntungan pribadi. 2. Pesan Dakwah dalam Novel “Hilda” Selain mencari pesan-pesan anti kekerasan terhadap perempuan, peneliti juga
menyerap pesan dakwah yang menjadi inti dari keseluruhan novel “Hilda”. Pesan
dakwah ini diperoleh dari beberapa narasi kutipan yang kemudian diinterpretasi
dengan memandang secara keseluruhan teks berdasarkan isu yang diangkat yaitu
“kekerasan seksual” terhadap perempuan. Muttaqien: E-ISSN : 2723-5963 | 35
Muyassarotul Hafidzoh adalah seorang penulis yang memiliki konsentrasi terhadap
isu tentang perempuan. Kepeduliannya tentang perempuan membuat ia peka
terhadap segala hal yang terjadi pada kaum perempuan. Dalam novel ‘Hilda’ ini
sangat terlihat upaya Muyas untuk memberdayakan kaum perempuan, khususnya
perempuan yang menjadi korban kekerasan seksual untuk kembali bangkit dan Muttaqien, Vol. 3. No. 1 Januari 2022, 15-41 menata kehidupan serta tidak kehilangan harapan. Pesan dakwah atau pesan
universal yang peneliti temukan dalam novel “Hilda” adalah pesan ‘akhlak kepada
perempuan’, lebih tepatnya akhlak untuk memanusiakan perempuan. Berikut
merupakan narasi dalam novel yang mengandung pesan akhlak kepada perempuan: 6 | Muttaqien: E-ISSN : 2723-5963 Narasi 1 : Dan apabila perempuan tidak berdaya untuk menolak dan terus dipaksa, maka
Allah Maha Pengampun dan Penyayang. Jadi perempuan tidak perlu merasa jijik
dengan dirinya sendiri, tidak perlu merasa tidak pantas hidup, dan tidak punya masa
depan. Selagi perempuan terus berusaha memperbaiki diri dan mendekatkan diri
kepada Allah, Maka Allah akan mengampuni segala dosanya. b. Memuliakan Perempuan dengan Menundukkan Pandangan b. Memuliakan Perempuan dengan Menundukkan Pandangan “Jadi, jika laki-laki tidak bisa menahan hasratnya ketika memandang perempuan, maka tundukkan
pandangannya dan berpuasa. Bukan malah memperkosa perempuan, meskipun dia tidak menutup
aurat.” Pesan dakwah dalam kutipan di atas yaitu tentang bagaimana seharusnya laki-laki
bersikap terhadap perempuan. Laki-laki hendaknya melihat perempuan sebagai
manusia seutuhnya, bukan sebagai objek seksual. Karena apabila laki-laki melihat
perempuan sebagai objek seksual, maka yang terjadi adalah pelecehan bahkan tidak 36 | Muttaqien: E-ISSN : 2723-5963 Analisis Pesan Dakwah … (Rina, dkk) perkosaan. Menundukkan pandangan adalah salah satu cara untuk memuliakan
perempuan. perkosaan. Menundukkan pandangan adalah salah satu cara untuk memuliakan
perempuan. Narasi 4 : “Oh iya mas, saya ingat satu hadis dalam Sunan Turmudzi, nomer 1195 yang berbunyi ‘An Abi
Hurairota radhiyallahu ‘anhu qala, qala Rosulullah SAW, akmalul mu’minina imanan ahsanuhum
khuluqan, wa khiyarukum linisaihim khuluqan, “kataku. “Oh iya mas, saya ingat satu hadis dalam Sunan Turmudzi, nomer 1195 yang berbunyi ‘An Abi
Hurairota radhiyallahu ‘anhu qala, qala Rosulullah SAW, akmalul mu’minina imanan ahsanuhum
khuluqan, wa khiyarukum linisaihim khuluqan, “kataku. “Sip, bahwa mukmin yang paling sempurna adalah mereka yang memiliki akhlak mulia dan sebaik-
baik kamu adalah dia yang berperilaku baik terhadap perempuan. Sedangkan paham ekstremis sangat
bertolak belakang dengan hadis tersebut, begitukan maksudmu?” Tanya Mas Wafa. “Sip, bahwa mukmin yang paling sempurna adalah mereka yang memiliki akhlak mulia dan sebaik-
baik kamu adalah dia yang berperilaku baik terhadap perempuan. Sedangkan paham ekstremis sangat
bertolak belakang dengan hadis tersebut, begitukan maksudmu?” Tanya Mas Wafa. Pesan dakwah dalam kutipan tersebut adalah tentang akhlak yang dicintai Nabi
SAW. Dalam hadis dijelaskan bahwa seorang mukmin yang sempurna adalah
mereka yang memiliki akhlak mulia, dan sebaik-baik akhlak itu adalah yang
berperilaku baik terhadap wanita. Dalam hal ini, Nabi mengajarkan kepada umatnya
untuk senantiasa melindungi dan memuliakan perempuan, karena perempuan
adalah simbol kasih sayang, dan jangan menjadikan mereka sebagai objek yang
dikorbankan. . Berperilaku Adil Kepada Istri dengan Monogami
Narasi 5 :
“Membandingkan dan menilai masa lalu dengan masa kini itu kurang arif. Rasulullah memiliki lebih
dari empat istri dan tentu tidak bisa kita bandingkan dengan umatnya, apalagi umatnya yang hidup di
zaman sekarang. Dulu, di zaman nabi, banyak sekali raja-raja yang memiliki selir yang jumlahnya tak
terbatas. Belum lagi budak perempuan yang juga jumlahnya tak terbatas. Jika kita bandingkan raja-raja
yang hidup pada zaman itu dengan Nabi, maka sangat jelas sekali perbedaannya. Nabi memang
memiliki lebih dari empat istri namun statusnya resmi sebagai istri Nabi, buka selir ataupun budak. Narasi 3 : “Jadi tolong sampaikan kepada para laki-laki jika ingin melihat perempuan, jangan jadikan mereka
objek seksual. Tapi lihatlah mereka sebagai manusia yang patut dimuliakan, bukan dilecehkan, patut
dicintai dan dikasihi bukan dinodai dan disakiti.” Hilda semakin tidak kuasa menahan tekanan
perasaannya ketika menyampaikan kalimat-kalimat terakhirnya ini. `Pesan dakwah dalam kutipan di atas hampir sama dengan narasi kedua, yakni
tentang bagaimana seharusnya laki-laki memandang perempuan. Perempuan adalah
makhluk yang patut dimuliakan, di hormati dan dicintai bukan malah sebaliknya
didominasi bahkan disakiti. c. Berperilaku Baik Terhadap Perempuan Adalah Akhlak Mulia Narasi 4 : Narasi 5 : “Membandingkan dan menilai masa lalu dengan masa kini itu kurang arif. Rasulullah memiliki lebih
dari empat istri dan tentu tidak bisa kita bandingkan dengan umatnya, apalagi umatnya yang hidup di
zaman sekarang. Dulu, di zaman nabi, banyak sekali raja-raja yang memiliki selir yang jumlahnya tak
terbatas. Belum lagi budak perempuan yang juga jumlahnya tak terbatas. Jika kita bandingkan raja-raja
yang hidup pada zaman itu dengan Nabi, maka sangat jelas sekali perbedaannya. Nabi memang
memiliki lebih dari empat istri namun statusnya resmi sebagai istri Nabi, buka selir ataupun budak. “Membandingkan dan menilai masa lalu dengan masa kini itu kurang arif. Rasulullah memiliki lebih
dari empat istri dan tentu tidak bisa kita bandingkan dengan umatnya, apalagi umatnya yang hidup di
zaman sekarang. Dulu, di zaman nabi, banyak sekali raja-raja yang memiliki selir yang jumlahnya tak
terbatas. Belum lagi budak perempuan yang juga jumlahnya tak terbatas. Jika kita bandingkan raja-raja
yang hidup pada zaman itu dengan Nabi, maka sangat jelas sekali perbedaannya. Nabi memang
memiliki lebih dari empat istri namun statusnya resmi sebagai istri Nabi, buka selir ataupun budak. Muttaqien: E-ISSN : 2723-5963 | 37 Muttaqien, Vol. 3. No. 1 Januari 2022, 15-41 Dari kisah itu, sesungguhnya Nabi sedang mengajarkan kepada para penguasa, para raja-raja saat itu
untuk memperlakukan perempuan dengan baik, tidak seenaknya mempermainkan perasaan mereka.” Dari kisah itu, sesungguhnya Nabi sedang mengajarkan kepada para penguasa, para raja-raja saat itu
untuk memperlakukan perempuan dengan baik, tidak seenaknya mempermainkan perasaan mereka.”
Pesan dakwah dalam narasi di atas yakni perintah untuk memperlakukan
perempuan dengan baik dan tidak mempermainkan perasaannya. Ketika Nabi SAW
menikahi lebih dari satu istri tujuannya adalah untuk menolong para wanita yang
pada saat itu kondisinya dalam kesulitan, seperti wanita janda yang ditinggalkan oleh
suaminya karena syahid di jalan Allah. Dari kisah ini, sesungguhnya poligami dalam
Islam sebenarnya menjadi aturan yang berlaku ketika terjadi darurat sosial, tidak
dalam kondisi normal. Semua perempuan pasti mengharapkan pernikahan
monogami bukan poligami. Dari kisah itu, sesungguhnya Nabi sedang mengajarkan kepada para penguasa, para raja-raja saat itu
untuk memperlakukan perempuan dengan baik, tidak seenaknya mempermainkan perasaan mereka.” e. Istri Seperti Ladang yang Harus Dijaga dan Dirawat e. Istri Seperti Ladang yang Harus Dijaga dan Dirawat Narasi 6 :
“Terimakasih atas pertanyaannya Mbak Hilda. Sebelum saya menjelaskan lebih dalam, saya ingin
bertanya kepada semua. Silahkan jawab pertanyaan saya; istri seperti ladang bagi suaminya, maka istri
harus…?” Para peserta saling pandang satu sama lain. Sebagian besar mereka menjawab bahwa istri harus patuh
pada suami. Sementara Hilda hanya diam dan tidak menjawab pertanyaan itu. Ia sendiri tidak setuju
atas jawaban sebagian besar peserta. j
g
p
“Maka istri harus diperlakukan dengan baik,” salah seorang peserta mengangkat tangannya dan
menjawab dengan lantang. “Nah, jawaban ini yang saya tunggu. Teks pada ayat ini sama sekali tidak ada kesalahan, dan Maha
Benar Allah dengan segala firman-Nya. Akan tetapi memahami teks tersebut kita butuh sebuah
perspektif. Jika memang ayat tersebut menggambarkan bahwa istri sebagai ladang yang bisa dicocok
tanami, maka perlakukan ladang tersebut dengan baik, dengan cara diberi pupuk, dibersihkan, tidak
disakiti, tidak dikotori dan lainnya.” Pesan dakwah dalam kutipan di atas yakni jika istri di metaforkan sebagai ladang,
maka ladang tersebut harus dijaga, dirawat dan diberi pupuk supaya dapat
mendatangkan kebaikan bagi yang menanamnya. Begitupula dengan seorang istri,
jika ia diperlakukan dengan baik, diberi kasih sayang dan dijaga oleh suaminya,
maka ia akan menjadi istri yang dapat mendatangkan kebaikan kepada suami dan
juga anak-anaknya. Kemuliaan manusia bukan semata-mata karena jenis kelamin melainkan karena
ketawaan dan amal solehnya, hal ini dijelaskan dalam QS. Al-Hujurat ayat 13 yang
berbunyi: َي اَ يُّهَا النَّا سُ اِنَّا خَلَقْن كُمْ م ِنْ ذَك ْرٍ وَّاُن َث ى و
جَعَلْن كُمْ شُعُوْ بًا وَّقَبَآئِلَ لِتَعَا رَفُوْ ا
ۗ
ْاِنَّ اَكْرَمَكُمْ عِنْدَ ّٰللاه ِ اَ تْق ٮكُم
ۗ
اِن
ِّٰللاه َ عَلِيْمٌ خَب ٌيْر 38 | Muttaqien: E-ISSN : 2723-5963 Analisis Pesan Dakwah … (Rina, dkk) Artinya : "Wahai manusia! Sungguh, Kami telah menciptakan kamu dari seorang laki-laki
dan seorang perempuan, kemudian Kami jadikan kamu berbangsa-bangsa dan bersuku-suku
agar kamu saling mengenal. Sungguh, yang paling mulia di antara kamu di sisi Allah ialah
orang yang paling bertakwa. Sungguh, Allah Maha Mengetahui, Maha Teliti." Artinya : "Wahai manusia! Sungguh, Kami telah menciptakan kamu dari seorang laki-laki
dan seorang perempuan, kemudian Kami jadikan kamu berbangsa-bangsa dan bersuku-suku
agar kamu saling mengenal. Sungguh, yang paling mulia di antara kamu di sisi Allah ialah
orang yang paling bertakwa. Sungguh, Allah Maha Mengetahui, Maha Teliti." Dari ayat di atas menjelaskan bahwa Allah SWT menciptakan perbedaan pada
manusia fungsinya adalah untuk saling mengenal, bukan untuk saling menyakiti atau
mendominasi. e. Istri Seperti Ladang yang Harus Dijaga dan Dirawat Dilanjutkan dalam ayat tersebut bahwa manusia yang paling mulia
yaitu yang paling bertakwa. Sejatinya derajat laki-laki dan perempuan itu sama
dihadapan Allah SWT, dan yang membedakan keduanya adalah tingkat
ketakwaannya. Dari ayat di atas menjelaskan bahwa Allah SWT menciptakan perbedaan pada
manusia fungsinya adalah untuk saling mengenal, bukan untuk saling menyakiti atau
mendominasi. Dilanjutkan dalam ayat tersebut bahwa manusia yang paling mulia
yaitu yang paling bertakwa. Sejatinya derajat laki-laki dan perempuan itu sama
dihadapan Allah SWT, dan yang membedakan keduanya adalah tingkat
ketakwaannya. Tugas manusia di dunia ini adalah untuk beribadah dan juga melakukan amar
ma’ruf nahi munkar. Beribadah hanya kepada Allah, dan pada hakikatnya amar
ma’ruf memiliki arti perilaku taat kepada Allah dan juga Rasulnya serta memuliakan
sesama manusia lainnya. Termasuk di antaranya ada salah satu akhlak yang
Rasulullah SAW memberikan pujian kepadanya yaitu akhlak baik seorang laki-laki
kepada perempuan. Islam telah mendobrak relasi ini dan menegaskan bahwa perempuan bukan hamba
laki-laki, sebab keduanya sama-sama memiliki status melekat sebagai hamba Allah
SWT. Laki-laki juga bukan patron (suri teladan) perempuan, sebab keduanya sama-
sama mengemban amanah melekat sebagai khalifah fil ardh sehingga harus jadi
mitra dalam memakmurkan bumi.(Nur Rofiah 2020) Hal ini sebagaimana
ditegaskan di dalam Q.S Al-An’am ayat 165: ا ِلْعِقَاب ُؤَاِنَه
ٌلَغَفُؤْرٌرَحِ يْم ا ِلْعِقَاب ُؤَاِنَه
ٌلَغَفُؤْرٌرَحِ يْم Artinya: “Dan Dialah yang menjadikan kalian penguasa-penguasa di bumi dan Dia
meninggikan sebahagian kalian atas sebahagian (yang lain) beberapa derajat, untuk mengujimu
tentang apa yang diberikan-Nya kepada kalian. Sesungguhnya Tuhan kalian amat cepat
siksaan-Nya, dan sesungguhnya Dia Maha Pengampun lagi Maha Penyayang.” Muttaqien: E-ISSN : 2723-5963 | 39 Muttaqien, Vol. 3. No. 1 Januari 2022, 15-41 Kata khalifah dalam ayat tersebut tidak menunjuk kepada salah satu jenis kelamin
atau kelompok etnis tertentu. Laki-laki dan perempuan mempunyai fungsi yang
sama sebagai khalifah, yang akan mempertanggungjawabkan tugas-tugas
kekhalifahan di bumi, sebagaimana halnya mereka harus bertanggung jawab sebagai
hamba Tuhan.(Nasaruddin Umar 1999) Kata khalifah dalam ayat tersebut tidak menunjuk kepada salah satu jenis kelamin
atau kelompok etnis tertentu. Laki-laki dan perempuan mempunyai fungsi yang
sama sebagai khalifah, yang akan mempertanggungjawabkan tugas-tugas
kekhalifahan di bumi, sebagaimana halnya mereka harus bertanggung jawab sebagai
hamba Tuhan.(Nasaruddin Umar 1999) Selain daripada itu, Muyas juga mengatakan bahwa, “Penafsiran nash atau teks
agama yang tidak adil, akan menimbulkan ketidakadilan. Contohnya perbedaan zina
dan perkosaan, jika ada yang menyamakan hukum zina dan perkosaan maka sebagai
korban akan merasa dirinya kotor, jijik, tidak layak menjadi istri. Tidak sedikit
korban kekerasan seksual berujung kematian. Lembaga pendidikan seharusnya
menjadi rumah aman bagi korban”. Itulah harapan dari penulis novel “Hilda”. 3. Penutup . Penutup
Berdasarkan uraian yang telah dipaparkan dalam pembahasan, maka dapat diambil
kesimpulan bahwa novel “Hilda” adalah novel yang membawa misi dakwah dan pesan
dakwah tentang bagaimana seharusnya memperlakukan perempuan, lebih tepatnya misi
pemberdayaan perempuan yang menjadi korban kekerasan seksual. Adapun pesan anti
kekerasan terhadap perempuan dalam novel tersebut yakni: Jangan menyamakan
perkosaan dengan perzinahan, derajat perempuan dan laki-laki adalah sama,
penghapusan segala bentuk kekerasan terhadap perempuan, keadilan bagi perempuan
dan memuliakan perempuan. Sedangkan pesan dakwah atau pesan universal dalam
novel adalah tentang pesan akhlak kepada perempuan. Adapun pesan dakwah tersebut
yakni: larangan melacurkan budak perempuan, memuliakan perempuan dengan
menundukkan pandangan, berperilaku baik pada perempuan adalah akhlak mulia,
berperilaku adil kepada istri dengan monogami, dan istri seperti ladang yang harus
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Aziz, Ali. 2004. “Edisi Revisi Ilmu Dakwah.” 444. Fitria, Rini, Rafinita Aditia. n.d. “Prosfek Dan Tantangan Dakwah Bil Qalam Sebagai
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English
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Stabilization of arbitrary structures in a doubly degenerate reaction-diffusion system modeling bacterial motion on a nutrient-poor agar
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Calculus of variations and partial differential equations
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cc-by
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Calc. Var. (2022) 61:108
https://doi.org/10.1007/s00526-021-02168-2 Calc. Var. (2022) 61:108
https://doi.org/10.1007/s00526-021-02168-2 Calculus of Variations Communicated by P. H. Rabinowitz. B Michael Winkler
michael.winkler@math.uni-paderborn.de Stabilization of arbitrary structures in a doubly degenerate
reaction-diffusion system modeling bacterial motion on a
nutrient-poor agar Michael Winkler1 Received: 17 June 2021 / Accepted: 22 December 2021 / Published online: 15 April 2022
© The Author(s) 2022 1
Institut für Mathematik, Universität Paderborn, Paderborn 33098, Germany Mathematics subject classification 35B36 (primary) · 35B40, 35K65, 35K59, 92C17
(secondary) Abstract A no-flux initial-boundary value problem for the doubly degenrate parabolic system ut = ∇·
uv∇u
+ ℓuv,
vt = v −uv,
(⋆) ut = ∇·
uv∇u
+ ℓuv,
vt = v −uv, is considered in a smoothly bounded convex domain ⊂Rn, with n ≥1 and ℓ≥0. The first
of the main results asserts that for nonnegative initial data (u0, v0) ∈(L∞())2 with u0 ̸≡0,
v0 ̸≡0 and √v0 ∈W 1,2(), there exists a global weak solution (u, v) which, inter alia,
belongs to C0(×(0, ∞))×C2,1(×(0, ∞)) and satisfies supt>0 ∥u(·, t)∥L p() < ∞for
all p ∈[1, p0) with p0 :=
n
(n−2)+ . It is next seen that for each of these solutions one can find
u∞∈
p∈[1,p0) L p() such that, within an appropriate topological setting, (u(·, t), v(·, t))
approaches the equilibrium (u∞, 0) in the large time limit. Finally, in the case n ≤5 a result
ensuring a certain stability property of any member in the uncountably large family of steady
states (u0, 0), with arbitrary and suitably regular u0 : →[0, ∞), is derived. This provides
some rigorous evidence for the appropriateness of (⋆) to model the emergence of a strikingly
large variety of stable structures observed in experiments on bacterial motion in nutrient-poor
environments. Essential parts of the analysis rely on the use of an apparently novel class of
functional inequalities to suitably cope with the doubly degenerate diffusion mechanism in
(⋆). is considered in a smoothly bounded convex domain ⊂Rn, with n ≥1 and ℓ≥0. The first
of the main results asserts that for nonnegative initial data (u0, v0) ∈(L∞())2 with u0 ̸≡0,
v0 ̸≡0 and √v0 ∈W 1,2(), there exists a global weak solution (u, v) which, inter alia,
belongs to C0(×(0, ∞))×C2,1(×(0, ∞)) and satisfies supt>0 ∥u(·, t)∥L p() < ∞for
all p ∈[1, p0) with p0 :=
n
(n−2)+ . It is next seen that for each of these solutions one can find
u∞∈
[1
) L p() such that within an appropriate topological setting (u(· t) v(· t)) is considered in a smoothly bounded convex domain ⊂Rn, with n ≥1 and ℓ≥0. 1 Introduction The question to which extent simple reaction-diffusion models can describe essential aspects
of structure evolution in nature, and especially in living systems, has been stimulating con-
siderable parts of the literature on parabolic problems over the past decades. Within the realm
of bounded solutions, a dissipation-induced trend toward asymptotic equilibration has rigor-
ously been confirmed to predominate in large classes of such systems ( [21], [22], [12], [14],
[2] [9]). In line with this, substantial efforts have been focused on the identification of cir-
cumstances ensuring existence and favorable stability features of suitably structured steady
states. Descriptions of structure-supporting properties on the basis of parabolic systems that
exclusively involve reasonably regular ingredients seem accordingly constrained by natural
limitations on richness of respectively relevant equilibria, despite a considerable collection
of exceptions documented in the literature which assert the occurrence of impressively subtle
stationary profiles especially near critical parameter settings (cf. [7], [8], [6], [4], [28], [24],
[5] and also [25] for a very incomplete selection of findings in this regard). The present study now addresses a modeling context which, despite a remarkable simpleness,
seems characterized by an exceptionally large variety of evolutionary target states. Specif-
ically, experimental findings reported in [11], [10] and [23] illustrate noticeably intricate
facets in the collective behavior of populations of Bacillus subtilis when exposed to nutrient-
poor environments; in particular, evolution into complex patterns, and even snowflake-like
population distributions, appears as a generic feature rather than an exceptional event in such
frameworks. Following experimentally gained indications for certain limitations of bacterial
motility near regions of small nutrient concentrations, as a mathematical description for such
processes the authors in [15] (cf. also [19] and [26]) propose the parabolic system
ut = D∇·
uv∇u
+ ℓuv,
vt = dv −ϑuv,
(1.1) (1.1) for the population density u = u(x, t) and the food resource distribution v = v(x, t). Forming the apparently most prominent ingredient herein, the diffusion operator in the first
equation does not only degenerate in a standard porous medium-like manner at small values
of u, but moreover accounts for said motility restrictions by containing a second degeneracy
that emerges as soon as v approaches the level zero, in view of the second equation clearly
expected to dominate at least on large time scales. Abstract The first
of the main results asserts that for nonnegative initial data (u0, v0) ∈(L∞())2 with u0 ̸≡0,
v0 ̸≡0 and √v0 ∈W 1,2(), there exists a global weak solution (u, v) which, inter alia,
belongs to C0(×(0, ∞))×C2,1(×(0, ∞)) and satisfies supt>0 ∥u(·, t)∥L p() < ∞for
all p ∈[1, p0) with p0 :=
n
(n−2)+ . It is next seen that for each of these solutions one can find
u∞∈
∈[1
) L p() such that, within an appropriate topological setting, (u(·, t), v(·, t)) u∞∈
p∈[1,p0) L p() such that, within an appropriate topological setting, (u(·, t), v(·, t))
approaches the equilibrium (u∞, 0) in the large time limit. Finally, in the case n ≤5 a result
ensuring a certain stability property of any member in the uncountably large family of steady
states (u0, 0), with arbitrary and suitably regular u0 : →[0, ∞), is derived. This provides
some rigorous evidence for the appropriateness of (⋆) to model the emergence of a strikingly
large variety of stable structures observed in experiments on bacterial motion in nutrient-poor
environments. Essential parts of the analysis rely on the use of an apparently novel class of
functional inequalities to suitably cope with the doubly degenerate diffusion mechanism in
(⋆). Mathematics subject classification 35B36 (primary) · 35B40, 35K65, 35K59, 92C17
(secondary) 123 123 108
Page 2 of 25 M. Winkler 1 Introduction (1.3) (1.3)
u0 ∈L
() is nonnegative with u0 ̸
0,
and
v0 ∈L∞() is nonnegative and such that v0 ̸≡0 and √v0 ∈W 1,2(). (1.3) It is fairly evident that in this context, any expedient analysis of (1.2) needs to appropriately
quantify the potentially weakened diffusive action therein. In our first step toward this, by
merely relying on basic L1 bounds for both components we shall use evolution properties
of the sublinear functionals
uq for q ∈(0, 1) to gain a priori estimates for expressions of
the form
T
0
uq−1v|∇u|2 within this range of q (Lemma 2.3), which will in turn imply
bounds, inter alia, for
T T
0
u
v |∇v|2
(1.4) (1.4) (Lemma 2.4). The core part of our analysis will then be concerned with the derivation of
higher L p bounds for u, where in the course of standard testing procedures a key requirement
will consist in suitably exploiting the respective diffusion-induced contributions, as quantified
through weighted expressions of type
u p−1v|∇u|2. 1 Introduction This latter peculiarity evidently brings about significant challenges beyond those mastered
by classical approaches to degenerate diffusion problems of porous medium type ( [31], [2]),
and also beyond those going along with the combination of two diffusion degeneracies due to
the inclusion of diffusion rates that vanish both at small densities and at small gradient sizes
of the diffusing quantity. In contrast to the situation in the latter class of doubly degenerate
diffusion problems, meanwhile also fairly well-explored ( [13], [16], [29], [30]), especially
the presence of a cross-degeneracy marks a considerable distinctiveness in (1.1). Accordingly,
already at the stage of questions from basic existence theory the available knowledge so far
seems limited to a statement on global solvability in a one-dimensional version ( [33]); built
on a certain rather fragile energy structure, however, the corresponding arguments in [33]
quite strongly rely not only on the presence of an additional cross-diffusive summand in the
respective first equation and on strict positivity of ℓ, but beyond this also on the integrability
assumption
ln u(·, 0) > −∞which especially rules out an analysis of solutions emanating
from compactly supported initial data. 123 Stabilization of arbitrary structures... 108 Page 3 of 25 Main results. The present manuscript now attempts to develop an approach which is
not only capable of launching a fundamental theory of general nonnegative solutions for
the simple system (1.1) in multi-dimensional settings, but which moreover also allows for
conclusions addressing qualitative questions in such cases, in particular with regard to aspects
related to stabilization of structures. For convenience setting D = d = ϑ = 1 throughout the
sequel, but keeping the parameter ℓas an arbitrary nonnegative parameter in order to include
the proliferation-free case ℓ= 0, we shall subsequently examine this in the framework of
the initial-boundary value problem ⎧
⎪⎪⎪⎨
⎪⎪⎪⎩
ut = ∇·
uv∇u
+ ℓuv,
x ∈, t > 0,
vt = v −uv,
x ∈, t > 0,
uv ∂u
∂ν = ∂v
∂ν = 0,
x ∈∂, t > 0,
u(x, 0) = u0(x),
v(x, 0) = v0(x), x ∈,
(1.2) (1.2) in a smoothly bounded domain ⊂Rn, with n ≥1, and with given initial data such that
u0 ∈L∞() is nonnegative with u0 ̸≡0,
and
u0 ∈L∞() is nonnegative with u0 ̸≡0,
and
v0 ∈L∞() is nonnegative and such that v0 ̸≡0 and √v0 ∈W 1,2(). 1 Introduction (1.5) (1.5) This will be achieved in Lemma 4.3, on the basis of the bounds known for the integral in
(1.4), by means of two functional inequalities of the form This will be achieved in Lemma 4.3, on the basis of the bounds known for the integral in
(1.4), by means of two functional inequalities of the form ∇
ϕα
ψ
2
L
2p⋆
p⋆+2α−1 () ≤C(K) ·
ϕq−1ψ|∇ϕ|2 +
ϕ
ψ |∇ψ|2
(1.6) ∇
ϕα
ψ
2
L
2p⋆
p⋆+2α−1 () ≤C(K) ·
ϕq−1ψ|∇ϕ|2 +
ϕ
ψ |∇ψ|2
(1.6)
ϕ pψ ≤C(K) ·
ϕq−1ψ|∇ϕ|2 +
ϕ
ψ |∇ψ|2 +
ϕψ
,
(1.7) (1.6) ∇
ϕα
ψ
2
L
2p⋆
p⋆+2α−1 () ≤C(K) ·
ϕq−1ψ|∇ϕ|2 +
ϕ
ψ |∇ψ|2
(1.6)
ϕ pψ ≤C(K) ·
ϕq−1ψ|∇ϕ|2 +
ϕ
ψ |∇ψ|2 +
ϕψ
,
(1.7) and
ϕ pψ ≤C(K) ·
ϕq−1ψ|∇ϕ|2 +
ϕ
ψ |∇ψ|2 +
ϕψ
,
(1.7) (1.7) valid with some C(K) > 0 for arbitrary positive ϕ ∈C1() and ψ ∈C1() fulfilling valid with some C(K) > 0 for arbitrary positive ϕ ∈C1()
ϕ p⋆≤K,
ϕ p⋆≤K, throughout certain ranges of the parameters α, p, q and p⋆(Lemma 4.1 and Lemma 4.2). Within the framework of an appropriately regularized variant of (1.2), the a priori information 123 Page 4 of 25 108 M. Winkler thereby generated will firstly enable us to make sure that for any fixed initial data fulfilling
(1.3), the problem (1.2) is solvable by functions inter alia belonging to C0( × (0, ∞)) ∩
C2,1( × (0, ∞)) and enjoying some time-independent bounds in L p() × L∞(): Theorem 1.1 Let n ≥1 and ⊂Rn be a bounded convex domain with smooth boundary,
and suppose that ℓ≥0 and that (1.3) holds. Then there exist functions
u ∈L∞
loc( × [0, ∞)) ∩C0( × (0, ∞))
and
v ∈L∞( × (0, ∞)) ∩L4
loc([0, ∞); W 1,4()) ∩C2,1( × (0, ∞))
(1.8) (1.8) such that u ≥0 and v > 0 in ×(0, ∞), and that (u, v) forms a global weak solution of (1.2)
in the sense of Definition 2.1 below. 1 Introduction Moreover, this solution has the additional boundedness
property that sup
t>0
∥u(·, t)∥L p() < ∞
for all p ∈
1,
n
(n −2)+
. (1.9) (1.9) By suitably taking into account respective dependences on time, our estimates gained in the
context of (1.4), (1.5), (1.6) and (1.7) will, besides implying the above, already pave the way
for our subsequent asymptotic analysis. By means of a duality-based argument, namely, the
latter can be seen to ensure a decay feature of the form ∞
0
∥ut(·, t)∥(W 1,∞())⋆dt ≤C ·
v0
λ
,
(1.10) (1.10) with C = C(u0, v0) and some λ > 0, and with (W 1,∞())⋆denoting the dual space of
W 1,∞() (Lemma 5.1), and to thereby constitute the essential step toward our verification of
the following statement on large time convergence of each individual among the trajectories
obtained in Theorem 1.1. with C = C(u0, v0) and some λ > 0, and with (W 1,∞())⋆denoting the dual space of
W 1,∞() (Lemma 5.1), and to thereby constitute the essential step toward our verification of
the following statement on large time convergence of each individual among the trajectories
obtained in Theorem 1.1. Theorem 1.2 Let n ≥1 and ⊂Rn be a bounded convex domain with smooth bound-
ary, let ℓ≥0, and suppose that (1.3) holds. Then there exists a nonnegative u∞∈
p∈[1,
n
(n−2)+ ) L p() such that as t →∞, the solution (u, v) of (1.2) from Theorem 1.1
satisfies (
satisfies v(·, t) →0
in L∞()
if n ≤3,
in L p()
for all p ∈[1, ∞) if n ≥4,
(1.11) (1.11) and with some γ ∈(0, 1), as t →∞we have and with some γ ∈(0, 1), as t →∞we have ⎧
⎨
⎩
u(·, t)⇀u∞in L p()
for all p ∈
1,
n
(n−2)+
if n ≤5,
uγ (·, t)⇀uγ
∞in L
p
γ ()
for all p ∈
1,
n
(n−2)+
if n ≥6. the solution (u, v) of (1.2) obtained in Theorem 1.1 satisfies ∥u(·, t) −u0∥(W 1,∞())⋆≤η
for all t > 0. ding limit function u∞from Theorem 1.2 we then hav
∥u
u ∥
≤η for the corresponding limit function u∞from Theorem 1.2 we then hav ticular, for the corresponding limit function u∞from Theorem 1.2 we then have ∥u∞−u0∥(W 1,∞())⋆≤η. (1.16) 2 Approximation by positive classical solutions The following solution concept to be pursued below seems to generalize notions of classical
solvability in a manner fairly natural in contexts in which due to the considered diffusion
degeneracy, no substantial first order information on the solution component u is expected. Definition 2.1 Let n ≥1 and ⊂Rn be a bounded domain with smooth boundary, let ℓ≥0,
and assume that u0 ∈L1() and v0 ∈L1() are nonnegative. Then a pair of nonnegative
functions
u ∈L2
loc( × [0, ∞))
and
v ∈L∞( × (0, ∞)) ∩L1
loc([0, ∞); W 1,1())
(2.1) (2.1) such that
u2∇v ∈L1
loc( × [0, ∞); Rn)
(2.2) such that such that
u2∇v ∈L1
loc( × [0, ∞); Rn)
(2.2) u2∇v ∈L1
loc( × [0, ∞); Rn)
(2.2) u2∇v ∈L1
loc( × [0, ∞); Rn) (2.2) will be called a global weak solution of (1.2) if will be called a global weak solution of (1.2) if −
∞
0
uϕt −
u0ϕ(·, 0) = 1
2
∞
0
u2∇v · ∇ϕ + 1
2
∞
0
u2vϕ + ℓ
∞
0
uvϕ
(2 3 (2.3) for all ϕ ∈C∞
0 ( × [0, ∞)) fulfilling ∂ϕ
∂ν = 0 on ∂ × (0, ∞), and if all ϕ ∈C∞
0 ( × [0, ∞)) fulfilling ∂ϕ
∂ν = 0 on ∂ × (0, ∞), and if ∞
0
vϕt +
v0ϕ(·, 0) =
∞
0
∇v · ∇ϕ +
∞
0
uvϕ
(2.4) (2.4) for any ϕ ∈C∞
0 ( × [0, ∞)). any ϕ ∈C∞
0 ( × [0, ∞)). In order to construct such solutions through some convenient approximation by classical
solutions also in the presence of possibly discontinuous initial data, given u0 and v0 merely
fulfilling (1.3) we first fix families (u0ε)ε∈(0,1) ⊂W 1,∞() and (v0ε)ε∈(0,1) ⊂W 1,∞() in
such a way that ⎧
⎪⎪⎪⎨
⎪⎪⎪⎩
0 < u0ε ≤∥u0∥L∞() + 1 in
for all ε ∈(0, 1),
ε
2 ≤v0ε ≤∥v0∥L∞() + ε
in
for all ε ∈(0, 1),
|∇v0ε|2
v0ε
≤
|∇v0|2
v0
+ 1
for all ε ∈(0, 1),
and that
u0ε →u0
and v0ε →v0
a.e. in
as ε ↘0. 1 Introduction (1.12) (1.12) Finally, the third of our main results provides some rigorous analytical evidence for the
suitability of (1.1) in the considered modeling context: Namely, by adequately tracing the
dependence of the constant C = C(u0, v0) in (1.10) on the initial data we can identify the
following stability property enjoyed by actually any member of the uncountable family of
function pairs (u0, 0), which for all suitably regular u0 indeed form steady states of (1.2): Finally, the third of our main results provides some rigorous analytical evidence for the
suitability of (1.1) in the considered modeling context: Namely, by adequately tracing the
dependence of the constant C = C(u0, v0) in (1.10) on the initial data we can identify the
following stability property enjoyed by actually any member of the uncountable family of
function pairs (u0, 0), which for all suitably regular u0 indeed form steady states of (1.2): Theorem 1.3 Suppose that n ≤5, and that ⊂Rn is a bounded convex domain with smooth
boundary, and that ℓ≥0 and K > 0. Then for each η > 0, there exists δ = δ(η, K) > 0
with the property that whenever (1.3) holds with ∥u0∥L∞() ≤K,
∥v0∥L∞() ≤K
and
|∇v0|2
v0
≤K
(1.13) (1.13) 123 Stabilization of arbitrary structures... Page 5 of 25
108 108 as well as as well as
v0 ≤δ,
(1.14)
v0 ≤δ, the solution (u, v) of (1.2) obtained in Theorem 1.1 satisfies 2 Approximation by positive classical solutions (2.5) ⎧
⎪⎪⎪⎨
⎪⎪⎪⎩
0 < u0ε ≤∥u0∥L∞() + 1 in
for all ε ∈(0, 1),
ε
2 ≤v0ε ≤∥v0∥L∞() + ε
in
for all ε ∈(0, 1),
|∇v0ε|2
v0ε
≤
|∇v0|2
v0
+ 1
for all ε ∈(0, 1),
and that
u0ε →u0
and v0ε →v0
a.e. in
as ε ↘0. (2.5) (2.5) 108
Page 6 of 25 108 M. Winkler Then for each ε ∈(0, 1), the regularized variant of (1.2) given by Then for each ε ∈(0, 1), the regularized variant of (1.2) given by Then for each ε ∈(0, 1), the regularized variant of (1.2) given by ε ∈(0, 1), the regularized variant of (1.2) given by ⎧
⎪⎪⎨
⎪⎪⎩
uεt = ∇·
uεvε∇uε
+ ℓuεvε,
x ∈, t > 0,
vεt = vε −uεvε,
x ∈, t > 0,
∂uε
∂ν = ∂vε
∂ν = 0,
x ∈∂, t > 0,
uε(x, 0) = u0ε(x),
vε(x, 0) = v0ε(x),
x ∈,
(2.6) ⎧
⎪⎪⎨
⎪⎪⎩
uεt = ∇·
uεvε∇uε
+ ℓuεvε,
x ∈, t > 0,
vεt = vε −uεvε,
x ∈, t > 0,
∂uε
∂ν = ∂vε
∂ν = 0,
x ∈∂, t > 0,
uε(x, 0) = u0ε(x),
vε(x, 0) = v0ε(x),
x ∈,
(2.6) (2.6) can be seen to actually inherit its initial non-degeneracy throughout evolution, and to thus
allow for the following statement on global classical solvability by functions enjoying some
rather expected basic features formally associated with (1.2): can be seen to actually inherit its initial non-degeneracy throughout evolution, and to thus
allow for the following statement on global classical solvability by functions enjoying some
rather expected basic features formally associated with (1.2): Lemma 2.2 Let n ≥1 and ⊂Rn be a bounded domain with smooth boundary, let ℓ≥0,
and assume (1.3) and (2.5). Then for each ε ∈(0, 1), there exist functions Lemma 2.2 Let n ≥1 and ⊂Rn be a bounded domain with smooth boundary, let ℓ≥0,
and assume (1.3) and (2.5). Then for each ε ∈(0, 1), there exist functions
uε ∈C0( × [0, ∞)) ∩C2,1( × (0, ∞))
and
vε ∈C0( × [0, ∞)) ∩C2,1( × (0, ∞))
(2.7) (2.7) such that uε > 0 and vε > 0 in × [0, ∞), and that (uε, vε) solves (2.6) in the classical
sense. 2 Approximation by positive classical solutions Again by comparison, we hence obtain that if we let yε ∈C1([0, ∞)) denote the solution of
y′
ε(t) = −c3(ε)ec2(ε)ℓt yε(t), t > 0, with yε(0) = minx∈ v0ε(x) > 0, then Again by comparison, we hence obtain that if we let yε ∈C1([0, ∞)) denote the solution of
y′
ε(t) = −c3(ε)ec2(ε)ℓt yε(t), t > 0, with yε(0) = minx∈ v0ε(x) > 0, then Again by comparison, we hence obtain that if we let yε ∈C1([0, ∞)) denote the solution of
y′
ε(t) = −c3(ε)ec2(ε)ℓt yε(t), t > 0, with yε(0) = minx∈ v0ε(x) > 0, then vε(x, t) ≥yε(t)
for all (x, t) ∈ × (0, Tmax,ε). (2.16) (2.16) Now assuming Tmax,ε to be finite for some ε ∈(0, 1), we could combine (2.14) with (2.15)
to infer that uεvε would then be bounded in × (0, Tmax,ε), in view of standard parabolic
regularity theory ( [17]) applied to the second equation in (2.6) implying that vε would belong
to C1+θ1, 1+θ1
2 ( × [ Tmax,ε
2
, Tmax,ε]) for some θ1 = θ1(ε) ∈(0, 1). Since uεvε would then
also be uniformly bounded from below by a positive constant in × (0, Tmax,ε) by (2.13)
and (2.16), and since thus the first equation in (2.6) would actually be uniformly parabolic in
× (0, Tmax,ε), we could employ the same token once again to infer from the first equation
in (2.6) that uε ∈C1+θ2, 1+θ2
2 ( × [ Tmax,ε
2
, Tmax,ε]) for some θ2 = θ2(ε) ∈(0, 1). As
thus uεvε would in in fact lie in Cθ3, θ3
2 ( × [ Tmax,ε
2
, Tmax,ε]) with ∇(uεvε) ∈Cθ3, θ3
2 ( ×
[ Tmax,ε
2
, Tmax,ε]; Rn) for some θ3 = θ3(ε) ∈(0, 1), again relying on uniform parabolicity
of both equations in (2.6) we could then invoke parabolic Schauder theory ( [17]) to obtain
θ4 = θ4(ε) ∈(0, 1) such that {uε, vε} ⊂C2+θ4,1+ θ4
2 ( × [ Tmax,ε
2
, Tmax,ε]), and that hence,
in particular, sup
t∈( Tmax,ε
2
,Tmax,ε)
∥uε(·, )∥C2() + ∥vε(·, t)∥C2()
< ∞. Together with (2.13) and (2.16), in view of (2.12) this would contradict the hypothesis that
Tmax,ε be finite. In consequence, (2.9) and (2.10) immediately result from (2.15) and (2.14),
while (2.8) and (2.11) directly follow upon integrating in (2.6). 2 Approximation by positive classical solutions ⊓⊔ Together with (2.13) and (2.16), in view of (2.12) this would contradict the hypothesis that
Tmax,ε be finite. In consequence, (2.9) and (2.10) immediately result from (2.15) and (2.14),
while (2.8) and (2.11) directly follow upon integrating in (2.6). ⊓⊔ ⊓⊔ Throughout the remaining part of this manuscript, unless otherwise stated we shall tacitly
assume that ⊂Rn is a bounded convex domain with smooth boundary, and that ℓ≥0, and
whenever (u0, v0) and families ((u0ε, v0ε))ε∈(0,1) fulfilling (1.3) and (2.5) have been fixed,
by Tmax,ε and (uε, vε) we shall exclusively mean the objects introduced in Lemma 2.2. 2 Approximation by positive classical solutions This solution satisfies
u0ε ≤
uε(·, t) ≤
u0ε + ℓ
v0ε
for all t > 0
(2.8) (2.8) and and ∥uε(·, t)∥L∞() ≤∥u0ε∥L∞() · eℓ∥v0ε∥L∞()·t
for all t > 0
(2.9) (2.9) and and ∥vε(·, t)∥L∞() ≤∥vε(·, t0)∥L∞()
for all t0 ≥0 and any t > t0
(2.10)
ell as ∥vε(·, t)∥L∞() ≤∥vε(·, t0)∥L∞()
for all t0 ≥0 and any t > t0
(2.10)
ll as
∞ as well as ∞
0
uεvε ≤
v0ε. (2.11) (2.11) Proof According to standard parabolic theory ( [1]), the regularity and positivity assumptions
on u0ε and v0ε in (2.5) guarantee the existence of Tmax,ε ∈(0, ∞] and positive functions
uε and vε from C0( × [0, Tmax,ε)) ∩C2,1( × (0, Tmax,ε)) such that (2.6) is satisfied in
× (0, Tmax,ε), and that either Tmax,ε = ∞,
or
lim sup
t↗Tmax,ε
∥uε(·, t)∥C2() + ∥vε(·, t)∥C2() +
1
uε(·, t)
L∞() +
1
vε(·, t)
L∞()
= ∞. (2.12) (2.12) By nonnegativity of uεvε, the comparison principle ensures that this solution satisfies By nonnegativity of uεvε, the comparison principle ensures that this solution satisfies uε(x, t) ≥c1(ε) := min
y∈
u0ε(y)
for all (x, t) ∈ × (0, Tmax,ε)
(2.13) and vε(x, t) ≤∥vε(·, t0)∥L∞()
≤c2(ε) := ∥v0ε∥L∞()
for all t0 ∈[0, Tmax,ε) and any (x, t) ∈ × (t0, Tmax,ε),
(2.14)
123 vε(x, t) ≤∥vε(·, t0)∥L∞()
≤c2(ε) := ∥v0ε∥L∞()
for all t0 ∈[0, Tmax,ε) and any (x, t) ∈ × (t0, Tmax,ε),
(2 1 vε(x, t) ≤∥vε(·, t0)∥L∞()
≤c2(ε) := ∥v0ε∥L∞()
for all t0 ∈[0, Tmax,ε) and any (x, t) ∈ × (t0, Tmax,ε),
(2.14) Page 7 of 25
108 Stabilization of arbitrary structures... 108 where the latter especially asserts that where the latter especially asserts that where the latter especially asserts that pecially asserts that uεt ≤∇· (uεvε∇uε) + c2(ε)ℓuε
in × (0, Tmax,ε). uεt ≤∇· (uεvε∇uε) + c2(ε)ℓuε
in × (0, Tmax,ε). Another comparison argument therefore shows that writing c3(ε) := ∥u0ε∥L∞(), we have uε(x, t) ≤c3(ε)ec2(ε)ℓt uε(x, t) ≤c3(ε)ec2(ε)ℓt
for all (x, t) ∈ × (0, Tmax,ε),
(2.15) and that hence, in particular, and that hence, in particular, vεt ≥vε −c3(ε)ec2(ε)ℓtvε
in × (0, Tmax,ε). 2.1 Fundamental first-order estimates. Basic decay properties of v The following simple observation forms a fundament for the main part of our first step toward
deducing further regularity properties of the solutions gained above, to be accomplished in
Lemma 2.4 below on the basis of the estimate in (2.17). Apart from that, (2.17) will later on
be utilized as a basic piece of information in our derivation of L p bounds for uε (Lemma
4.3), and (2.18) as well as (2.19) will prove useful in the course of our large time analysis in
Lemma 5.5 and Lemma 5.7. 123 123 108
Page 8 of 25 M. Winkler Lemma 2.3 Let n ≥1, assume (1.3) and (2.5), and let q ∈(0, 1). Then Lemma 2.3 Let n ≥1, assume (1.3) and (2.5), and let q ∈(0, 1). Then ∞
0
uq−1
ε
vε|∇uε|2 ≤
||1−q
q(1 −q) ·
u0ε + ℓ
v0ε
q
for all ε ∈(0, 1)
(2.17) and and ℓ
∞
0
uq
εvε ≤||1−q
q
·
u0ε + ℓ
v0ε
q
for all ε ∈(0, 1),
(2.18) (2.18) and furthermore we have
uq
ε(·, t) ≥
uq
0ε
for all t > 0 and any ε ∈(0, 1). (2.19) (2.19) Proof Let ε ∈(0, 1). Then using that uε is positive in × (0, ∞), we may test the first
equation in (2.6) against uq−1
ε
to see that 1
q
d
dt
uq
ε = (1 −q)
uq−1
ε
vε|∇uε|2 + ℓ
uq
εvε
for all t > 0,
(2.20) (2.20) which firstly implies (2.19) as an immediate consequence of the restriction q ∈(0, 1). After
an integration in time, we moreover infer from (2.20) that thanks to the Hölder inequality, (1 −q)
t
0
uq−1
ε
vε|∇uε|2 + ℓ
t
0
uq
εvε = 1
q
uq
ε(·, t) −1
q
uq
0ε
≤1
q
uq
ε(·, t)
≤||1−q
q
·
uε(·, t)
q
for all t > 0.
uε ≤
u0ε +ℓ
v0ε for all t > 0 by (2.8), this establishes both (2.17) and (2.18). ⊓⊔ As
uε ≤
u0ε +ℓ
v0ε for all t > 0 by (2.8), this establishes both (2.17) and (2.18). 2.1 Fundamental first-order estimates. Basic decay properties of v ⊓⊔ Now in the course of a standard testing procedure performed on the second equation in
(2.6), an application of (2.17) to q := 1
2 enables us to control the respective interaction-
driven contribution by means of conveniently decaying quantities. In particular, the following
conclusion can therefore be drawn in such a way that besides providing first order regularity
features, due to its temporally global nature it furthermore already includes some information
on temporal decay in the second solution component; this latter aspect will be of essential
importance to our argument asserting L p bounds for uε (see Lemma 4.3). The following
lemma is the only place in this manuscript in which convexity of is explicitly made use
of. Lemma 2.4 Let n ≥1. Then for all K > 0 there exists C(K) > 0 such that whenever (1.3),
(2.5) and (1.13) hold, we have ∞
0
|∇vε|4
v3ε
≤C(K)
for all ε ∈(0, 1)
(2.21) (2.21) and and ∞
0
uε
vε
|∇vε|2 ≤C(K)
for all ε ∈(0, 1). (2.22) (2.22) 123 Stabilization of arbitrary structures... Page 9 of 25
108 5
108 108 Proof As vε is positive in × (0, ∞) and actually belongs to C3( × (0, ∞)) according to
standard parabolic regularity theory, on the basis of the second equation in (2.6) we compute Proof As vε is positive in × (0, ∞) and actually belongs to C3( × (0, ∞)) according to
standard parabolic regularity theory, on the basis of the second equation in (2.6) we compute 1
2
d
dt
|∇vε|2
vε
=
∇vε
vε
· ∇vεt −1
2
|∇vε|2
v2ε
vεt
=
1
vε
∇vε · ∇vε −
1
vε
∇vε · ∇(uεvε)
−1
2
1
v2ε
|∇vε|2vε + 1
2
uε
vε
|∇vε|2
=
1
vε
∇vε · ∇vε −1
2
1
v2ε
|∇vε|2vε
−1
2
uε
vε
|∇vε|2 −
∇uε · ∇vε
for all t > 0 and ε ∈(0, 1). 2.1 Fundamental first-order estimates. Basic decay properties of v Winkler 108 Since by twice employing Young’s inequality we can estimate Since by twice employing Young’s inequality we can estimate Since by twice employing Young’s inequality we can estimate Since by twice employing Young’s inequality we can estimate −
∇uε · ∇vε ≤1
4
vε
√uε
|∇uε|2 +
√uε
vε
|∇vε|2
≤1
4
vε
√uε
|∇uε|2 + c1
2
|∇vε|4
v3ε
+ 1
2c1
uεvε
for all t > 0 and ε ∈(0, 1), upon an integration in (2.24) we thus infer that due to (2.25), (2.26) and (2.27),
|∇vε(·, t)|2
vε(·, t)
+ c1
t
0
|∇vε|4
v3ε
+
t
0
uε
vε
|∇vε|2
≤
|∇v0ε|2
v0ε
+ 1
2
t
0
vε
√uε
|∇uε|2 + 1
c1
t
0
uεvε
≤K + 1 + c2(K)
2
+ (K + 1)||
c
for all t > 0 and ε ∈( and conclude as intended. and conclude as intended. ⊓⊔ 2.1 Fundamental first-order estimates. Basic decay properties of v (2 23 (2.23) Here we may combine the outcome of a straightforward rearrangement ( [32, Lemma 3.2])
with a known functional inequality ( [32, Lemma 3.3]) to see for all ϕ ∈C3() such that
ϕ > 0 in and ∂ϕ
∂ν = 0 on ∂, writing c1 :=
1
(2+√n)2 and using that ∂|∇ϕ|2
∂ν
≤0 on ∂ by
convexity of ( [20]) we have Here we may combine the outcome of a straightforward rearrangement ( [32, Lemma 3.2])
with a known functional inequality ( [32, Lemma 3.3]) to see for all ϕ ∈C3() such that
ϕ > 0 in and ∂ϕ
∂ν = 0 on ∂, writing c1 :=
1
(2+√n)2 and using that ∂|∇ϕ|2
∂ν
≤0 on ∂ by
convexity of ( [20]) we have −
1
ϕ ∇ϕ · ∇ϕ + 1
2
1
ϕ2 |∇ϕ|2ϕ =
ϕ|D2 ln ϕ|2 −1
2
∂
1
ϕ
∂|∇ϕ|2
∂ν
≥
ϕ|D2 ln ϕ|2
≥c1
|∇ϕ|4
ϕ3
. Therefore, (2.23) implies that 1
2
d
dt
|∇vε|2
vε
+ c1
|∇vε|4
v3ε
+ 1
2
uε
vε
|∇vε|2 ≤−
∇uε · ∇vε
for all t > 0 and ε ∈(0, 1). (2.24) and to derive the claimed conclusion from this, we now assume that (1.13) holds with some
K > 0, observing that then and to derive the claimed conclusion from this, we now assume that (1.13) holds with some
K > 0, observing that then
u0ε ≤(K + 1)||
and
v0ε ≤(K + 1)|| as well as
|∇v0ε|2
v0ε
≤K + 1
(2.25) (2.25) due to (2.5). Therefore, an application of Lemma 2.3 to q := 1
2 shows that ∞
0
vε
√uε
|∇uε|2 ≤c2(K) := 4
(1 + ℓ)(K + 1)||
for all ε ∈(0, 1),
(2.26)
while (2.11) entails that ∞
0
vε
√uε
|∇uε|2 ≤c2(K) := 4
(1 + ℓ)(K + 1)||
for all ε ∈(0, 1),
(2.26) while (2.11) entails that while (2.11) entails that ∞
0
uεvε ≤c3(K) := (K + 1)||
for all ε ∈(0, 1). (2.27) 123 108
Page 10 of 25 M. 3 Global existence Page 11 of 25
108 108 whereobservingthatv0ε →v0 in L1()asε ↘0dueto(2.5)andthedominatedconvergence
theorem, thanks to well-known regularization features of e
τ
4 we see that whereobservingthatv0ε →v0 in L1()asε ↘0dueto(2.5)andthedominatedconvergence
theorem, thanks to well-known regularization features of e
τ
4 we see that e
τ
4 v0ε →e
τ
4 v0
in L∞()
as ε ↘0. e
τ
4 v0ε →e
τ
4 v0
in L∞()
as ε ↘0. e
τ
4 v0ε →e
τ
4 v0
in L∞()
as ε ↘0. Since our overall assumption v0 ̸≡0 ensures that c2(τ) ≡c2(τ; u0, v0) := e
τ
4 v0 is positive
throughout by the strong maximum principle, we can thus pick ε1 = ε1(τ; u0, v0) ∈(0, 1)
in such a way that e
τ
4 v0ε ≥c2(τ)
2
in
for all ε ∈(0, ε1), so that, in particular, so that, in particular, e
τ
4 v0ε ≥c3(τ) ≡c3(τ; u0, v0) := min
c2(τ)
2
, ε1
2
in
for all ε ∈(0, 1),
(3.6)
because by the comparison principle and (2.5), e
τ
4 v0ε ≥e
τ
4 ( ε
2) = ε
2 in for all ε ∈(0, 1). Now a second application of (3.4), again accompanied by a comparison argument, shows
that for arbitrary t ∈( τ
4, T ), e
τ
4 v0ε ≥c3(τ) ≡c3(τ; u0, v0) := min
c2(τ)
2
, ε1
2
in
for all ε ∈(0, 1),
(3.6) because by the comparison principle and (2.5), e
τ
4 v0ε ≥e
τ
4 ( ε
2) = ε
2 in for all ε ∈(0, 1). Now a second application of (3.4), again accompanied by a comparison argument, shows
that for arbitrary t ∈( τ
4, T ), vε(·, t) = e−c1(T )·(t−τ
4 )e(t−τ
4 )vε
·, τ
4
in
for all ε ∈(0, 1), vε(·, t) = e−c1(T )·(t−τ
4 )e(t−τ
4 )vε
·, τ
4
in
for all ε ∈(0, 1), so that combining (3.5) with (3.6) we find that for any such t, so that combining (3.5) with (3.6) we find that for any such t, vε(·, t) ≥e−c1(T )·(t−τ
4 )e(t−τ
4 )
e−c1(T )· τ
4 c3(τ)
= c3(τ)e−c1(T )·t
≥c4(τ, T ) ≡c4(τ, T ; u0, v0) := c3(τ)e−c1(T )·T
in
for all ε ∈(0, 1). 3 Global existence Besides on the boundedness properties documented in (2.9), (2.10) and (2.21), our con-
struction of a solution to (1.2) through a limit procedure in Lemma 3.2 will rely on some
further information on significantly enhanced regularity within finite time intervals bounded
away from the initial instant. Due to its temporally local nature, the following statement in
this regard, based on a combination of comparison arguments and standard parabolic theo-
ries, can apparently not be used in our large time analysis below, and hence does not trace
dependencies of the obtained constants on the size of the initial data. Lemma 3.1 Let n ≥1, and assume (1.3) and (2.5). Then for all τ > 0 and T > τ, there exist
θ = θ(τ, T ; u0, v0) ∈(0, 1) and C(τ, T ) = C(τ, T ; u0, v0) > 0 such that ∥uε∥
Cθ, θ
2 (×[τ,T ]) ≤C(τ, T )
for all ε ∈(0, 1)
(3.1) (3.1) and ∥vε∥
C2+θ,1+ θ
2 (×[τ,T]) ≤C(τ, T )
for all ε ∈(0, 1)
(3.2) as well as vε(x, t) ≥C(τ, T )
for all (x, t) ∈ × (τ, T ) and any ε ∈(0, 1). (3.3)
Proof We fix τ > 0 and T > τ and recall (2.9) to find c1(T ) > 0 such that
uε ≤c1(T ) in × (0, T )
for all ε ∈(0, 1), and that thus vεt ≥vε −c1(T )vε
in × (0, T )
for all ε ∈(0, 1). (3.4) vεt ≥vε −c1(T )vε
in × (0, T )
for all ε ∈(0, 1). (3.4) vεt ≥vε −c1(T )vε
in × (0, T )
for all ε ∈(0, 1). (3.4) (3.4) According to a comparison argument, this firstly implies that if we let (et)t≥0 denote the
Neumann heat semigroup on , then According to a comparison argument, this firstly implies that if we let (et)t≥0 denote the
Neumann heat semigroup on , then Neumann heat semigroup on , then vε
·, τ
4
≥e−c1(T )· τ
4 e
τ
4 v0ε
in
for all ε ∈(0, 1),
(3.5) (3.5) 123 Stabilization of arbitrary structures... 3 Global existence Proof According to the equicontinuity properties asserted by Lemma 3.1, the Arzelà-Ascoli
theorem provides (ε j) j∈N ⊂(0, 1) as well as functions u ∈C0( × (0, ∞)) and v ∈
C2,1( × (0, ∞)) such that ε j ↘0 as j →∞, and that uε →u in C0
loc( × (0, ∞))
and vε →v in C2,1
loc ( × (0, ∞)) as ε = ε j ↘0. Clearly, u is nonnegative, while strict
positivity of v in × (0, ∞) follows from (3.3). Apart from that, the estimates in (2.9)
and (2.10) together with the Vitali convergence theorem imply that uε →u and vε →v in
L p
loc( × [0, ∞)) for all p ≥1 as ε = ε j ↘0, and that u belongs to L∞
loc( × [0, ∞)) and
v lies in L∞( × (0, ∞)). As (2.21) along with (2.10) ensures boundedness of (∇vε)ε∈(0,1)
in L4( × (0, ∞)), we readily obtain that also v ∈L4
loc([0, ∞); W 1,4()), and that (3.10)
holds as ε = ε j ↘0. Proof According to the equicontinuity properties asserted by Lemma 3.1, the Arzelà-Ascoli
theorem provides (ε j) j∈N ⊂(0, 1) as well as functions u ∈C0( × (0, ∞)) and v ∈
C2,1( × (0, ∞)) such that ε j ↘0 as j →∞, and that uε →u in C0
loc( × (0, ∞))
and vε →v in C2,1
loc ( × (0, ∞)) as ε = ε j ↘0. Clearly, u is nonnegative, while strict
positivity of v in × (0, ∞) follows from (3.3). Apart from that, the estimates in (2.9)
and (2.10) together with the Vitali convergence theorem imply that uε →u and vε →v in
L p
loc( × [0, ∞)) for all p ≥1 as ε = ε j ↘0, and that u belongs to L∞
loc( × [0, ∞)) and
v lies in L∞( × (0, ∞)). As (2.21) along with (2.10) ensures boundedness of (∇vε)ε∈(0,1)
in L4( × (0, ∞)), we readily obtain that also v ∈L4
loc([0, ∞); W 1,4()), and that (3.10)
holds as ε = ε j ↘0. 3 Global existence Now the regularity requirements in (2.1) and (2.2) clearly result from the properties in (1.8)
just asserted, and a verification of (2.3) and (2.4) can be achieved on the basis of (2.6) in a
straightforward manner, using that due to (3.8) and (3.10) we have u2
ε∇vε⇀u2∇v
in L1
loc( × [0, ∞))
as ε = ε j ↘0, and that by (3.8) and (3.9), u2
εvε →u2v
and uεvε →uv
in L1
loc( × [0, ∞)) u2
εvε →u2v
and uεvε →uv
in L1
loc( × [0, ∞)) as ε = ε j ↘0. as ε = ε j ↘0. ⊓⊔ 3 Global existence Winkler We may therefore draw on classical parabolic Schauder theory ( [17]) to finally find θ3 =
θ3(τ, T ; u0, v0) ∈(0, 1) and c7(τ, T ) = c7(τ, T ; u0, v0) > 0 such that We may therefore draw on classical parabolic Schauder theory ( [17]) to finally find θ3 =
θ3(τ, T ; u0, v0) ∈(0, 1) and c7(τ, T ) = c7(τ, T ; u0, v0) > 0 such that ∥vε∥
C2+θ3,1+ θ32 (×[τ,T ]) ≤c7(τ, T )
for all ε ∈(0, 1), which precisely establishes (3.2), while (3.1) has already been contained in (3.7). ⊓⊔ ⊓⊔ A weak solution with the additional properties announced in (1.8) can now be obtained by
means of a straightforward extraction using the Arzelà-Ascoli theorem. Lemma 3.2 Let n ≥1, and assume (1.3) and (2.5). Then there exist (ε j) j∈N ⊂(0, 1) and
functions u and v fulfilling (1.8) such that ε j ↘0 as j →∞, that u ≥0 and v > 0 in
× (0, ∞), that Lemma 3.2 Let n ≥1, and assume (1.3) and (2.5). Then there exist (ε j) j∈N ⊂(0, 1) and
functions u and v fulfilling (1.8) such that ε j ↘0 as j →∞, that u ≥0 and v > 0 in
× (0, ∞), that uε →u
in L p
loc( × [0, ∞)) for all p ≥1 and in C0
loc( × (0, ∞)),
(3.8)
vε →v
in L p
loc( × [0, ∞)) for all p ≥1 and in C2,1
loc ( × (0, ∞)),
and
(3 9) (3.8) ∇vε⇀∇v
in L4( × (0, ∞))
(3.10) ∇vε⇀∇v
in L4( × (0, ∞))
(3.10) (3.10) as ε = ε j ↘0, and that (u, v) forms a global weak solution of (1.2) in the sense of Definition
2.1. as ε = ε j ↘0, and that (u, v) forms a global weak solution of (1.2) in the sense of Definition
2.1. as ε = ε j ↘0, and that (u, v) forms a global weak solution of (1.2) in the sense of Definition
2.1. 3 Global existence vε(·, t) ≥e−c1(T )·(t−τ
4 )e(t−τ
4 )
e−c1(T )· τ
4 c3(τ)
vε(·, t) ≥e−c1(T )·(t−τ
4 )e(t−τ
4 )
e−c1(T )· τ
4 c3(τ = c3(τ)e−c1(T )·t
≥c4(τ, T ) ≡c4(τ, T = c3(τ)e−c1(T )·t
≥c4(τ, T ) ≡c4(τ, T ; u0, v0) := c3(τ)e−c1(T )·T
in
for all ε ∈(0, 1). 3( )
≥c4(τ, T ) ≡c4(τ, T ; u0, v0) := c3(τ)e−c1(T )·T
in
for all ε ∈(0, 1). Besides trivially implying (3.3), in conjunction with (2.9), (2.10) and (2.5) this shows that in
the identity Besides trivially implying (3.3), in conjunction with (2.9), (2.10) and (2.5) this shows that in
the identity uεt = ∇·
aε(x, t)uε∇uε
+ ℓbε(x, t),
(x, t) ∈ × (0, ∞), ε ∈(0, 1),
the functions aε := vε and bε := uεvε satisfy c4(τ, T ) ≤aε ≤∥v0∥L∞() + 1 in ×
τ
4 , T
for all ε ∈(0, 1) and and |bε| ≤
∥u0∥L∞() + 1
· eℓ·(∥v0∥L∞()+1)·T ·
∥v0∥L∞() + 1
in ×
τ
4 , T
for all ε ∈(0, 1), whence a standard result on Hölder regularity in porous medium type scalar parabolic
equations ( [27]) becomes applicable so as to provide θ1 = θ1(τ, T ; u0, v0) ∈(0, 1) and
c5(τ, T ) = c5(τ, T ; u0, v0) > 0 such that ∥uε∥
Cθ1, θ12 (×[ τ
2 ,T ]) ≤c5(τ, T )
for all ε ∈(0, 1). (3.7) (3.7) As quite a similar reasoning yields an analogous Hölder bound for the solutions vε of vεt =
vε −bε(x, t), this entails that with some θ2 = θ2(τ, T ; u0, v0) ∈(0, 1) and c6(τ, T ) =
c6(τ, T ; u0, v0) > 0 we moreover have ∥bε∥
Cθ2, θ22 (×[ τ
2 ,T ]) ≤c6(τ, T )
for all ε ∈(0, 1). 123 108
Page 12 of 25 108
Page 12 of 108 M. 4.1 Two functional inequalities Up to this point, temporally global bounds which exclusively refer to uε, without any pres-
ence of weight functions involving vε as an expectedly decaying quantity such as in (2.18), 123 Stabilization of arbitrary structures... Page 13 of 25 108 seem limited to the L1 boundedness feature in (2.8). A key step toward a more substan-
tial description of the large time behavior in (1.2), and especially toward an exclusion of
asymptotic Dirac-type mass accumulation, will now consist in an adequate control of the
diffusion degeneracy in the first equation from (2.6), and particularly its part stemming from
the presence of the factor vε therein. In the framework of standard L p testing procedures, to
be performed in Lemma 4.3, this specifically amounts to appropriately estimating integrals
of the form seem limited to the L1 boundedness feature in (2.8). A key step toward a more substan-
tial description of the large time behavior in (1.2), and especially toward an exclusion of
asymptotic Dirac-type mass accumulation, will now consist in an adequate control of the
diffusion degeneracy in the first equation from (2.6), and particularly its part stemming from
the presence of the factor vε therein. In the framework of standard L p testing procedures, to
be performed in Lemma 4.3, this specifically amounts to appropriately estimating integrals
of the form
u p−1
ε
vε|∇uε|2
(4.1) (4.1) from below, and to thereby control the temporal growth of
u p
ε , as potentially driven by
the forcing term ℓuεvε in (2.6). from below, and to thereby control the temporal growth of
u p
ε , as potentially driven by
the forcing term ℓuεvε in (2.6). Of crucial importance to our approach in this direction will be the following observation on
how far expressions of the form in (4.1), when added to integrals of the type appearing in
(2.22), dominate gradients of certain products involving uε and vε. Lemma 4.1 Let n ≥1, α > 1
2, p⋆≥1 and K > 0. 4.1 Two functional inequalities Then there exists C(α, p⋆, K) > 0 with
the property that whenever ϕ ∈C1() and ψ ∈C1() are positive in and such that
ϕ p⋆≤K,
(4.2) (4.2) for any choice of q ∈[0, 2α −1] the inequality any choice of q ∈[0, 2α −1] the inequality ∇
ϕα
ψ
2
L
2p⋆
p⋆+2α−1 () ≤C(α, p⋆, K) ·
ϕq−1ψ|∇ϕ|2 +
ϕ
ψ |∇ψ|2
(4.3) (4.3) holds. holds. Proof We first observe that for any such ϕ and ψ, the pointwise estimate ∇
ϕα
ψ
2p⋆
p⋆+2α−1 =
αϕα−1
ψ∇ϕ + 1
2
ϕα
√ψ ∇ψ
2p⋆
p⋆+2α−1
≤c1ϕ
2p⋆(α−1)
p⋆+2α−1 ψ
p⋆
p⋆+2α−1 |∇ϕ|
2p⋆
p⋆+2α−1 + ϕ
2p⋆α
p⋆+2α−1 ψ−
p⋆
p⋆+2α−1 |∇ψ|
2p⋆
p⋆+2α−1
(4.4)
2 √ψ
≤c1ϕ
2p⋆(α−1)
p⋆+2α−1 ψ
p⋆
p⋆+2α−1 |∇ϕ|
2p⋆
p⋆+2α−1 + ϕ
2p⋆α
p⋆+2α−1 ψ−
p⋆
p⋆+2α−1 |∇ψ|
2p⋆
p⋆+2α−1
(4.4) (4.4) holds throughout with c1 ≡c1(α, p⋆) := (2α)
2p⋆
p⋆+2α−1 . Since p⋆+2α−1
p⋆
> 1 thanks to our
assumption that α > 1
2, we may employ the Hölder inequality and rely on (4.2) to control
the integral of the second summand on the right of (4.4) according to holds throughout with c1 ≡c1(α, p⋆) := (2α)
2p⋆
p⋆+2α−1 . Since p⋆+2α−1
p⋆
> 1 thanks to our
assumption that α > 1
2, we may employ the Hölder inequality and rely on (4.2) to control
the integral of the second summand on the right of (4.4) according to
ϕ
2p⋆α
p⋆+2α−1 ψ−
p⋆
p⋆+2α−1 |∇ψ|
2p⋆
p⋆+2α−1 =
ϕ
ψ |∇ψ|2
p⋆
p⋆+2α−1 · ϕ
p⋆(2α−1)
p⋆+2α−1
≤
ϕ
ψ |∇ψ|2
p⋆
p⋆+2α−1
·
ϕ p⋆
2α−1
p⋆+2α−1
≤K
2α−1
p⋆+2α−1 ·
ϕ
ψ |∇ψ|2
p⋆
p⋆+2α−1
. (4.5) p⋆
2α−1 =
ϕ
ψ |∇ψ|2
p⋆
p⋆+2α−1 · ϕ
p⋆(2α−1)
p⋆+2α−1
≤
ϕ
ψ |∇ψ|2
p⋆
p⋆+2α−1
·
ϕ p⋆
2α−1
p⋆+2α−1
≤K
2α−1
p⋆+2α−1 ·
ϕ
ψ |∇ψ|2
p⋆
p⋆+2α−1
. (4.5) (4.5) 123 108
Page 14 of 25 M. 4.1 Two functional inequalities Winkler Similarly, Similarly,
ϕ
2p⋆(α−1)
p⋆+2α−1 ψ
p⋆
p⋆+2α−1 |∇ϕ|
2p⋆
p⋆+2α−1 =
ϕq−1ψ|∇ϕ|2
p⋆
p⋆+2α−1 · ϕ
p⋆(2α−q−1)
p⋆+2α−1
≤
ϕq−1ψ|∇ϕ|2
p⋆
p⋆+2α−1
·
ϕ
p⋆(2α−q−1)
2α−1
2α−1
p⋆+2α−1
,(4.6) where using that the restrictions q ≥0 and q ≤2α −1 warrant that 0 ≤p⋆(2α−q−1)
2α−1
≤p⋆,
we may invoke Young’s inequality along with (4.2) to see that where using that the restrictions q ≥0 and q ≤2α −1 warrant that 0 ≤p⋆(2α−q−1)
2α−1
≤p⋆,
we may invoke Young’s inequality along with (4.2) to see that
ϕ
p⋆(2α−q−1)
2α−1
≤
(ϕ p⋆+ 1) ≤K + ||. In conjunction with (4.6) and (4.5), this shows that (4.4) implies the inequality In conjunction with (4.6) and (4.5), this shows that (4.4) implies the inequality ∇
ϕα
ψ
2p⋆
p⋆+2α−1
L
2p⋆
p⋆+2α−1 ()
≤c1 · (K + ||)
2α−1
p⋆+2α−1 ·
ϕq−1ψ|∇ϕ|2
p⋆
p⋆+2α−1
+K
2α−1
p⋆+2α−1 ·
ϕ
ψ |∇ψ|2
p⋆
p⋆+2α−1
, from which (4.3) immediately follows if we let from which (4.3) immediately follows if we let C(α, p⋆, K) := 2
p⋆+2α−1
p⋆
· max
c
p⋆+2α−1
p⋆
1
· (K + ||)
2α−1
p⋆, K
2α−1
p⋆
, C(α, p⋆, K) := 2
p⋆+2α−1
p⋆
· max
c
p⋆+2α−1
p⋆
1
· (K + ||)
2α−1
p⋆, K
2α−1
p⋆
,
instance. for instance. for instance. ⊓⊔ Combined with suitable Sobolev embedding properties, the latter entails the following class
of interpolation inequalities appropriate for our purposes. Combined with suitable Sobolev embedding properties, the latter entails the following class
of interpolation inequalities appropriate for our purposes. Lemma 4.2 Let n ≥1, p⋆≥1 and Lemma 4.2 Let n ≥1, p⋆≥1 and p :=
p⋆+ 1 if n = 1,
2p⋆+n
n
if n ≥2. (4.7) (4.7) Then for all K > 0 one can find C(p⋆, K) > 0 such that for any q ∈[0, min{p⋆, 2p⋆
n }] and
for each ϕ ∈C1() and ψ ∈C1() fulfilling ϕ > 0 and ψ > 0 in as well as
ϕ p⋆≤K,
(4.8) (4.8) we have
ϕ pψ ≤C(p⋆, K) ·
ϕq−1ψ|∇ϕ|2 +
ϕ
ψ |∇ψ|2 +
ϕψ
. 4.1 Two functional inequalities If n = 1, we proceed quite similarly, replacing (4.10) with the inequality
ρ2 ≤c3∥ρx∥2
L1() + c3∥ρ∥2
L1(),
ρ2 ≤c3∥ρx∥2
L1() + c3∥ρ∥2
L1(), valid for some c3 > 0 and all ρ ∈W 1,1(), substituting (4.12) by the observation that for
all positive ϕ ∈C1() and ψ ∈C1() we have ϕ
p⋆+1
2
ψ
2
L1() =
ϕψ · ϕ
p⋆
2
2
≤
ϕψ
·
ϕ p⋆
, ϕ
p⋆+1
2
ψ
2
L1() =
ϕψ · ϕ
p⋆
2
2
≤
ϕψ
·
ϕ p⋆
,
ying Lemma 4.1 to α := p⋆+1
2
. ϕ
p⋆+1
2
ψ
2
L1() =
ϕψ · ϕ
p⋆
2
2
≤
ϕψ
·
ϕ p⋆
,
and applying Lemma 4.1 to α := p⋆+1
2
. ⊓⊔ and applying Lemma 4.1 to α := p⋆+1
2
. ⊓⊔ 4.1 Two functional inequalities (4.9) (4.9) Proof We first consider the case n ≥2, in which W 1, 2n
n+2 () is continuously embedded into
L2(), so that we can pick c1 > 0 such that
ρ2 ≤c1∥∇ρ∥2
L
2n
n+2 ()
+ c1∥ρ∥2
L
2n
n+2 ()
for all ρ ∈W 1, 2n
n+2 (). (4.10) (4.10) We may then apply Lemma 4.1 to α := 2p⋆+n
2n
> 1
2 to infer the existence of c2(p⋆, K) > 0
such that whenever ϕ and ψ are positive functions from C1() which satisfy (4.8), for any We may then apply Lemma 4.1 to α := 2p⋆+n
2n
> 1
2 to infer the existence of c2(p⋆, K) > 0
such that whenever ϕ and ψ are positive functions from C1() which satisfy (4.8), for any 123 123 bilization of arbitrary structures... Page 15 of 25
108 Stabilization of arbitrary structures... Page 15 of 25
108 Stabilization of arbitrary structures... Page 15 of 25
108 108 choice of q ∈[0, 2α −1] we have choice of q ∈[0, 2α −1] we have choice of q ∈[0, 2α −1] we have ∇
ϕ
2p⋆+n
2n
ψ
2
L
2n
n+2 () ≤c2(p⋆, K) ·
ϕq−1ψ|∇ϕ|2 +
ϕ
ψ |∇ψ|2
. (4.11) ∇
ϕ
2p⋆+n
2n
ψ
2
L
2n
n+2 () ≤c2(p⋆, K) ·
ϕq−1ψ|∇ϕ|2 +
ϕ
ψ |∇ψ|2
. (4.11) (4.11) Apart from that, we note that as a consequence of the Hölder inequality, any such pair (ϕ, ψ)
satisfies Apart from that, we note that as a consequence of the Hölder inequality, any such pair (ϕ, ψ)
satisfies ϕ
2p⋆+n
2n
ψ
2
L
2n
n+2 () =
ϕ
2p⋆+n
n+2 ψ
n
n+2
n+2
n
=
(ϕψ)
n
n+2 · ϕ
2p⋆
n+2
n+2
n
≤
ϕψ
·
ϕ p⋆
2
n
≤K
2
n
ϕψ (4.12) because of (4.8). Together with (4.11) and (4.10), this already establishes (4.9) with
C(p⋆, K) := c1 · max
c2(p⋆, K) , K
2
n
in this case, for the restriction q ∈[0, 2p⋆
n ] clearly
warrants that q ≤2α −1. 4.2 Global Lp estimates for u. Proof of theorem 1.1 Having Lemma 4.2 at hand, we are now prepared for our derivation of the following statement
on L p boundedness of uε, yet conditional in presupposing the existence of corresponding
bounds in L p⋆with some p⋆≥1, by means of the announced testing-based argument. Lemma 4.3 Let n ≥1, p⋆≥1 and K > 0, and let p be as defined in Lemma 4.2. Then there
exists C(p⋆, K) > 0 such that if (1.3), (2.5) and (1.13) hold, and if
u p⋆
ε (·, t) ≤K
for all t > 0 and ε ∈(0, 1),
(4.13)
then
u p
ε (·, t) ≤C(p⋆, K)
for all t > 0 and ε ∈(0, 1)
(4.14)
and
∞
0
u p−1
ε
vε|∇uε|2 ≤C(p⋆, K)
for all ε ∈(0, 1)
(4.15)
u p⋆
ε (·, t) ≤K
for all t > 0 and ε ∈(0, 1),
(4.13) (4.13) then then and 123 108
Page 16 of 25 M. Winkler 108 as well as as well as ∞
0
u p
ε vε ≤C(p⋆, K)
for all ε ∈(0, 1). (4.16) (4.16) Proof We fix any q ∈(0, 1) such that q ≤min{p⋆, 2p⋆
n }, and combine Lemma 2.3 with
Lemma 2.4 and (2.11) to see that thanks to the hypothesis (1.13) we can find c1(K) > 0
fulfilling ∞
0
uq−1
ε
vε|∇uε|2 +
∞
0
uε
vε
|∇vε|2 +
∞
0
uεvε ≤c1(K)
for all ε ∈(0, 1). (4.17) (4.17)
On the basis of (4.13) and our selection of p and q, from Lemma 4.2 we thus infer the
existence of c2(p⋆, K) > 0 such that (
)
On the basis of (4.13) and our selection of p and q, from Lemma 4.2 we thus infer the
existence of c2(p⋆, K) > 0 such that ∞
0
u p
ε vε ≤c2(p⋆, K)
for all ε ∈(0, 1). 4.2 Global Lp estimates for u. Proof of theorem 1.1 (4.18) (4.18) As a multiplication of the first equation in (2.6) by u p−1
ε
, followed by an integration by parts,
shows that As a multiplication of the first equation in (2.6) by u p−1
ε
, followed by an integration by parts,
shows that 1
p
d
dt
u p
ε + (p −1)
u p−1
ε
vε|∇uε|2 = ℓ
u p
ε vε
for all t > 0 and ε ∈(0, 1), and that hence and that hence 1
p
u p
ε (·, t) + (p −1)
t
0
u p−1
ε
vε|∇uε|2
≤1
p
u p
0ε + ℓ
t
0
u p
ε vε
≤1
p ·
∥u0∥L∞() + 1
p · || + c2(p⋆, K)
for all t > 0 and ε ∈(0, 1) according to (2.5) and (4.18), the claim directly follows. ⊓⊔ according to (2.5) and (4.18), the claim directly follows. ⊓⊔ Upon repeated application of the latter, we obtain bounds in all the L p spaces appearing
in the claim from Theorem 1.1. Apart from that, the additional decay features expressed in
(4.20) and (4.21) will play essential roles in our derivation of stabilization in the first solution
component in the next section (see Lemma 5.1). Corollary 4.4 Let n ≥1, p ∈(1,
n
(n−2)+ ) and K > 0. Then there exists C(p, K) > 0 such
that if (1.3), (2.5) and (1.13) are satisfied, then
u p
ε (·, t) ≤C(p, K)
for all t > 0 and ε ∈(0, 1)
(4.19)
and
∞
0
u p−1
ε
vε|∇uε|2 ≤C(p, K)
for all ε ∈(0, 1)
(4.20)
as well as
∞
0
u p
ε vε ≤C(p, K)
for all ε ∈(0, 1). (4.21)
u p
ε (·, t) ≤C(p, K)
for all t > 0 and ε ∈(0, 1)
(4.19)
u p
ε (·, t) ≤C(p, K)
for all t > 0 and ε ∈(0, 1)
(4.19) (4.19) and and ∞
0
u p−1
ε
vε|∇uε|2 ≤C(p, K)
for all ε ∈(0, 1)
(4.20) as well as ∞
0
u p
ε vε ≤C(p, K)
for all ε ∈(0, 1). (4.21) Proof We recursively define (pk)k≥0 by letting p0 := 1
and
pk+1 :=
pk + 1,
k ≥0, if n = 1,
2pk+n
n
,
k ≥0, if n ≥2. 4.2 Global Lp estimates for u. Proof of theorem 1.1 123 123 ation of arbitrary structures... Page 17 of 25
108 Stabilization of arbitrary structures... Page 17 of 25
108 108 Assuming (1.3), (2.5) and (1.13) to hold, from Lemma 4.3 we then immediately obtain
(ck(K))k≥1 ⊂(0, ∞) such that for each k ≥1,
u pk
ε (·, t) ≤ck(K)
for all t > 0 and ε ∈(0, 1)
(4.22) (4.22) as well as ∞
0
u pk−1
ε
vε|∇uε|2 +
∞
0
u pk
ε vε ≤ck(K)
for all ε ∈(0, 1). (4.23) (4.23) Since it can readily be verified that pk ↗
n
(n−2)+ as k →∞, for arbitrary p ∈(1,
n
(n−2)+ ) the
estimates in (4.19), (4.20) and (4.21) easily result from (4.22) and (4.23) upon an interpolation
using the basic bounds provided by (2.8), (2.17) and (2.11). ⊓⊔ In particular, the latter completes our reasoning with regard to solvability and global L p
regularity in (1.2): Proof of Theorem 1.1 The part concerning existence and regularity has been completely cov-
ered by Lemma 3.2. The additional boundedness feature in (1.9) immediately follows from
(4.19) when combined with (3.8). ⊓⊔ Remark For initial data enjoying regularity and positivity features beyond those in (1.3),
the existence result from Theorem 1.1 can be supplemented by a corresponding uniqueness
statement by a straightforward combination of the reasoning from Lemma 2.2 with the
standard theory developed in [1]: Indeed, if beyond (1.3) it was required that both u0 and v0
belong to
q>n W 1,q() and satisfy u0 > 0 and v0 > 0 in , then (1.2) could actually be
seen to admit a global classical solution which is unique in the class of functions fulfilling
{u, v} ⊂
q>n C0([0, ∞); W 1,q())∩C2,1(×(0, ∞)).Underthepresentmildhypotheses
in (1.3) on the initial data, however, we do not expect solutions to be uniquely determined
by the requirements in Definition 2.1, nor by the additional regularity features obtained in
Lemma 3.2 which are yet fairly poor near the initial instant; we cannot even rule out the
possibility that different choices of approximate initial data in (2.5) may lead to different
limits. 5.1 Large time convergence of u in dual Sobolev spaces when n ≤5 Fortunately, in all physically relevant space dimensions the respective restrictions on p in
Corollary 4.4 are mild enough so as to allow for the following conclusion on large time decay
of uεt on the basis of (4.20), (4.21) and, again, the basic integrability property from (2.11). Lemma 5.1 Let n ≤5. Then there exists λ = λ(n) > 0 such that for all K > 0 one can find
C(K) > 0 with the property that if (1.3), (2.5) and (1.13) hold, we have ∞
0
∥uεt(·, t)∥(W 1,∞())⋆dt ≤C(K) ·
v0ε
λ
for all ε ∈(0, 1). (5.1) (5.1) 123 108
Page 18 of 25 108 Proof Since n ≤5, we have 3 −
n
(n−2)+ <
n
(n−2)+ , so that it is possible to pick p = p(n) ∈
(1, 2) fulfilling Proof Since n ≤5, we have 3 −
n
(n−2)+ <
n
(n−2)+ , so that it is possible to pick p = p(n) ∈
(1, 2) fulfilling
n
n Proof Since n ≤5, we have 3 −
n
(n−2)+ <
n
(n−2)+ , so that it is possible to pick p = p(n) ∈
(1 2) fulfilling p <
n
(n −2)+
and
3 −p <
n
(n −2)+
,
(5.2) (5.2) where using a continuity argument we may rely on the latter inequality in choosing r =
r(n) ∈(0, 1) suitably small such that where using a continuity argument we may rely on the latter inequality in choosing r =
r(n) ∈(0, 1) suitably small such that 3 −p −r
1 −r
<
n
(n −2)+
. (5.3) (5.3) Noting that the restriction p < 2 warrants that then 3−p−r
1−r
> 1, given K > 0 we can draw
on Corollary 4.4 to see that according to (5.3) and the first inequality in (5.2) we can fix
c1(K) > 0 and c2(K) > 0 such that whenever (1.3), (2.5) and (1.13) holds, ∞
0
u p−1
ε
vε|∇uε|2 ≤c1(K)
for all ε ∈(0, 1)
(5.4) (5.4) and and ∞
0
u
3−p−r
1−r
ε
vε ≤c2(K)
for all ε ∈(0, 1). 5.1 Large time convergence of u in dual Sobolev spaces when n ≤5 (5.5) (5.5) Under these hypotheses, we now use the first equation in (2.6) to find that for all t > 0 and
any ψ ∈W 1,∞() such that ∥ψ∥W 1,∞() ≡max
∥ψ∥L∞() , ∥∇ψ∥L∞()
≤1, Under these hypotheses, we now use the first equation in (2.6) to find that for all t > 0 and
any ψ ∈W 1,∞() such that ∥ψ∥W 1,∞() ≡max
∥ψ∥L∞() , ∥∇ψ∥L∞()
≤1,
uεtψ
=
−
uεvε∇uε · ∇ψ + ℓ
uεvεψ
≤
uεvε|∇uε| + ℓ
uεvε
for all ε ∈(0, 1), so that ∥uεt∥(W 1,∞())⋆≤
uεvε|∇uε| + ℓ
uεvε
for all t > 0 and ε ∈(0, 1). (5.6) (5.6) ince for all T > 0 and ε ∈(0, 1) we have Since for all T > 0 and ε ∈(0, 1) we have Since for all T > 0 and ε ∈(0, 1) we have T
0
uεvε|∇uε| ≤
T
0
u p−1
ε
vε|∇uε|2
1
2
·
T
0
u3−p
ε
vε
1
2
=
T
0
u p−1
ε
vε|∇uε|2
1
2
·
T
0
(uεvε)r · u3−p−r
ε
v1−r
ε
1
2
≤
T
0
u p−1
ε
vε|∇uε|2
1
2
·
T
0
uεvε
r
2
·
T
0
u
3−p−r
1−r
ε
vε
1−r
2 due to the Hölder inequality, from (5.4), (5.5) and (5.6) we thus infer that thanks to (2.11), due to the Hölder inequality, from (5.4), (5.5) and (5.6) we thus infer that thanks to (2.11), T
0
∥uεt(·, t)∥(W 1,∞())⋆dt ≤c
1
2
1 (K)c
1−r
2
2
(K) ·
T
0
uεvε
r
2
+ ℓ
T
0
uεvε
≤c
1
2
1 (K)c
1−r
2
2
(K) ·
v0ε
r
2
+ ℓ
v0ε
for all T > 0 and ε ∈(0, 1). 123 Page 19 of 25
108 Stabilization of arbitrary structures... 5.1 Large time convergence of u in dual Sobolev spaces when n ≤5 108 As As As As
v0ε ≤
v0ε
r
2
·
∥v0ε∥L∞() · ||
1−r
2
≤
v0ε
r
2
· (K + 1)
1−r
2 ||
1−r
2
for all ε ∈(0, 1) by (1.13) and (2.5), this implies the claim with λ(n) := r
2 and C(K) := c
1
2
1 (K)c
1−r
2
2
(K) +
ℓ· (K + 1)
1−r
2 ||
1−r
2 . ⊓⊔ ⊓⊔ As (5.1) involves L1 norms with respect to the time variable only, in order to avoid any
discussion of possibly measure-valued parts appearing in the time derivatives ut of the cor-
responding limits we prefer to formulate a conclusion of Lemma 5.1 for u which is sufficient
for our subsequent reasoning in a version including temporal BV norms only, and hence
exclusively involving the zero-order expression u itself: Corollary 5.2 Let n ≤5 and K > 0, let λ = λ(n) and C(K) be as in Lemma 5.1, and assume
(1.3), (2.5) and (1.13). Then for any (tk)k∈N ⊂[0, ∞) such that tk+1 ≥tk for all k ∈N, we
have ∞
k=1
u(·, tk+1) −u(·, tk)
(W 1,∞())⋆≤C(K) ·
v0
λ
,
(5.7) (5.7) where we have set u(·, 0) := u0. where we have set u(·, 0) := u0. Proof Fixing any such nondecreasing sequence (tk)k∈N, under the present hypotheses we
infer from Lemma 5.1 that for each N ∈N, N
k=1
uε(·, tk+1) −uε(·, tk)
(W 1,∞())⋆=
N
k=1
tk+1
tk
uεt(·, t)dt
(W 1,∞())⋆
≤
N
k=1
tk+1
tk
uεt(·, t)
(W 1,∞())⋆dt
≤C(K) ·
v0ε
λ
for all ε ∈(0, 1),
(5 (5.8) because (tk, tk+1) ∩(tl, tl+1) = ∅for k ∈N and l ∈N with k ̸= l. Since from (2.5) and
Lemma 3.2 we know that with (ε j) j∈N as provided by the latter we have uε(·, tk) →u(·, tk)
in L1() →(W 1,∞())⋆as ε = ε j ↘0 for each k ∈N, we obtain (5.7) from (5.8) upon
taking ε = ε j ↘0 and then N →∞there. ⊓⊔ because (tk, tk+1) ∩(tl, tl+1) = ∅for k ∈N and l ∈N with k ̸= l. 5.1 Large time convergence of u in dual Sobolev spaces when n ≤5 ⊓⊔ Proof As the number λ(n) provided by Corollary 5.2 is positive, the claim immediately
results upon applying the latter to (tk)k∈N defined by t1 := 0 and tk := t for k ≥2 and any
fixed t > 0. ⊓⊔ 5.1 Large time convergence of u in dual Sobolev spaces when n ≤5 Since from (2.5) and
Lemma 3.2 we know that with (ε j) j∈N as provided by the latter we have uε(·, tk) →u(·, tk)
in L1() →(W 1,∞())⋆as ε = ε j ↘0 for each k ∈N, we obtain (5.7) from (5.8) upon
taking ε = ε j ↘0 and then N →∞there. ⊓⊔ because (tk, tk+1) ∩(tl, tl+1) = ∅for k ∈N and l ∈N with k ̸= l. Since from (2.5) and
Lemma 3.2 we know that with (ε j) j∈N as provided by the latter we have uε(·, tk) →u(·, tk)
in L1() →(W 1,∞())⋆as ε = ε j ↘0 for each k ∈N, we obtain (5.7) from (5.8) upon
taking ε = ε j ↘0 and then N →∞there. ⊓⊔ When considering individual trajectories, we may here yet ignore any of the information
about dependencies on initial data enclosed in (5.1), and to thereby obtain the following as
a particular consequence. Corollary 5.3 Letn ≤5,andsupposethat(1.3)and(2.5)hold.Thenthereexistsanonnegative
u∞∈(W 1,∞())⋆such that u(·, t) →u∞in
W 1,∞()
⋆
as t →∞. (5.9) (5.9) 123 108
Page 20 of 25 M. Winkler 108 Proof We only need to observe that whenever (tk)k∈N ⊂(0, ∞) is nondecreasing and
unbounded, u(·, tk+m) −u(·, tk)
(W 1,∞())⋆≤
k+m−1
l=k
u(·, tl+1) −u(·, tl)
(W 1,∞())⋆
for all k ≥1 and m ≥1, for all k ≥1 and m ≥1, and that thus (5.7) particularly asserts that (u(·, tk))k∈N forms a Cauchy sequence in
(W 1,∞())⋆. ⊓⊔ ⊓⊔ Beyond this, however, the particular quantitative form of the right-hand side in (5.1) enables
us to also make sure that initially small v0 enforce solutions to remain near u0 in their first
component. Corollary 5.4 Let n ≤5 and K > 0. Then given any η > 0, one can find δ = δ(K, η) > 0
such that whenever (1.3), (2.5) and (1.13) hold as well as
v0 ≤δ,
(5.10) (5.10) we have we have ∥u(·, t) −u0∥(W 1,∞())⋆≤η
for all t > 0. (5.11) (5.11) Proof As the number λ(n) provided by Corollary 5.2 is positive, the claim immediately
results upon applying the latter to (tk)k∈N defined by t1 := 0 and tk := t for k ≥2 and any
fixed t > 0. 5.2 Stabilization of u in dual Sobolev spaces for all n ≥6 and some ∈(0, 1) Since here T
0
uγ
ε vε|∇uε| ≤
T
0
u2γ −1
ε
vε|∇uε|2 +
T
0
uεvε
for all T > 0 and ε ∈(0, 1) by Young’s inequality, the claim readily results upon combining (2.17) with (2.18) and (2.11),
because both γ and 2γ belong to (0, 1). ⊓⊔ After all, convergence of such powers uγ for each fixed trajectory can be asserted within this
topological setting: Corollary 5.6 Let n ≥6 and γ > 0 be as in Lemma 5.5, and assume that (1.3) and (2.5)
hold. Then there exists a nonnegative u∞∈(W 1,∞())⋆such that uγ (·, t) →uγ
∞in
W 1,∞()
⋆
as t →∞. Proof This can be concluded from Lemma 5.5 in much the same manner as Corollary 5.3
was derived from Lemma 5.1. ⊓⊔ ⊓⊔ 5.2 Stabilization of u in dual Sobolev spaces for all n ≥6 and some ∈(0, 1) In higher-dimensional cases in which the estimates provided by Corollary 4.4 seem insuf-
ficient for the above argument, with regard to large time asymptotics we rather resort to an
analysis of certain sublinear powers of u. Indeed, for suitably small γ the expressions uγ
ε
can be seen to enjoy a stabilization feature similar to that in Lemma 5.1, albeit apparently
without information about comparably favorable effects induced by small v0 here: Lemma 5.5 There exists γ ∈(0, 1) with the property that whenever n ≥6 and (1.3) as well
as (2.5) hold, there exists C = C(u0, v0) > 0 such that Lemma 5.5 There exists γ ∈(0, 1) with the property that whenever n ≥6 and (1.3) as well
as (2.5) hold, there exists C = C(u0, v0) > 0 such that ∞
0
∂tuγ
ε (·, t)
(W 1,∞())⋆dt ≤C
for all ε ∈(0, 1). (5.12) (5.12) Proof We fix any γ ∈(0, 1
2) and observe that then for all t > 0 and each ψ ∈W 1,∞(),
according to (2.6) we have Proof We fix any γ ∈(0, 1
2) and observe that then for all t > 0 and each ψ ∈W 1,∞(),
according to (2.6) we have
∂tuγ
ε · ψ = γ (1 −γ )
uγ −1
ε
vε|∇uε|2ψ −γ
uγ
ε vε∇uε · ∇ψ
+γ ℓ
uγ
ε vεψ
for all ε ∈(0, 1), from which it follows that with some c1 > 0, T
0
∂tuγ
ε (·, t)
(W 1,∞())⋆dt ≤c1 ·
T
0
uγ −1
ε
vε|∇uε|2 +
T
0
uγ
ε vε|∇uε| + ℓ
T
0
uγ
ε vε
T
0
∂tuγ
ε (·, t)
(W 1,∞())⋆dt ≤c1 ·
T
0
uγ −1
ε
vε|∇uε|2 +
T
0
uγ
ε vε|∇uε| + ℓ
T
0
uγ
ε vε
123 n of arbitrary structures... Page 21 of 25
108 Stabilization of arbitrary structures... Page 21 of 25
108 108 for all T > 0 and ε ∈(0, 1). Since here for all T > 0 and ε ∈(0, 1). Since here > 0 and ε ∈(0, 1). 5.3 L1 decay of v By now making use of the lower bound for sublinear Lq quasi-norms from Lemma 2.3,
irrespective of the spatial dimension we can derive the following decay property of v from
(2.11) and (2.21). Lemma 5.7 Let n ≥1 and assume (1.3) as well as (2.5). Then Lemma 5.7 Let n ≥1 and assume (1.3) as well as (2.5). Then
v(·, t) →0
as t →∞. (5.13) (5.13) Proof We fix any q ∈(0, 1) such that 4q
3
≤1, and from (2.8) we then obtain c1 =
c1(u0, v0) > 0 such that ∥uε∥
L
4q
3 () ≤c1
for all t > 0 and ε ∈(0, 1). (5.14) We moreover employ a Poincaré inequality to pick c2 > 0 fulfilling We moreover employ a Poincaré inequality to pick c2 > 0 fulfilling We moreover employ a Poincaré inequality to pick c2 > 0 fulfilling ϕ −1
||
ϕ
L4()
≤c2∥∇ϕ∥L4()
for all ϕ ∈W 1,4(),
(5.15) (5.15) and abbreviating c3 :=
1
||
uq
0 > 0, given η > 0 we choose t0 = t0(η; u0, v0) > 1 large
enough such that
1
c3
t
t−1
uqv ≤η
2
for all t > t0
(5.16) and abbreviating c3 :=
1
||
uq
0 > 0, given η > 0 we choose t0 = t0(η; u0, v0) > 1 large
enough such that
1 t 1
c3
t
t−1
uqv ≤η
2
for all t > t0
(5.16) and and and
c1c2
c3
·
t
t−1
|∇v|4
1
4
≤η
2
for all t > t0;
(5.17) c1c2
c3
·
t
t−1
|∇v|4
1
4
≤η
2
for all t > t0;
(5.17) here we note that such a selection is possible due to the fact that (2.11), Lemma 2.4 and (2.10)
together with Lemma 3.2 and Fatou’s lemma guarantee that both
∞
0
uv and
∞
0
|∇v|4 here we note that such a selection is possible due to the fact that (2.11), Lemma 2.4 and (2.10)
together with Lemma 3.2 and Fatou’s lemma guarantee that both
∞
0
uv and
∞
0
|∇v|4 108
Page 22 of 25 M. 5.3 L1 decay of v With (ε j) j∈N taken from Lemma 3.2, we let ε = ε j ↘0 here to infer from (2.5) and the
locally uniform convergence properties of (uε j ) j∈N, (vε j ) j∈N and (∇vε j ) j∈N asserted by 123 Page 23 of 25
108 Stabilization of arbitrary structures... 108 Lemma 3.2 that in line with our definition of c3, Lemma 3.2 that in line with our definition of c3, Lemma 3.2 that in line with our definition of c3,
v(x, t)dx ≤1
c3
t
t−1
uqv + cq
1c2
c3
·
t
t−1
|∇v|4
1
4
≤η
2 + η
2 = η
for all t > t0. ⊓⊔ As η > 0 was arbitrary, this establishes the claim. 5.3 L1 decay of v Winkler 108 are finite, and that hence, by the Hölder inequality and again due to (2.10) and Lemma 3.2, are finite, and that hence, by the Hölder inequality and again due to (2.10) and Lemma 3.2, t
t−1
uqv ≤
∥v0∥L∞() + 1
1−q
t
t−1
uqvq
≤
∥v0∥L∞() + 1
1−q||1−q ·
t
t−1
uv
q
→0
as t →∞. For t > t0 and ε ∈(0, 1), we may then decompose For t > t0 and ε ∈(0, 1), we may then decompose For t > t0 and ε ∈(0, 1), we may then decompose t
t−1
uq
εvε =
t
t−1
uq
ε(x, s) ·
1
||
vε(y, s)dy
dxds
+
t
t−1
uq
ε (x, s) ·
vε(x, s) −1
||
vε(y, s)dy
dxds,
(5.18) (5.18) by the Hölder inequality, (5.15) and (5.14), where by the Hölder inequality, (5.15) and (5.14),
t
t−1
uq
ε(x, s) ·
vε(x, s) −1
||
vε(y, s)dy
dxds
≤
t
t−1
∥uε(·, s)∥q
L
4q
3 ()
·
vε(·, s) −1
||
vε(·, s)
L4()
ds
≤cq
1c2
t
t−1
∥∇vε(·, s)∥L4()ds
≤cq
1c2 ·
t
t−1
|∇vε|4
1
4
. Since furthermore Since furthermore t
t−1
uq
ε(x, s) ·
1
||
vε(y, s)dy
dxds
=
1
||
t
t−1
uq
ε(x, s)dx
·
vε(y, s)dy
ds
≥
1
|| ·
uq
0ε
·
vε(y, t)dy according to Lemma 2.3 and the evident nonpositivity of d
dt
vε throughout (0, ∞), from
(5.18) we altogether conclude that according to Lemma 2.3 and the evident nonpositivity of d
dt
vε throughout (0, ∞), from
(5.18) we altogether conclude that 1
|| ·
uq
0ε
·
vε(x, t)dx ≤
t
t−1
uq
εvε
+cq
1c2 ·
t
t−1
|∇vε|4
1
4
for all t > t0 and ε ∈(0, 1). 5.4 Proofs of theorems 1.2 and 1.3 Large time convergence in the claimed topological settings can now be obtained from Corol-
lary 5.3, Corollary 5.6 and Lemma 5.7 by suitable interpolation using the boundedness
features asserted by Theorem 1.1. Proof of Theorem 1.2 As supt>0 ∥v(·, t)∥L∞() is finite according to Theorem 1.1, for n ≥4
the convergence statement in (1.11) can be derived from Lemma 5.7 by a simple interpolation
based on the Hölder inequality. In the case n ≤3, we once again use that thanks to (2.21)
and Lemma 3.2, the integral
∞
0
|∇v|4 is finite, so that with some c1 > 0 and some
(tk)k∈N ⊂(0, ∞) fulfilling tk →∞as k →∞we have ∥v(·, tk)∥W 1,4() ≤c1 for all k ∈N. Since for any such n the Gagliardo-Nirenberg inequality provides c2 > 0 fulfilling ∥ϕ∥L∞() ≤c2∥ϕ∥
4n
3n+4
W 1,4()∥ϕ∥
4−n
3n+4
L1()
for all ϕ ∈W 1,4(), ∥ϕ∥L∞() ≤c2∥ϕ∥
4n
3n+4
W 1,4()∥ϕ∥
4−n
3n+4
L1() in view of Lemma 5.7 this implies that ∥v(·, tk)∥L∞() ≤c
4n
3n+4
1
c2∥v(·, tk)∥
4−n
3n+4
L1() →0
as k →∞, and that thus (1.11) also holds in this case, because being a classical solution of its respective
sub-problem of (1.2) in × [1, ∞) by Theorem 1.1, thanks to the maximum principle the
function v has the property that 0 < t →∥v(·, t)∥L∞() is nonincreasing. To deduce (1.12), we only need to combine the outcomes of Corollary 5.3 and Corollary 5.6
with the observation that for each κ > 0 and any p ∈(1,
n
(n−2)+ ), the family (uκ(·, t))t>0 is
bounded in L
p
κ () by (1.9), and hence relatively compact with respect to the weak topology
in this space. ⊓⊔ Our main result on stability of arbitrary equilibria in (1.2) has actually been covered by
Corollary 5.4 in its essence: Proof of Theorem 1.3 It is sufficient to apply Corollary 5.4, and to note that with (ε j) j∈N
taken from Lemma 3.2 we have ∥v(·, t) −v0∥L1() ≤
lim
ε=ε j ↘0
vε(·, t) +
v0ε
≤2
lim
ε=ε j ↘0
v0ε = 2
v0
for all t > 0
due to the evident nonincrease of 0 ≤t →
vε(·, t) for all ε ∈(0, 1). 5.4 Proofs of theorems 1.2 and 1.3 ⊓⊔ ∥v(·, t) −v0∥L1() ≤
lim
ε=ε j ↘0
vε(·, t) +
v0ε
≤2
lim
ε=ε j ↘0
v0ε = 2
v0
for all t > 0 ∥v(·, t) −v0∥L1() ≤
lim
ε=ε j ↘0
vε(·, t) +
v0ε
≤2
lim
ε=ε j ↘0
v0ε = 2
v0
for all t > 0
due to the evident nonincrease of 0 ≤t →
vε(·, t) for all ε ∈(0, 1). ⊓⊔ e to the evident nonincrease of 0 ≤t →
vε(·, t) for all ε ∈(0, 1). ⊓⊔ ⊓⊔ due to the evident nonincrease of 0 ≤t →
vε(·, t) for all ε ∈(0, 1) Acknowledgements The author warmly thanks the anonymous reviewer and Frederic Heihoff for numerous
insightful comments which significantly improved this manuscript. Acknowledgements The author warmly thanks the anonymous reviewer and Frederic Heihoff for numerous
insightful comments which significantly improved this manuscript. Funding Open Access funding enabled and organized by Projekt DEAL. He furthermore acknowledges sup-
port of the Deutsche Forschungsgemeinschaft in the context of the project Emergence of structures and
advantages in cross-diffusion systems (Project No. 411007140, GZ: WI 3707/5-1). 123 108
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24. Montecchiari, P., Rabinowitz, P.H.: On the existence of multi-transition solutions for a class of elliptic
systems. Ann. Inst. H. Poincaré – Anal. Non Linéaire 33, 199–219 (2016) 123 Stabilization of arbitrary structures... 108 Page 25 of 25 26. Plaza, R.G.: Derivation of a bacterial nutrient-taxis system with doubly degenerate cross-diffusion as the
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219–268 (2021) 123
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Studien iiber die Pest.
II. Teil.
Die r a t i o n e l l e
Kur der Pest.
Serumtherapie oder sofortiger chirurgischer Eingriff.
~on
Prof. Dr. Camillo Terni,
Privatdezent der Hygiene in ~ailand.
(llierzu Tar. X.)
Obgleich die in Indien gemaohten interessanten Forschungen der verschiedenen wissenschaftlichen Kommissionen in Yervollst~ndigung der ersten
Studien yon K i t a s a t o (1) und Yersin (2) das Problem der ~tiologie
und Pathologie der Pest vollkommen gelSst haben, wurden bis jetzt doeh
nur geringe Vorteile ffir die Kur erreicht, da wit verleitet wurden, die
Pest ausschliei31ich als eine septik~mische Infektion mit so raschem Verlauf zu betrachten, dab die lokalen und allgemeinen Manifestationen sich
vermischten. Die ganze spezitisehe Kur wurde daher ausschlieBlich auf
die Serumtherapie basiert, auf tier man mit irrtfimliehen Ansichten bestand, aueh wenn d e r MiBeffolg nut zu augenscheinlich war. Der lobenswerte Wunsch, auch fiir die Pest die Erfolge zu erreichen, die die
Sertuntherapie his jetzt nut fiir die Diphtherie gegeben hat, daft uns
nicht so welt hinreil3en, andere Kurmittel auszuschliet3en, die in der groBen
~Iehrzahl der F~lle die Genesung herbeiffihren oder alas Serum wirksam
unterstfitzen kSnnen, um so mehr, wenn dieses sich als schwaeh oder
ungenfigend erweist.
Deshalb sehien es mir nfitzlich, naeh diesen Probejahren, in denen
das antipestSse Serum ausgedehnte und fast ausschliei~liehe •
in d~r Kur der Pest gehabt hat, einige Betraehtungen zu erSrtern, wie
sie aus meinen objektiven Beobachtungen zahlreicher klinischer Pesti~lle
hervorgehen, die mit verschiedenen Kurmethoden behandelt worden sind.
Zeitsehr. f. Hygiene.
LIu
25
386
CamT,Lo TERZ~I:
I. Antipest~se Sera und ihr kurative.r Wert.
In einer frfiheren Arbeit (3) babe ich Gelegenheit gehabt hervorzuheben, dab der Pestbazillus im Menschen sich nicht wirklich wie ein
dem Milzbrand vergleichbares septik~misches Bakterium benimmt und dab
das Eindringen und die u
in den Organismus niemals direkt
dureh die Blutbahneu effolgt. Alle klinischen und experimentellen Tatsachen best~tigen, dab die Pest anfangs im Lymphsystem lokalisiert ist
und besonders in den Drfisen, wo der Pestbazillus die gfinstigsten Lebensund Entwieklungsbedingungen findet, indem er im sogen. Bubo einen
Entzfindungs- und Nekroseprozefl hervorruft und die Bildung /~uBerst
giftiger Substanzen, welche, wenn sie absorbiert werden, die schwersten
und charakteristischsten Symptome dieser Infektion hervorrufen. Die
Gegenwart der Bazillen im Blute erfolgt immer in einer sehr sp/~ten
Periode und bildet ein nebens~chliches Faktum im Verlaufe der Krankheit,
w/ihrend der Tod gewShnlich eintritt, noch ehe die Bazillen aus den
Lymphbahnen in die Blutbahnen fibergehen, infolge der schweren Intoxikation dutch die in das Blur gelangten Produkte, welche haupts~ichlich eine
paxalysierende Wirkung auf die Kapillarzirkulation ausfiben. In vielen F/illen
kann auch noch nach erfolgter Infektion des Blutes die spontane Genesung
erfolgen, wenn die Symptome tier allgemeinen Intoxikation weniger ernst
sind, eine Tatsache, die auch yon A l b r e c h t und Ghon und andern festgestellt wurde (4). In tier Pest h~ngt also die grS•ere Gefahr ffir das
Leben des Individuums yon der Menge der Gifte ab, die die Bazillen in
den bevorzugten Herden des Lymphsystems oder in besf.immten Geweben
bereiten, wie es z. B. in F~illen yon prim/irer Pestpneumonie vorkommt,
in denen der plStzliche Tod durch Herz- und Kapillargef/il3nervenl~hmung
tier gewShnliche Ausgang ist, wahrend die lokalen L~sionen den Verlust
des Kranken noch nicht erkl/iren kSnnen. [Lutz (5), W. E. Kossack (6),
L. F. Childe (7).]
Unter diesen Umst/inden ist es natfirlich, dat~ das Serum, wenn es
in der spezifischen Behandlung der Pest genfigen soll, eine bedeutende
antitoxische Wirksamkeit besitzen mul3, besonders in schweren F/illen,
wenn die Entzfindung im Lokalisationspunkte der Bazillen die nekrotische
Phase erreicht hat. Hier kann sehr schwer eine aktive ZerstSrung der
Bazillen durzh die yore Serum angeregte phagozyt~re Wirkung ausgefibt
werden, w~hrend in der Masse des toten, ffir Phagozyten unzug/inglichen
Gewebes die Produktion der Toxine und die Absorption mit groBer Gefahr
ffir den Kranken fortf~hrt.
Diese yon mir zuerst erSrterten wesenflichen Bedingungen des pathologischen Prozesses der Pest wurden vSUig best~tigt yon der englischen
STU])~:EN ~ E R
D~
38,7
PEST.
Kommission in !adieu (8), die .sehr angemessen hervorhebt~ da~ in der
Pest ein k o m b i n i e r t e r Prozel3 y o n b a k t e r i s c h e r I n v a s i o n u n d
I n t o x i k a t i o n stattfindet und dal~ es daher augenscheinlich ist, dal3 zwei
verschiedene Quaht~ten therapeutischer Substanzen mSglichst in der Kur
der Pest angewandt werden mfissen. In erster Linie a n t i b a k t e r i s c h e
S u b s t a n z e n , die f~hig sind die Bazfllen zu tSten oder ihre Entwicklung
zu ersehweren; in zweiter Linie a n t i t o x i s c h e S u b s t a n z e n zu dem
Zweck, die yon den bakterischen Giften verursaehten Symptome zu entfernen oder zu mildern, dutch die dernattirliche Widerstan4 des Organismus
gegen die Infektion haupts~chlich gef~hrdet wird.
Entsprechen die heute gebr~uchlichen antipestSsen Heilsera den eben
erSrterteu fundamentalen therapeutischen Bedingungea? Sicher nioht.
Die ersten u
Yersins kSnnen wit jetzt als tibereilte Hoffnungen
betrachten gegentiber den in tier Folge stets best~tigten ]~il~erfolgen.
Auch die den Vorsehl~gen C a l m e t t e s und Borrels und sparer Roux' (9)
entsprechende ~,nderung der Immunisierungsmethode der Tiere (gauze
uud virulente Kulturen in stufenweis zunehmender ~Ienge den Pferden
inokulieren) nfitzte nichts, und das haupts~ehlioh, weft man das ttauptfaktum, welches die Pestinfektion charakterisiert, nicht gegenw~rtig hielt,
d. h. den Komplex tier Intoxikationssymptome, welehe ersoheinen, sobald
tier Bazillus sich in den Lymphdrfisen festgesetzt hat und deft die Bildung
des Bubos veranla~te. Jeder Techniker, der sich mit tier Serumtherapie
der Pest besch~ftigt hat, mul3 jetzt iiberzeugt sein, da~ kein ftir die
Serumbereitung gewShnlich zur Verfiigung stehendes Tier ein antitoxisches
Serum produziert, das den ffir die Kur des ]~[enschen wfinschenswertesten
Bediugungen entspricht, und dab man nicht einmal eine so hohe antibakterische Kraft erreichen kann, um sichere und best~ndige Resultate
zu erhalten auch nur in der •
der Infektion, wenn die
Symptome der Intoxikation nooh nicht oder fast nicht offenbar sind.
Mit der yon L u s t i g (10)v0rgeschlagenen ]~ethode schien es theoretiseh
leichter den Zweck zu erreichen, da er das Tier mit grSBtenteils 15sliohen
Proteinen immunisierte, die voraussichtlieh durch die bei der Bereitung
erlittene chemische Behandlung leichter assimilierbar geworden sind: aber
leider befinden wir uns auch hier vet einer Entt~iusehung, da es bewiesen
ist, dab die Pferde nur in geringem Grade die pestSsen Nucleoproteide
zerstSren und keine wirksamen kurativen Substanzen in ihrem Serum
hervorbringen. Von diesem Standpunkte aus ist es also noch besser, der
Methode mit der Inokulation ganzer und virulenter Kulturen zu folgen,
wodurch man eine grSBere anregende Wirkung der Phagozyten erreicht
25*
388
CAmT.LO T ~ I :
und die Produktion yon antibakterisehen Substanzen, die, .obgleich sehwach,
doch im frischen Serum ziemlich akt~v sind, besonders wenn ffir die
Bereitung ~[aulesel, Esel und Ochsen statt der Pferde angewandt werden.
Wenn Pferde benutzt werden, l~uft man zu h~ufig Gefahr, dab das Serum
start der kurativen eine toxische Wirkung hat wegen des Exzesses yon
nieht zerstSrten, noch aktiven und im Serum vorhandenen bakterisehen
Giften. Deshalb hat sich in einigen in Indien gemaehtenBeobachtungen
ergeben, dab die ]~ortalit~t grSBer war unter den mit Serum behandelten
Individuen, obgleioh die Kur am ersten Tage der Krankheit begonnen
war, also unter den gfinstigsten Umst~nden, um ein befriedigendes Resultat
zu erwarten.
Die englische Kommission kam nach jahrelangen Beobaohtungen und
sehr demonstrativen Experimenten bezfiglich der Frage der antipestSsen
Sera zu den folgenden Schl~issen:
,,1. Wir sind der Meinung, dab die Serumtherapie der Pest nieht
yon therapeutischen Erfolgen gekrSnt worden ist, die in irgend einer Weise
mit den bei der Diphtherie erreichten zu vergleiohen w~ren; trotzdem ist
die Serumtherapie in der Pest wie in anderu Infektionskranl~heiten die
einzige Methode, die die Aussicht auf definitiven Erfolg bietet.
2. Die Behandlung mit Serum hat nieht genfigende Resultate gegeben, um die Ausdehnung seines Gebrauches in den gegenwiirtigen Verh~ltnissen auf alle yon der Pest betroffenen Lokalit~ten anzuraten. Es
scheint uns, dab die Unvollkommenheiten der gegenw~rtigen Bereitungsund Anwendungsmethoden v o l l k o m m e n a n e r k a n n t w e r d e n mfiBten,
und dab der leitende Gedanke, um Fortsehritte zu maehen, nieht in der
Anwendung der gegenw~rtig zur Verfi~gung stehenden Sera bei einer
m6gliehst groBen Anzahl yon Kranken liegen muB, sondern in dem
S t u d i u m der Modifikationen, welche in dem Blute tier zur Bereitung
des Serums bestimmten Tiere vet sioh gehen, was die Assimilation der
pestSsen Toxine und die Bereitung antitoxischer und antibakterischer
Substanzen betrifft. Ebenso milBten die Beschaffenheit des Blutes der
Pestkranken und die infolge der Yerabreichung des Serums in demselben
vet sioh gehenden Modifikationen mit Aufmerksamkeit studiel:t werden."
Naeh diesen Ansichten hat sioh eben das serumtherapisehe Laboratorium
in Messina geriehtet, als wir uns mit der Serumtherapie der Pest besoh~ftigen muBten.
Die MSgliehkeit, mit aaderen Methoden ein antipestSses Serum yon
heher antitoxischer Wirkung zu bereiten, war unsere best~ndige Serge, und
in einer andern Arbeit habe ich die angestellten Forschungen und die
STUDIEN I~BER DIE PEST.
389
erreichten Resultate mitgeteilt, die zweifellos denjenigen des yon Yersin
und L u s t i g - G a l e o t t i bereiteten Serums fiberlegen sind, sowohl in der
Kur des Menschen wie in den Experimenten an Tieren. Die u
heir der Bereitungsmethode der Sera mit dem Yers in- und L u s t i g - G a l e ottiprozel~ ist nieht derartig, dal3 sie groBe Unterschiede i n tier Qualit~it der
Produkte und in den kurativen Wirkungen bedingen kSnnte, denn der erste
inokuliert den Pferden entweder Filtrate yon Kulturen oder ganze Kulturen;
der zweite bediente sioh der auf ehemischem Wege dem KSrper der Bakterien
entzogen~n Proteine. Weder mit der einen noch mit der andern BIethode
ist es mSgheh, ein Serum yon bemerkenswerter antitoxiseher Wirkung zu
erhalten, besonders yon Pferden, und alas Resultat in tier Kur ist lediglich
eine Anregung der phagozyt~en T~tigkeit, nicht immer entsohieden bemerkbar, selbst nicht bei intravenSsen Inokulationen; bemerkbar ist nut
ein leiehter Vorteil zugunsten des Lustig-GaleottirSerums, wie wit aus den
S~atistiken des Arthur Road-Hospitals in Bombay ersehen kSnnen, welche
fibrigens nicht frei yon Fehlern in der ~[ethode und in der Berechnung sind.
Xhnliche Resultate kann man aueh mit den kfinstlichen Sera yon
H a j e m und F o d o r erreiehen, und besser noch mit den yon Baccelli (11)
vorgeschlagenen Sublimatinokulationen wegen der groBen anregenden
Wirkung des Queeksilberalbuminat auf die Leukozyten. [Gaglio (12),
J. Ross (13), A r n o z a n und M o n t e l (14), Stassano (15), Bald o n i (16) u. a.]
Sehon aus den Kontrollexperimenten i n v i t r o und bei Tieren
(M~use, Ratten, Meerschweinehen, Kaninohen) kann man mit Leichtigkeit
ersehen, dab das mit den gewShnlichen Immunisierungsprozessen yon
Pferden bereitete antipest6se Serum nur geringe oder gar keine kurative
Wirksamkeit besitzt.
Die antibakterischen Substanzen (Agglutinine, Bakterizidine, Bakteriolysine) finder man in so schwachem Grade darin, dab das Experiment
best~ndig unsicher ist. Naoh Kolle (17) wirkt das antipestSse Serum
nur pr~ventiv und ist weder rein antitoxisch, wie das Tetanus- und
Diphtherieserum, nooh rein bakterizid, wie das Cholera- und Typhusserum: seine Wirkung zeigt sich vorzugsweise mittels der Bakteriolysine
und a n d e r e r S u b s t a n z e n , deren b i o l o g i s e h e M e r k m a l e m i t u n s e r n
j e t z i g e n F o r s c h u n g s m e t h o d e n n i c h t f e s t g e s t e l l t werden k S n n e n .
Es sind gerade diese vorl~ufig unbekannten Substanzen, welche dem
antipestSgen Serum jene preventive Wirksamkeit verleihen, die man mit
Yersuchen in einer groBen Anzahl yon Tieren beweisen kann. Sie wirken
haupts~chlich als Anreger der Phagozyten und bilden nicht ein spezifisehes
Merkmal des Serums, da man mit anderen Mitteln ebenso gute Resultate
erreichen kann.
390
CA~aZ_LT,OTv.mr
Die Bakteriolysine zeigen sieh wirksam nur bei Anwesenheit schon
abgeschwiichter Pestbazillen EMarkl (18)], w~hrend die ZerstSrung der
virulenten Bazillen im Organismus fast g~nzlich duroh die Phagozyten
erfolgt, in den Versuohen mit Serum an Tieren befinden wir uns Tat,
saohen gegenfiber, die keine Erkltirung finden, wenn wit uns yon diesem
genau yon der yon jedem Vorurtefl freien Beobaohtung hergeleiteten
Prinzip entfernen.
Wena man aueh im antipest5sen Serum Ambozeptoren nachweisen
kann, die einer grol3en Aflinitiit ffir die Rezeptorenelemente der Bakterien
ftihig sind, so fehlt dooh die bakterizide Wirkung. Die Tiere unterliegen
der Infektion~ trotzdem sie hShere Serumdosen als die als wirksam erwiesene Durchsehnittsdose erhalten hubert; andere dagegen genesen auch
mit geringeren Dosen, tr0tzdem alle anderen Bedingungea des Versuches
streng beobachtet werden.
Wie Kolle bemerkt, kann man in solchen F~llen nicht den SchluB
ziehen~ dab der Tod der Tiere, welehe hohe Serumdosen erhalten haben,
dutch einen Uberschul3 an Ambozeptoren erfolgt nach E h r l i c h s Seitenkettentheorie, wodurch die Yerankerung des ImmunkSrpers verhinder~
wfirde und sich die sogen. Komplementablenkung nach Neisser und
W e c h s b e r g zeigt; noch kann er yon der verschiedenen Virulenz der
Bazillen abhtingen. Kolle nimmt daher an, dal3 dieses unbestiindige Yerhalten des Serums im TierkSrper zusammen mit anderen unbekannten
Faktoren yon der individuellen Pradisposition der einzelnen Tiere fiir die
Pestinfektion abhtingt, welche in vielen Ftillen auch nicht mit den
hSchsten Serumdosen besiegt werden kann.
Wenn man aufmerksam im Organismus die Wirkung des mit Minimaldoseu yon Virus allmtihlich in verschiedenen Zwisehenr~umen inokulierten
Pestserums verfolgt, kann man sehr g~t feststellen, dab in den dem Tode
geweihten Tieren sich die Phagozytose nicht oder fast nicht im Stiohpunkte
oder in den nahen Lymphbahnen zeigt, w~hrend sie dagegen sehr aktiv
in den tier Genesung zuschreitenden Tieren ist. Aber aueh in den toten
Tieren, obgleich sie mit Serum inokuliert sind, zeigt sieh die sehou yon
Kolle hervorgehobene Tatsache, dab der Tod nicht mit den Merkmalen
einer wirkliehen Septiktimie erfolgt, wie in den Kontrolltieren, und selten
finden sich Bazillen im Blutlauf. Sie bleiben im Stichpunkt und in den
nahen Lymphdrfisen lokalisiert; abet das Tier unterliegt dutch Intoxikation.
Das, was man bis jetzt bezfiglich der therapeutischen Wirkung des
Serums erreichen kann, sowohl in den Tieren wie im Menschen, ist eine
u
der K r a n k h e i ~ und nieh~ die Genesung.
Alle diese Tatsachen, welehe beziiglich der Wirkung des Serums in
den Versuchstieren und im kranken Menschen vollkommen tibereinstimmen,
STUDIEN ~BER DrY, PEST.
391
beweisen, dab der Kliniker yon dem mit den angegebenen Methoden
bereiteten antipestSsen Serum nut eine anregende Wirlmng auf die
Phagozytose hoffen kann, die abet unsicher ist in dem Sinne, dab
wegen b e s o n d e r e r i n d i v i d u e l l e r B e d i n g u n g e n n i c h t alle T i e r e
auoh d e r s e l b e n A r t diese A n r e g u n g in gleicher Weise ffihlen
und im Y e r h M t n i s zur i n o k u l i e r t e n S e r u m m e n g e .
Es mul3 auBerdem beaehtet werden, dal~ die dem Serum eigentfunliohen Substanzen, die eine positive Chemotaxis gegen die Pestbazillen
hervorrufen, sehr schwach sind, und dab im antipestSsen Serum fast immer
noch ~berreste yon noeh nicht neutralisierten bakterisehen Giften existieren,
so daB, wenn die chemotaktische Wirkung fehlt, die toxischen Produkte
in Thtigkeit bleiben und sicher dazu beitragen, den Ausgang der Krankher zu beeintraohtigen.
Infolge des yon allen Versuchen hergeleiteten Beweises, dab die
Wirkung des antipestSsen Serums fast ausschlieBlieh auf die Anregung
der Phagozytose beschr~nkt ist und ohne spezifischen Charakter, schien
es mir nfitzlich, in den Tieren wie fin Menschen den Gebrauoh des Sublimats zu versuchen, sei es in der yon Baccelli vorgeschlagenen Formel,
sei es im Verhaltnis yon 0: I promille dem Serum immunisierter Tiere
hinzugeffigt zu dem Zweok, auBer einer ~5Beren uad bestandigeren an.
regenden T~itigkeit der Phagozyten auch ein Hindernis zu erhalten ffir
die Entwicklung der Bazillen lungs der Lymphbahnen und in den Organen
(Leber, Milz, Nieren), wo das Queoksilber sich mit u
festsetzt.
Aus den Resultaten der Experimente geht unzweifelhaft hervor, dab
das Quecksilber als eine energische Beihilfe zur Wirkung des Serums betrachtet werden muB, weft es den phagozytaren ProzeB in T~tigkeit setzt
in einer betr~chtlich hSheren Zahl yon Fallen im u
mit denen,
die nut mit Serum behandelt werden, so dab eine grSBere Verlangerung
des Lebens und eine u
der Mortalit~t erfolgt.
Es ist jedoch immer augenscheinlich, dab eine groBe Zahl yon Tieren
infolg~e yon Intoxikation unterliegt, auch wenn eine sehr lebhafte Tatigkeit
tier Phagozyten naohweisbar ist, welche im Stichpunkt und in den Lymph,
driisen erscheinen, fiberladen mit in AuflSsung begriffenen Bazillem Man
mSchte sagen, dab in solchen Fallen dem Organismus die Kraft fehlt, die
yon Leukozyten als caput mortuum ihrer Digestion ausgeschiedenen
bakterischen Gifte zn zerstSren, oder dab den Leukozyten die Faknltat
fehR, die yon der AuflSsung tier bakterischen KSrper herriihrenden Gifte
zu assimilieren oder zu zerstSren.
Auf alle Falle best~tigt aueh diese Serie yon Beobachtungen immer
mehr, dab im infek~iven Proze~ der Pest zwei Faktoren mitwirken: die Invasion und fortschreitende Verbreitung der Bazfllen, und die Intoxikatioa
392
CA~.~LLO TERNI:
dutch die yon denselben im bevorzugten Punkte erzeugten Produkte
und dutch die toxischen -~berreste ihrer AuflSsung. Diesen wesentlichen
l~aktoren der Krankheit kann die Serumtherapie bisher nut einen geringen
Teil yon Aktivit~t entgegensetzen, die fast ausschlie~lich auf die Anregung
der Phagozytose besehr~nkt ist; es bleibt nun noch zu sehen, ob es mSglich
ist, die bakterizide Kraft zu erhShen, so dal~ sie eine gewisse Sicherheit
ffir die Genesung gew~hrt, oder besser die Bereitung eines antitoxischen
und polyvalenten Serums zu erreichen, da man in den meisten Pestf~llen
beim Menschen mehr die Symptome und Folgen der Intoxikation als die
Septik~mie ffirchten muB.
Von den hier mitgeteilten Betrachtungen ausgehend, naoh zahlreichen
Forschungen und Experimenten, in denen ioh haupts~ehlich die toxische
Wirkung der pathogenen pestSsen Produkte des Mensohen und der Tiere
mit den Toxinen der kfinstlichen Kulturen verglich, habe ich reich yon
der Niitzlichkeit fiberzeugt~ die Tiere mit den dem kranken Organismus
entnommenen Produkten (Peritonealexsudat pest5ser l~Ieerschweinchen,
Saft yon Bubonen usw.), start mit kfinstlichen Kulturen zu immunisieren.
Da die Pferde sich der Wirkung solcher Produkte gegenfiber als sehr
wenig widerstandsf'~hig erwiesen, so wurden sie dutch ~Iaultiere und
Oohsen ersetzt mit sehr versprechenden Resultaten, denn der antibakterische und antitoxische Wert des so erhaltenen Serums im Yergleich
mit dem Serum yon mit Kulturen oder Nueleoproteiden (Lustig) immunisierten Pferden variiert im Verh~iltnis yon 50:1.
Auf diese Weise konnte man ein polyvalentes Antiserum erhalten, besonders aktiv gegen die spezifischen pest5sen Toxine, die in den prim~ren
Bubonen vorhanden und durch die Einwirkung der Bazillen auf die dem
Gewebe der Lymphdrfise eigenen Elemente hervorgerufen werden und
vielleicht i n besonderer Weise durch die Lymphe, durch die folgende
l~ekrose des Parenchyms und der Leukozyten und dutch die ZerstSrung
tier Bazillen. Auf keine andere Weise ist es mSglioh ein Antiserum zu
bereiten, das f~hig w~re in v i t r o die filtrierten Extrakte des prim~ren
Bubos zu neutralisieren und den Tod der Tiere dutch Intoxikation zu
verhindern, der best~ndig an den Kontrolltieren erfolgt.
Das naeh dieser ~[ethode bereitete Serum wurde in ausgedehntem
Mal3e in Brasilien angewandt im Vergleich mit den Sera Lustig und
Yersin. Die Resultate jedoch, obgleich sie entsehieden zugunsten der
neuen Methode waren, da die totale Mortalit~it der mit diesem Serum
behandelten Kranken auf 25 Prozent herabgesetzt wurde, lassen immer
~och Raum zu Zweifeln, sowohl wegen der Schwierigkeit soloher Versuche
STUDIEN ~J-BEI%DIE PEST.
393
bei relativ besehr~nkter Zahl yon Kranken und in Epidemien, die, was
Intensit~t u n d klinisohe F~lle anbetrifft, oft zu versohieden sind, als aueh
weil mein Serum frisoh bereitet ~var, w~hrend die anderen beiden viel
friiheren Datums waren. Die grebe Unbest~ndigkeit der antibakterisohen
und antitoxisohen Substanzen des antipestSsen Serum% einerlei wie es
bereitet ist, - - Substanzen, die zum grSBten Teil nicht spezifisch s i n d macht es sehr sohwer, vergleiohbare statistisehe Daten zu haben, wenn
man nicht frisch bereitete Sera gebraucht und in Epidemien yon gleioher
]ntensit~t.
Die Experimente an Tieren, in denen der Verlauf der Pest rasch
einen septik~mischen Charakter annimmt und in denen also haupts~ehlieh
die antibakterisehen Substanzen des antipestSsen Serums wirken miiBten,
beweisen, dab auch diese Substanzen raseh ausgeschieden werden, so dab
die Resultate manohmal in auffallender Weise verschieden sind mit
ein und demselben Serum, je nachdem es frisch oder erst nach 4 bis
5 Tagen gebrauoht wird. Dies wurde aueh yon der englisehen Kernmission festgestellt mit dem nach Lustigseher und Yersinsoher ~[ethode
bereiteten Serum (19).
Diesem Hauptfibelstande muB ich auoh das unsichere Resultat zuschreiben, das mit meinem Serum in Bombay erreioht warde. Es wurde
an etwa 300 Kranken unter tier direkten Kontrolle yon J. H a f f k i n e
versucht, dem ich bier meinen besten Dank ausspreehe. Es hande]te
sich um eine Partie Serum, dessen Gebraueh um fast 6 Monate verzSgert
wurde und das nicht rechtzeitig dureh anderes frischeren Datums ersetzt
werden konnte.
•
alle F~lle ist der Charakter der Unbest~ndigkeit tier therapeutischen Substanzen des antipestSsen Serums eine andere direkte Best~tigung seiner geringen spezifischen Wirkung, die man, wie wit sehen
werden, leicht wahrnehmen kann, wenn man sie aufmerksam im Kranken
verfolgt.
Ich ffige bier eine vergleichende Tafel bei fiber die in Indien mit
den verschiedenen Sera gemachten Beobachtungen. Die Untersuchungsbedingungen waren die strengsten; denn in diesen Serien yon u
waren die Kontrollkranken, was klinisehe Form anbetrifft, genau fibereinstimmend mit den mit Serum behandelten F~llen. In diesen Experimenten
wurden die geringsten klinischen Einzelheiten im Verlauf der Krankheit
hervorgehoben und in sorgffaltigen Anmerkungen im erw~hnteu Bericht yon
B a n n e r m a n n (20) registriert; sie haben also einen absoluteu Wert, um
die spezifisohe kurative Wirkung der Sera am Menschen zu beurteilen:
394
C~_LLO TERNI:
MR Serum behandelte Fiille
Durchschnitt
~]er Tage fiber
die normale
Dauer der
Kontrolle
I=
Datum und
Art und ~
Ort d e r
Weise tier
Bereitung Inokulation
RouxYersln
.~
;
N
Krankheit
~r
L681
hinaus in den
mit Serum
behandelten
FMlen
friseh,
subkutan 22~
58 74.33 231 63 68 70.56 i
und
Nha-trang
(Tonking) in~raven5s
und Paris
(aus Pferde.)
Lusfig
frisch,
desgl. 60~ ~36 172 71.71 609 t82 127 79.14
Bombay
(aus Pferden)
Terni !6 Monate bis subkutan l t 0 89 21 80-90
1 ]ahr alt,
Messina
I
!(aus hlauleseln
a n d Oehsen)
70 5~ 12 82.85
Brazil 6 Monate alt, subkutan
und
S. Paulo
I (Brasilien) intraven5s
i(aus Pferden)
nach YersinMethode
8"38
1 "13
.o 90[ 31.s,!
0-34
706010857
.1
0.31
In den yore Dr. Godinho an den Pestkranken des Isolierspitals in
Santos (Brasilien) gemachten Versuchen mit dem Serum yon Messina
und demjenigen von R o u x - Y e r s i n wurden die folgenden Resultate erreicht:
Q u a l i ~ der Sera
~
T e r n i , Messina.
Polyvalentes Serum
(2 Monate alt)
R o u x - Y e r s i n , Paris.
Antibakteriseh-monovalent.
Serum (2 Monate alt)
20
.~. I ~
"'-~
5
3
720
71
125
12
8
1875
116
60
I
i 37.5
;
40
;
Wie ersichtlich, stimmen die beiden Sera iiberein in den Wirkungen,
obgleich yon dem Serum aus ~essina um ein Drittel und mehr kleinere
Dosen angewandt wurden als yon dem Serum Roux-Yersin, und obgleich
es nut hypodermisch verabreicht wurde.
STUDZEN UBER DIE PEST
395
In der noch in den peripherisehen LymphdrSsen begrenzten Bubonen.
pest sehen wir dieselben Verh~ltnisse sich reproduzieren, die im Typhus'
sieh zeigen:, emer anderen Krankheit mit infektivem und toxisehem
Charakter , die ffir eine gewisse Zeit im Lymphsystem der Eingeweide
und des Mesenteriums Iokalisiert bleibt. Und man begreift,, da~ auch
beim TyphUs wir nie auf eine wirksame K u r hoffen kSnnen yon einem
nut antibakterisehen Serum (Chantemesse), bereitet mit kfinstliehen
Kulturen, in denen die spezifischen Toxine nicht produziert werden, die
den klinischer~ Typus der Krankheit im ~Ienschen charakterisieren und
yon den Bazillen nur im Parenchym der Drtisen und in den lympbatischen
Follikeln erzeugt werden.
Die YOn mir vorgeschlagene und bei der Bereitung yon Antisera in
~ihnlichen Krankheiten angewandte N[ethode entspricht den exaktesten
Kenntnissen fiber die ~tiologie und Natur dieser Infektionen und verdient vor alien anderen bisher gebr~uchliche n ~Iethoden ffir weitere
Versuche und Forschungen empfohlen z u werden.
Aus allen an Mensehen gemaehten Versuchen kSnnen wit die folgenden Schlfisse fiber die kurative Wirksamkeit der antipestSsen Sera zlehen:
1. Das yon Pferden mit kfinstlichen Kulturen gewonnene Serum besitzt
ffir eine gewisse Zeit (etwa einen Monat) eine antibakterische Kraft, die
sich im Kranken in einer Anregung der Phagozytose zeigt, so da6 wir
eine V e r l ~ n g e r u n g der K r a n k h e i t e r r e i c h e n , aber n i c h t die
G e n e s u n g , weft die antitoxische Wirkung fehlt.
2. Das antiproteidisehe Serum (Methode L u s t i g - G a l e o t t i ) besitzt
in sehr geringem Grade antibakterische und antitoxische Substanzen yon
ephemerer Dauer (etwa einer Woche).
3. Das nach der yon mir vorgeschlagenen ~[ethode bereitete polyvalente
Serum verspricht, was Weft, Qualit~t und Dauer der immunisierenden
Substanzen anbetrifft, alas beste, was man bis jetzt yon der Serumtherapie
der Pest hoffen kann.
Doch auch mit diesem Serum kann man die Serumtherapie:der Pest
noch nieht als gelSst erkl~ren und in den schwereren F~llen ist noch die
Uhzul~inglichkeit der kurativen Kraft des Serums bemerkbar wegen der
sehwachen und unbest~ndigen antitoxisehenWirkung. Mit den gewShnlich
in den Laboratorien zur Bereitung der Sera im gro6eu verffigbaren Tieren
ist es bis jetzt nieht m5gheh, ein antipestSses Serum zu erhalten, dessen
antitoxische kurative Wirkung auch nur im entferntesten mit der des
antidiphtherischen Serums zu vergleichen w~re~ und diesem Mangel mfissen
wit die vergebens mit statistischen Ziffern yon gewShnlieh zweifelhaftem
Wert oder mit der Schwere der der Kur unterworfenen F~ille verdeckten
396
C~TT.T.O TF_a~I:
MiBerfolge der Serumtherapie in der :Pest zuschreiben. Nut yore Menschen,
der yon schweren Pestformen Rekonvaleszent ist, oder yon Affen (~Iaoacus
resus) nnd yon den Ratten (Mus decumanus) ist es mSglich, ein sehr
aktives antitoxisches und antibakterisches Serum zu erhalten, indem die
normal% ffir die Kur genfigende Dosis auf 20 bis 30 corn herabgesetzt
wird, auch in F~illen mi~ schweren broncho-pulmonaren Komplikationen,
die bisher jedem anderen antipestSsen Serum widerstanden haben. Aber
die Schwierigkeit,. sich solche Sera zu verschaffen, macht das Resultat
dieser Beobachtungen praktisch nutzlos, besonders wenn groBe Mengen
des Materials nStig sind, um den Anforderungen einer Epidemie zu
genfigen.
Das Problem der spezifischen Kur der Pest bleibt daher noch zum
grSBten Teil ungel6st. Die gegenw~irtig mit tier Serumtherapie der Pest
erreichten Vorteile sind sehr beschr~nkt und mfissen als ein gutes Versprechen ffir die Zukunft betrachtet werden, nicht aber als der vollst~indige und sichere Erfolg, den wir noch yon weiteren Forschungen
erwarten mfissen.
II. Chirurgischer Eingriff.
Wie wird sich unter solchen Umst~nden, da man yon der Serumtherapie keinen entscheidenden Vorteil in der Kur der Pest erwarten
kann, die Tiitigkeit des Arztes dem Kranken gegenfiber ~uBern miissen?
Es geniigt, Gelegenheit gehabt zu haben, einen Pestbubo i n der
kritischen Periode der Krankheit (3. bis 5. Tag) zu untersuchen, um
sich zu fiberzeugen, dal3 es nicht mSglich ist, yon der Serumtherapie
allein die Beseitigung der nekrotisierten Masse der Lymphdrfisen zu erhoffen. Kann sich doch schon die Phagozytose nicht wirksam entwickeln
in diesem abgestorbenen Gewebe, wahrend die Pestbazillen sich darin
rasch entwickeln und fortfahren Toxine auszuscheiden, denen sioh andere
15sliche Gifte tier in AuflSsung befindiichen bakterischen KSrper und die
durch eventuelle andere Bakterien hervorgerufenen hinzuffigen, die fast
immer im prim~ren Bubo gegenw~rtig sind zusammen mit den spezifischen
Keimen.
Die natfirliche Entwicklung der Krankheit zeigt an, welches die
rationelle Kur sein mul].
In den F[~llen spontaner Heilung fiillt das Fieber durch K r i s i s
nach 24 bis 48 Stunden, und die Infektion bleibt stehen noch vor der
Bildung eines wirklichen Bubos nnd beschr~nkt sich auf die Entzfindung
eider oder zweier Drtisen. In anderen Fiillen kann die spontane Heilung
in mehr vorgesehrittener Periode der Krankheit vorkommen, wean tier
STUDIEN D~BER DIE PEST.
397
Bubo vollst~ndig entwickelt ist, d.h. wenn die Entzfindung den grSBten
Teil oder alle Driisen einer besthnmten Region ergriffen hat; abet in diesen
F~llen f~ngt der Bubo schon an sioh fluktuierend zu zeigen nach der
kritischen Periode ~3. bis 5. Tag) und dann. folgt die Erweichungs- und
Eiterungsphase mit der spontanen AusstoBung des Pus gegen den 10. bis
15. Tag, wenn die Inzision nicht rechtzeitig gemacht wird. Deshalb mul~
man als yon guter Prognose alle die F~lle betrachten, in denen de~ Pestbazillus sich in den Bubonen' mit pyogenen Staphylokokken zusammen
befindet - - und Zwar nicht, weft diese mildernd auf die Virulenz und
Toxizit~t der Produkte wirken, wie einige Beobachter m e i n t e n - - , sondern
weil alas Eingreifen dieser Bakterien in die Eiterungsphase die L5sung
der Krankheit mit der rascheren Ausleerung des Infektionsherdes nach aul3en
beg~nstigt. Wenn dagegen der Eiter unter anderen Umst~nden nicht
rechtzeitig ausgeschieden wird, so ist tier Tod dutch allgemeine Infektion
oder dutch langsame Intoxikation und dutch sie veru.rsachte Kachexie die
Sichere Folge. Die Wohltat tier raschen Ausleerung tier im Bubo angeh~uften infizierenden und toxisohen Produkte mittels der dutch pyogene
Staphylokokken hervorgerufenen Eiterung ist so augenscheinlich, dab seit
den ~iltesten Zeiten die Kur der Pest gerade darin bestand, mit allen
~Iitteln die Eiterung und 0ffaung tier Bubonen zu erreichen. Und noch
lr0rzlich erkl~irte einer der angesehensten i~rzte Alexandriens in ~gypten
ffir nfitzlich, sogar die Inokulation yon pyogenen Staphylokokkenkulturen
in die Pestbubonen als Kurmittel zu versuchen, wenn diese keine Tendenz
zu.r Eiterung zeigen (21).
Anders verh~lt es sich, wenn der Pestbazftlus sich mit septik~mischen
Bakterien (Diplokokken) oder mit dem Streptococcus zusammen befindet,
weft sich dann schon yon Anfang an um die den Bubo bildenden Drfisen
eine Zone yon 5dematSser Infiltration zeigt, und mit der Teilnahme der
umliegenden Gewebe und der Haut am EntziindungsprozeB die Verbreitung
der Pestbazillen fiber die anfangs infekten Dr0sen hinaus und folglich die
allgemeine Infektion leichter und rascher effolgt.
Die Umst~nde, unter denen w i t die spoatane Kur der Pest sich vollziehen sehen, geben die ~Iethode an, welcher wit in tier Behandlung
derselben folgen miissen; e n t w e d e r der Versuch, den I n f e k t i o n s prozel3 in seinem A n f a n g a u f z u h a l t e n , was wit in e i n i g e n sehr
l e i c h t e n FMlen mit der S e r u m t h e r a p i e e r r e i o h e n kSnnen, oder
tier c h i r u r g i s c h e E i n g r i f f , wenn die progressive E n t w i c k l u n g
tier B u b o n e n und die Schwere der I n t o x i k a t i o n s s y m p t o m e beweisen, dab die spezifische B e h a n d l u n g mit dem S e r u m a l l e i n
ffir die G e n e s u n g nioht genfigt. Und dies ist gerade gewShnlich der
Fall bei jenen Pestkranken, die in den Hospit~lern Aufnahme suchen,
.398
CA~r~LLO TERI~I:
wenn die Krankheit sohon zu weit vorgeschritten ist, und in den schwers~en
F~llen, was Lokalisation, Verlauf und Virulenz der Keime anbetrifft
(Pestis major).
Abgesehen yon der Art, wie einige Pestffalle auftreten und yon der
manchmal aul3erordentliohen Rapidit~it des infektiven Prozesses bei (ter
Bubonenform, kSnnen wir yore pathologisohen und kurativen Standpunkte
aus das, was bei der Pest vorkommt, nicht anders betraohten als das, was
sonst bei Lymphangitis und Lymphadenitis mit bSsartigem Charakter
vorf~llt, wo kein Arzt daran denken wfirde, Serum als Antidot anzuwenden, bevor er mit eiuem Operationsakt eingreift. Der einzige Unterschied besteht darin, dab bei der Pest die lokalen PhSnomene weniger
augensoheinlich sind gegenfiber den allgemeinen Ersoheinungen, welohe
dutch die yore prim~ren Herd ausgehende Intoxikation bestimmt sind,
w~ihrend in den anderen Lymphadenitis die lokalen Verh~ltnisse leiohter
die Aufmerksamkeit des Beobachters auf sieh ziehen und zum chirurgisohen
Eingriff veranlassen, sohon ehe die Syrup.tome der Intoxikation und I m
fektion sich zeigen.
Aus der Praxis, die ich mir in der Kur der Pest, besonders im Seehospital in Rio Janeiro erworben babe, kann ich ohne ZSgern die Behauptung aufstellen, daf~ die grol3e Sterblichkeit, weIche wir in den
Pesthospit~lern beobachten, yon dem Mangel oder der VerzSgerung des
chirurgisohen Eingriffes herriihrt, denn die Infektion bleibt fiir eine
Periode yon 3 bis 5 oder mehr Tagen immer im prim'~ren Bubo und in
den nahen Lymphbahnen lokalisiert und kann mit der Aussoheidung des
infekten Teiles iiberwunden werden, wenn die Kur mit dem Serum allein
augenscheinlich wirkungslos bleibt.
Das Resultat zahlreicher mikroskopischer und bakteriologischer Untersuchungen, ausgeffihrt unter Mitwirkung der DDr. Gomes u. G u i m a r a e s ,
um den Verbreitungsweg der Bazillen yore Eintrittspunkte in den Bubo
an und in den verschiedenen Entwicklungsstadien desselben festzustellen,
fiberzeugte mich yon tier Niitzlichkeit des sofortigen chirurgischen Eingriffes bei der Kur der Pest und zwar mit einer radikalen Operation - tier E x s t i r p a t i o n der Bubonen.
Bei 82 Pestkranken, die Phlykt~en oder Furunkel oder andere
prim~tre Hautl~sionen zeigten, war es nicht mSglieh, in der l~ngs des
Verlaufes der Lymphgef~iBe yon tier primKren L~sion an bis zum Initialbubo entnommenen Lymphe Bazillen zu finden, nooh in den umliegenden
Geweben aagerhalb der Kapsel der Driisen.
Aus d i e s e n R e s u l t a t e n miissen wir sohlief~en, dab der P e s t bazillus in den L y m p h g e f ~ B e n keine giinstigen E n t w i c k l u n g s b e d i n g u n g e n finder, und dab n u r in den Drfisen sieh der wirk-
STUDIEN i~BEI~ DIE PEST.
399
l i t h e I n f e k t i o n s h e r d bildet. Im Bubo selbst geht die Verbreitung
der Bazillen stufenweise yon Drfise zu Drfise vor sioh und dehnt sich
allm~hlich auf alle Drfisen einer Region aus, bevor sic in eine andere
fibergeht, und immer verl{iuft sic in der Richtung der Zirkulation der
Lymphe yon den oberfl~chlichsten in die ~iefsten Teile, niemals dutch
die Blutbahnen oder mit Entzfindung der intermedi~ren Lymphgef~iBe,
es mfiBten denn zusammen mit dem Pestbazillus sieh noch andere Bakterien (Diplococcus, Streptococcus) befinden, in welQhem ~alle man immer
schon yon der prim~ren Liision an Lymphangitis, Phlebitis und mehr
oder weniger ausgedehntes 0dem beobachtet.
Wenn z. B. tier prim~ire Bubo femoral und an der Spitze des Dreiecks
yon Scarpa lokalisiert ist, wie es gewShnlioh vorkommt, teilt sich die
Infektion allen oberfl~chlichen Inguinaldrfisen mit, ehe die tiefen vor dem
Forum crurale liegenden Drfisen ergriffen werden, und nut auf diesem
Wege dringbn die Bazillen dann in die Drfisen der Beckenkavitiit, so dab
wit im Anfang mit der mikroskopischen und bakteriologischen Untersuchung die Infektion der inguino-cruralen Drfisen feststellen kSnnen,
wenn die retroperitonealen noch intakt sind. Ebenso wenn der Bubo
axill~r und dutch die Schwellung der Drfise gebildet wird, welche am
vorderen inneren Winkel der Axfllarkavit~t hinter dem auBeren Rande
des groBen Pektoralmuskels liegt, teilt sich die Infektion allen Drfisen tier
Gegend mit, ehe sic die sub-klavikul~re Drfisenplejade :angreift. In den
Cervikalbubonen, obgleich dutch prim~ire Pestamygdalitis hervorgerufen,
babe ich festgestellt, dal~ tier InfektionsprozeB sich tagelang auf eine oder
zwei Drfisen der hinteren hxill~ir, oder oberen Cervikalgegend beschriinkte,
ohne sich fiber die mittlere und untere Plejade der Hals- oder Superklavikul~rgegend zu verbreiten.
Aus der histo-pathologischen Untersuchung der Gewebe ist es leicht
Zu ersehen, dal~ die Art der Infektion des Pestbazillus in den Drfisen
selbst, wie in'anderen Geweben, in denen die rasche und allgemeine u
breitung des Prozesses leichter erscheinen mfil~te, immer stufenweise vor
sich geht, in kleinen Herden zuerst in den Lymphr~umen lokalisiert,
sparer mit der fortschreitenden ZerstSrung des Gewebes zusammenfliel3end
[Aoyama (22), A l b r e c h t und Ghon (23), Banal und S t a g n i t t a (24),
Powel White (25), F l e x n e r (26), Babes und Levaditi (27), Dfirck (28),
Hassan H a m d i (29)].
Die Infektion der Drfise geht immer yore sinus aus und entwickelt
sich aus einem oder mehreren Bazillenherden, die alhn~hlich die ~[asse
des Parenchyms fiberfiuten, alas mit gewShnhch polym~leierten Leukozyten
infiltriert ist, die sich an der.Peripberie der Follikel und in dem Rindenteil der Markstrahlen u m die verletzten Stellen anh~ufen. Im Gewebe
400
CAMrr,LO TER~I:
folgen dann die Htimorrhagien~ die dazu beitragen, das adenoide Gewebe
vollsttindig oder fast vollsttindig zu zerstSren, weshalb man yon der normalen
Struktur der Drfisen nichts mehr erkennen kann. Die Kolonien der im
Gewebe eingenisteten Bazillen werden grSiler~ verschmelzen ineinander,
indem sie sich nach der Peripherie bin ausdehnen, wfihrend die rfickst~ndige Masse in 1%krose verf~llt; und erst dann fangen im Kranken
die schwersteu Ph~nomene der Infektion mit tier Intoxikation an. Der
typische ProzeB der fibrill~ren Koagulation, dem g~nzlich oder fast gtinzlich das Fibrin fehlt, und der sich in und um die GeftiBe in tier Masse
der nekrotisierten Pestdrfise b i l d e t - so vorzfiglich yon A l b r e c h t und
Ghon studiert und yon ihnen als charakteristisehe L~sion des prim~iren
Pestbubos angesehen--, ist ein Anzeichen und Produkt dieser spezifischen
Intoxikationsphase, denn er fehlte vollst~ndig in den Drfisen tier leichteren
F~lle~ die tier spontanen Heilung entgegengehen, und in denjenigen mit
septiktimischem Verlauf infolge yon gastro-instestinaler Infektion.
Diese ffir die Unterscheidung des prim~ren Bubos yon den sekundtiren so charakteristisehe Lokahsation und die Ver~nderungen der
prim~ren Herde in anderen Organen (Tonsillen, Lunge) stellen die
erste Anpassung des Virus in dem neuen Wirt dar, und sind die
Stellen, wo die Bazillen die besten Bedingungen finden, um sich die
Virulenz anzueignen und die folgende toxische und infektive T~tigkeit z u
entwickeln. Deshalb sehen wir, dab diese primtiren Ltisionen in den
Lymphbahnen experimentell leichter reproduziert werden kSnnen mit Kulturen yon abgeschwtichter Virulenz, wtihrend sie sich nicht mehr zeigen,
wenn die Bazillen durch verschiedene ~bertragungen in derselben Tierart
die hSchste Aktivittit erreicht haben, weft wir dann eine fast sofortige
allgemeine Diffusion haben~ immer jedoch noch anfangs augenscheinlicher
im Lymphsystem~ ehe sie ins Blur fibergehen, aber ohne den Charakter
der Lokalisation in Initialherden.
Das anatomiseh-pathologische Ergebnis~ auch yon A l b r e c h t und
G h o n und H a s s a n H a m d i beobachtet, dab besonders in den nekrotischen
Herden der Prim~rbubonen die Anhtiufung der Bazillen um und in den
Venen, in der Kapsel und dem perikapsultiren Gewebe bemerkt wurde, kann
meine Beobachtungen nicht entkrtiften, die an Bubonen im Anfang der
Krankheit ausgeffihrt wurden~ als das septik~mische Stadium noch nicht
erreicht war. Auch die histo-pathologische Untersuchung best~tigt, dab
der Ubergang der Bazillen aus der Drfise in die Blutbahnen mittels L~sionen der Gef~Bwand in einer sehr sp~ten Periode erfolgt. Die Drfise
stellt also einen wirkhchen Filter ffir die durch die Haut eingedrungenen
Pestbazillen dar, und ffir eine mehr oder weniger lange Zeit h~lt sie ihren
fortschreitenden Marsch auf. Das entsprieht fibrigens dem Resultat der
STC~)~EN ~v.R Dim P]~ST.
401
bakteriologischen Untersuchangen, welehe in augenscheinlichster Weise darlegen~ dab selten vor dem dritten Tage die Bazillen im Blutkreislauf
gefunden werden kSnnen, aach wenn ffir die Kalturen bedeutende Mengen
Blur genommen werden (bis zu 20 ~r in meinen Experimenten) und aus
den u
die in direkter Beziehung zum Prim~rbubo sind.
Aus diesen Beobachtungen geht die augenscheinhche Notwendigkeit
hervor, die sofortige Exstirpation des primiiren Babes vorzunekmen, und
man begreift~ dab die MSglichkeit der Kur in einer K_rankheit yon so
rasch infizierendem und toxisehem Charakter wie die Pest um so sicherer
sein wird, je rascher und sorgf~ltiger die Ausscheidung des infekten Teiles
ist, der die primate Lokalisatioa der Bazillen im 0rganismus und den
Ausgangspankt der allgemeinen Infektion darstellt. Zu denselben SchluBfolgerungen sind auch A l b r e c h t und Ghon (30) yon der 5sterreichischen
Kommission ffir alas Studium der Pest in Indien gekommen and sprachen
die hleinung aus, dab in der Kur der Pest niemals die E x s t i r p a t i o n
des p r i m ~ r e n Bubos v e r g e s s e n w e r d e n dfirfte trotz tier A n w e n d u n g des Serums.
J a m a g i w a (31) hat ebenfalls nachgewiesen, dab die rasche Exstirpation der infekten Drfisen rationell und wohltuend ist. B a n d i (32) hat
in einigen yon mir vorgeschlagenen Experimenten gute Resultate bei
Tieren erreicht~ obgleich die Schwierigkeit einer aufmerksamen Medikation
and der wegen tier Quantit~t und u
des inokulierten ~iateriales
raschere Verlauf der Infektion die Wirkungen der Operation bei ihnen
weniger augenscheinlich machen.
In allen neueren khnischen Studien fiber die Pest wurde nicht genfigend Gewicht gelegt auf das~ was die empirische Praxis der Vergangenheir bezfiglieh der chirurgischen Kur derselben erworben butte in Epidemien, die~ was Virulenz der Keime und Schwere der F~lle anbetrifft, viel
gef~hrlicher waren als die gegenw~rtigen. Seit den filtesten Studien fiber
die Pest sehen wit alas Prinzip aufgestellt, dab die Genesung yon der
raschen Ausleerung der Bubonen bedingt sein muB. Um Hamorrhagien
zu vermeiden, riet man den Gebrauch yon Rtzmitteln oder des Brenneisens und zerstSrte die Gewebe bis zu den gesunden. In allen alten
Abhandhngen fiber die Kur der Pest ist angegeben, im Anfang die Eiterung hervorzurufen, aber n i c h t zu l a n g e zu warten~ wenn diese zSgern
sollte sich einzustellen, und rasch mit der Inzision and mit s
einzugreifen, wenn die Eiterung sich am 2. bis 3. Tage noeh nieht zeigte.
Die ersten Kenntnisse fiber die chirurgische Kur der Pest gehen bis
zu H i p p o k r a t e s (33) und dem yon Galen (34) erw~hnten A r c h i g e n e s ,
aber besonders zu den arabisehen "~rzten ( E b u - S i n a - A v i c e n n a , Isaac
Zelfschr. f. Hygiene, LI~r.
26
402
CAMmLO TEa~X:
J u d e u s , Beitar, Rhazes) zurfick, und wurden yon unseren Arzten des
Mittelalters zur Zeit der Kreuzzfige naoh dem Orient erfahren, we noch
heute die eingeborenen Empiriker die Pestbubonen mit tiefen Inzisionen,
mit ~tzmitteln und mit dem glfihenden Eisen behandeln.
In den Epidemien, welche Frankreieh 1500 heimsuchten, behauptete
sich die ehirurgische Kur der Pest besonders dutch Ambrois Par~ (35)
und seine Schule als die einzige positive, wirksame Methode inmitten
aller anderen extravaganten veto Empirismus vorgeschlagenen Heilmittel.
Gleiehe Resultate erreichten Settala (36) und Tadino (37) in der berfihmten Pest yon Mailand 1630. Sp~iter in tier Epidemie yon Marseille 1720
sehen wir mit den besseren Kenntnissen der Praxis und des Studiums
der Anatomie auBer der der Eiterung vorhergehenden Inzision und tier
Medikation mit den antiseptisehen ~Iitteln jener Zeit (Sahwasser und
Essig) aueh die Exstirpation der Bubonen eingeffihrt nach der yon
Manger (38) angegebenen Methode, welche der gegenw~rtig vorgeschlagenen entspricht.
Bemerkenswert ist das best~ndige Urteil aller alten Forscher, da~
der husgang der Kur der Pest wesentlieh yon zwei Momenten bedingt
wird, die yon S e t t a l a sehr gut in diesem Rat zusammengefaBt sind:
a u f alle F~lle und so raseh wie mSglich die Materie ausscheiden,
damit sie, wenn sie z u r f i e k g e h a l t e n wird, ihr Gift n i e h t fiber
den g a n z e n KSrper verbreite.
Die Notwendigkeit und Wirksamkeit eines sofortigen chirurgischen
Eingriffes, auch bevor die Eiterung eintritt, anerkannt yon Chirurgen, die
in so schweren Epidemien operierten, gewinnen um so grSBeren Wert dureh
die Tatsache, dab die Methode ausschlieBlieh ffir die Pest angeraten wurde,
w~ihrend man ffir alle anderen entziindlichen Geschwfilste (wie Furunkel,
Anthrax und Bubonen anderer Natur) zu aufweiehenden Kataplasmen
griff und den Ausgang der Eiterung mehr oder weniger sp~t erwartete.
&Is Vervollstiindigung dieses kurzen Hinweises auf die Beobaehtungen
der Vergangenheit will ich aueh das yon den franzSsischen ~irzten wfihrend
des Eroberungskrieges in Pal~stina (1799) ausgeffihrte Experiment erw~ihnen, dutch welches als allgemeine Kunnethode der Pestkranken die
I n z i s i o n aller B u b o n e n , die noch n i c h t die •
der
E i t e r u n g zeigten, f e s t g e s t e l l t wurde, und das um die Krisis
zu e r l e i e h t e r n . Der Privatarzt Napoleons in St. Helena, O']~eara (39),
berichtet, daB, bevor dieser Befehl gegeben wurde, Napoleon auf den Rat
der :&rzte einen Versuch an einer gewissen Zahl yon Kranken anstellen
liel~, wiihrend eine gleiehe Zahl nach den gewShnliohen Methoden (Kataplasmen usw.) behandelt wurde. Das Resultat war, da~ viel m e h r yon
den ersten als yon den zweiten genasen.
STUDnZN OB~ DZE PEST.
40~
Wenn also so gfinstige und best~ndige Resultate in frfiheren Zeiten
erreicht wurden, als die Bedingungen der Krankheit I was Virulenz und
Komplikationen anbetrifft, viel ernsterer Art waren, und w~hrend alle
technisohen und wissenschaftlichen Hilfsmittel der modernen ChirUrgie
fehlten, ersoheint es wirklioh sonderbar, dab man noch an tier Wirksamkeit des chirurgischen Eingriffes bei der Bubonenpest zweifeln will und
der Evidenz der Tatsachen theoretisehe Folgerungen entgegensetzt, die
gar keinen Wert haben, weft sie im Widerspruch sind mit den neuen in
den letzten Jahren erworbenen Kenntnissen fiber die Pathogenie und
Klinik der Pest und mit allem was t~glioh die moderne Chirurgie im
Kampfe gegen die in den Organen lokalisierten Infektionen feststellt,
auch in den F~llen, we man sie schon fast als verallgemeinert erkl~ren
kann. (Exstirpation des Uterus infolge yon septiseher Endometritis, die
intestinale Sutur bei den veto Typhus hervorgerufenen Perforationen, die
Inzision des Herdes und die Exstirpation tier Drfisen beim mflzbrandigen
Anthrax tier Rinder, bei der ansteckenden Adenitis der Pferde, bei der
septischen, tuberkulSsen usw. Lymphadenitis des l~Ienschen und der Tiere.)
Die Tradition tier chirurgischen Kur tier Pest wurde immer mit
Erfolg fortgesetzt, auoh nach den oben erw~hnten Epidemien, wie wit aus
den yon P r o u s t (40), besonders bezfiglich der im Seelazarett Farol in
Marseille vorgekommenen F~lle und yon Cabanas (41) gesammelten
Daten ersehen kSnnen. Es ist auch Cantlies (42) u
in diesen
letzten Jahren yon neuem die Aufmerksamkeit tier ~rzte auf die Vorteile
gelenkt zu haben, die man aus der ohirurgischen Kur tier Pestkranken
ziehen kann, um so mehr gegenfiber tier Unzul~nglichkeit al!er anderen
bisher angewandten Kurmethoden.
Die Exstirpation der Bubonen, besonders wenn sie sich nooh in der
Anfangsperiode befinden, bietet ffir den Chirurgen keine Schwierigkeit
und wird auch ohne Narkose yon den Kranken ertragen, da die lokale
An~sthesie genfigt, besonders wenn der Babe oberfl~chlioh ist.
Der Operationsakt beschr~nkt sioh inden meisten F~llen auf die Inzision
der Haut und der oberfi~ohlichen Aponeurosis; die Isolierung des Bubos
yon den umgebenden Geweben, die man mit den Fingern und tier Sonde
unter groBer Yorsicht ausffihrt, um die Inzision und das Zerreil3en der
grol~en Gef~13e und Nerven zu vermeiden; die Exzision des Bubos; das
Aufsuchen der nahegelegenen Lymphdrfisen, haupts~chlieh wenn sie
hfimorrhagisch, geschwollen und sehmerzhaft sind. Wenn man mit Vorsicht vorgeht, ist selten nStig, die grol3en Gef~13e zu binden, und der
Verlust an Blur ist unbedeutend.
26*
404
CAMm~o TEXNI:
Wenn das Feld yon allen infekten Driisen befreit ist, w~scht man
mit reichlichen antiseptischen LSsungen aus, macht eine feuchte Medikation, die man in den folgenden Tagen erneuert, in denen man auch
zum zweiten Male eingreifen kann, falls noch Uberreste des infekten
Teiles zurfickgeblieben sein sollten.
Die im Pesthospital in Rio Janeiro w~hrend der Epidemien yon
1900 bis 1901 ausgeffihrten Operationen beliefen sich auf 642, mi~ einer
durchschnittli0hen Mortalit~t yon 10 bis 15 Prozent, je nach den F~illen
in bezug auf die Zahl und Lokalit~t der Bubonen und die Dauer der
Krankheit, bevor der chirurgische Eingriff erfolgte. Die meisten Todesfiille ereigneten sich unter den Kranken mit mehr als 5 Krankheitstagen,
in denen die allgemeine Infektion dutch andere Komplikationen schon
zu vermuten war.
Die Exstirpation der Bubonen fief auch in diesen yore Tode gefolgten
F~llen immer gfinstige Wirkung auf den Verlauf der Krankheit hervor
ffir mehr oder weniger lange Zeit, so dab man eine gfinstige Prognose
stellen konnte, lind immer haben wir bei der Autopsie feststellen kSnnen,
dab der ProzeB schon vorher die Lymphdriisen tier Beckenkavit~t erreicht
hatte, besonders diejenigen, die an der hinteren 0ffnung des Inguinalringes liegen, und die nicht rechtzeitig operiert wurden, endweder weil
sie fiir noch nicht infekt gehalten wurden, oder weil der Kranke sich
dagegen auflehnte. In einem solchen Falle haben wit nach fast einem
Monate miihsamer Rekonvaleszenz den Tod eines Kranken durch septische
Peritonitis feststellen kSnnen, veranlaBt durch Ausleerung der ichorSsen
Ansammlung einer Beckendriise in die BauchhShle. Dieser Fall war einer
der typischsten, um die Unzul~nglichkeit des antipestSsen Serums, auch
nur, was seine antibakterische Wirkung aubetrifft, zu beweisen, denn es
wurden mehr als 300 ~cm verbraucht, und die Bazillen blieben noch im
nekrotischen Herde der Driise lebend und virulent.
Die Operationen wurden alle in sehr schweren, gewShnlich tSdl i c h e n F~llen ausgefiihrt (multiple Bubonen in verschiedenen Gegenden:
axill~r und inguinal, doppelt inguinal und Becken, zervikal, parotitis) und
das erreichte Resultat l~Bt keinen Zweifel fiber die Wirksamkeit der
Methode.
Angesichts der im Hospital yon Rio Janeiro mit der chirurgischeu
Behandlung erreichten Erfolge gegenfiber allen anderen Kurmethoden,
ffihle ich reich berechtigt, die Zweifel als unbegrfindet zu betrachten, die
yon denjenJgen entgegengesetzt werden, welche vorziehen, den sichern
Tod des Kranken zu erwarten, statt die Kur mit dem sichersten Erfolge
zu versuchen. Ich bfn auch fiberzeugt, dab die AusschlieBung dieser
~ethode yon der geringen klinischen Kenntnis der Krankheit abh~ingt
STUDIEN UBER DIE PEST.
405
und yon einer eigentiimliehen Pr~iokkupation der Arzte beziiglich des
Einflusses des chirurgischen Eingriffes auf das Lymphsystem, eher als yon
reiflich erwogenen wissenschaftlichen Griinden.1 In allen Operierten, aueh
in denjenigen, denen fast alle Inguino-kruraldrfisen beider Seiten und die
axill~ren exstirpiert wurden, zeigten sioh niemals ~-belst~nde irgend welcher
Art, weder beim Operationsakt noeh bei der folgenden Medikation, nooh
sparer durch ZirkulationsstSrungen der Lymphe und des Blutes und in
den Funktionen der GliedmaBen. Die Kranken verlieBen das Hospital in
den besten Verh~ltnissen, um ihre Besoh~ftigungen wieder aufzunehmen
und ermutigten die Neuangekommenen, die Operation zu fordern, um
raseher v0n den Qualen der Krankheit befreit zu werden. Der grSl3te
Vorteil der Exstirpation der Bubonen ist gerade, dab man in wenigen
Stunden das Wohlbefinden des Kranken erreicht und den Anfang der
Rekonvaleszenz.
9Aus den Kurven (Taf. X), die typisehen nach verschiedenen Methoden
behandelten Pestfiillen entnommen sind, ersieht man, dab in den Operierten
alas Fieber sofort dutch Krisis f~llt, und dal3 gleichzeitig alle sohweren
Symptoms der Intoxikation (Delirium, Tachykardie, Dyspnoe) aufhSren, die
noeh lange Zeit fortdauern~ wenn die Kur nut auf das Serum besohr~nkt
bleibt, auch in den leiehtesten yon spontaner Genesung gefolgten F~llen.
Um bis zur Augenscheinlichkeit die Notwendigkeit des chirurgisehen
Eingriffes in der rationellen Kur der Pest mit einem experimentellen Versuoh am ]~[enschen zu zeigen, hubert wir oft in F~illen yon Doppelbubonen
die Exstirpation nur eines Bubos ausgefiihrt und zugleieh die Serumkur
angewandt. Die Besserung der Kranken war sofort augenscheinlich, aber
in den folgenden Tagen stieg die Temperatur wieder fiber 39 ~ und der
9 sub-typhische Zustand, die Tachykardie und das Delirium dauerten fort.
Wenn wit dann die Kur mit der Exstirpation des zweiten Bubos ve~vollst~ndigten, hSrten in wenigen Stunden die Ph~nomene der Intoxikation
auf und der Kranke befand sioh in vSlliger Rekonvaleszenz.
Schlul~folgerungen.
1. Im Pesthospital in Rio Janeiro verhielt sich die Mortalit~t unter
den nltr mit antipestSsem Serum behandelten Kranken zwisohen 25 bis
50 Prozent, je nach den F~llen und der Qualit~t der inokulierten Sera.
Wit mfissen iibrigens in Betracht ziehen, dab in der Statistik zugunsten
der Serumtherapie alle leichtesten F~ille einbegriffen sind, die gewShnlich
1 Die yon mir vorgeschlagene chirurgisehe Beh~ndlung wurde auch nach dem
Mfl~erfolge des Serums Yersln in den 1902 u. 1903 in Neapel vorgekommenen Pestf~llen mit sehr gutem Erfolge angewandt.
406
CAI~ILLO TWRI~I:
ohne Kur genesen. Was auBerdem die Schatzung der kurativen Wirkung
der spezifischen Sera unsicher macht, ist die auBerordentliche Uabestandigkeit der Dosen in, was Form und kliaischen Verlauf anbetrifft, identischen
Fallen. Die bisher angewandten Sera erwiesen sich auf alle F~ille durohaus
wirkungslos in den septikamischen F~llen (Infektion auf gastro-intestinalem
Wege) und in der pestSsen Pneumonie, w o e s gerade am nStigsten ware,
ein tIeilmittel zu verabreichen, das fahig ist, das spezifische Virus im
Organismus des Kranken unschadlioh zu machen.
2. Die kurative Wirkungs]osigkeit der jetzt gebrauchlichen antipestSsen
Sera hangt you der Unzulanglichkeit der antibakterischen Kraft und yon
dem fast g~inzlichen Mangel antitoxischer Substanzea ab, well die zur Bereitung benutzten Tiere nicht fiihig sind, die Gifte des Pestbazillus zu
assimilieren und zu zerstSren und im eigenea Blute antibakterische und
antitoxische Substanzen in ffir die Kur des lllenschen genfigender Menge
anzuh~ufen.
In dieser Hinsicht kann man ffir die Serumtherapie der Pest die
besten Resultate erreichen, wenn man langsam Maulesel, Esel und Ochsea
imamnisiert, indem man ihnen Safte pathologischer Produkte yon pestSsen
Tieren start kfinstlicher Kulturen inokuliert.
3. Mit den kfinstlichen Sera yon H a j e m und F o d o r erreichen wir
auch gfinstige Resultate, abet der ~belstand, dab man groBe Mengen
Fliissigkeit auf intravenSsem Wege inokulieren muB, veraala~te uns, die
Anwendung solr
Kurmethode auf wenige F~lle zu beschranken.
4. Mit den intravenSsen Inokulationen yon Sublimat, wie Baocelli
vorschliigt, schwankte die Sterblichkeit fast in denselben Grenzen der
besten spezifischen Sera, zwischen 30 bis 40 Prozent, und wie ich schon
frfiher erwahnt habe, ist diese Kurmethode vor allen anderen zu empfehlen,
wenn man keine guten Sera zur u
haben kann, und wean es
unmSglich ist, zeitig die chirurgische Kur anzuwenden. Das Sublimat
wirkt als starkes Reizmittel der Phagozytose (Gaglio) und bietet den
Vorteil, dab es leicht jedem Arzte zur Verffigung steht auch in Gegenden,
wo man nicht immer hoffen kann, andere so schwer zu konservierende
Medikamente wie die Sera zu haben. Es ist auBerdem bekannt, da~ das
Quecksilber sich vorwiegend in den Lymphozvten der Lymphdrfisen und
im Plasma festsetzt und so einen ffir die Entwicklung des Pestbazillus
ungfinstigen Zustand bildet, gerade in den yon diesem infizierenden Keime
vorgezogenen Geweben. Aus diesem Grunde halte ich die Anwendung des
Sublimats fiir vorteilhafter als die der Karbolsaure, wie Dr. Seymour(43)
empfiehlt, besonders in Fallen, in denen wir schon die Gegenwart der
Bazil]ea im Blute nachweisen kSnnen.
STUDIEN 0-BER DIE PEST.
407
5. In den schwersten F~llen (Pestis major), in denen wit yon der
Serumtherapie oder yon anderen lokalen Kuren keinen Erfolg erwarten
kSnnen, b l e i b t als einziges rationelles H i l f s m i t t e l ffir die K u r
der operative Akt mit der E x s t i r p a t i o n der Bubonen.
In meinen ErSrterungen fiber die chirurgisehe Kur babe ieh in erster
Linie die Exstirpation des Bubos in Betracht gezogen start der anderen
lokalen Kuren, da die Praxis, die ich mir erworben hab% reich fiberzeugt,
grS{~eres Vertrauen in diese radikale Operation zu setzen.
Die einfache Inzision des Bubos mit tier Ausleerung des Pus und
des nekrosierten hIaterials ist wohltuend, hat abet nicht so rasche und
dauernde Wirkung den Gang der Infektion aufzuhalten, weft an der Stelle
immer die yon Bazillen infiltrierten ~berreste des Gewebes bleiben und
die direkte Wirkung der lokalen Medikation langsamer und weniger leioht wird.
Als lokale Kur, um die Verbreitung des Prozesses beim Auftreten
der Bubonen zu beschriinken, ist au]er den Kompressen mit lauwarmen
desinfizierenden LSsungen (Sublimat, Karbols~nre) die Einspritzung dieser
LSsungen (Sublimat 1 promiUe, Karbols~ure 1 bis 2 Prozent) in und um die
Masse des Bubos angezeigt, besonders wenn man yon der umgebenden Geschwulst oder tier adh~siven Periadenitis gleich einen kombinierten infektiven
Prozel3 des Pestbazillus mit anderen Bakterien (Streptococcus, Diplococcus)
vermuten kann. Es ist ratsam, zu diesen Mitteln zu greifen, wenn die
radikale Operation nicht mSghch ist, oder man zu lange damit warten muB.
•
anderen lokalen Kuren mfissen eher ftir schhdhoh als nfitzhch
gehalten werden, weft sie gar keine Wirkung auf die im Gewebe der
Lymphdrtise lokahsierten Bazillen ausfiben kSnnen, und lassen eine kostbare Zeit verlieren, start die rationellste und sicherste Kur anzuwenden.
Es ist ein unverzeihlicher Irrtum, die Eiterung des Bubos abzuwarten,
ehe man den chirurgischen Eingriff beschlie~t, denn entweder unterhegt
der Kranke infolge des raschen Fortschrittes tier Infektion oder infolge
der Wirkung der toxischen Produkte, welche durch die kurative Wirhmg
des Serums nicht neutralisiert werden kSnnen. Man daft weder die
Konstitution des Individuums in Betracht ziehen,, noch zu sehr auf den
individuellen Widerstand des Kranken reehnen: solange der Bubo bleibt~
werden die Kurbedingungen immer sehwieriger und gefhhrlioher und fast
immer tritt die Notwendigkeit ein, die Operation sparer unter sehwierigeren
Verhaltnissen auszuffihren, sowohl wegen der Entkr~ftung des Kranken
uud weft die vorgeschrittene Geschwulst die anatomisehen Beziehungen
tier Region zerstSrt, als auch wegen der Komplikationen~ wie Phlebitis,
Lymphangitis, ichorSsen Infiltrationen l~ngs der ~[uskelscheiden mit Gefahr
des Eindringens in die Kavit~t.
408
C ~ m ~ o Tv.R~:
Wenn es nicht mSglich ist, in tier Wohnung des Kranken for die
ohirurgisohe Kur zu sorgen, mul3 man wenigstens deft die intravenSsen
Inokulationen yon spezifisohem Serum (20 bis 40 ~m) oder yon Sublimat
(1 bis 2 ~ der Baocellisehen LSsung) vornehmen, und zugleioh mit der
grSBten Eile den Kranken ins Hospital schaffen, we die passende Behandlung angewandt werden wird. Die Pflege tier Pestkranken verlangt
daher besondere praktische Kenntnisse der Krankheit bei den mit den Besuchen und Inspektionen im Domizil beauftragten "~rzten und ~bung in
den intravenSsen und hypodermischen Seruminokulationen. Im Hospital
ist die Gegenwart eines gesohickten Chirurgen und eines tfiohtigen Gehilfen
unerl~Blich.
Mit den eben erSrterten Systemen, die, yon rigorSsen Laboratoriumsexperimenten begleitet, yon einer langen klinischen Praxis der Pest hergeleitet sind, wurden die Kranken des Seehospitals in Rio Janeiro behandelt,
und die erreichten Resultate im Vergleich mit denjenigen anderer Lokalithten
bes~tigen vollkommen die in dieser Arbeit mitgeteilten Betrachtungen. Ich
kann mit vo]lkommener Sicherheit behaupten, dab sich die Mortalitht,
wenn die Pest nach den oben angegebenen Methoden behandelt wird, auf
'die Verhiiltnisse und Grenzen aller anderen infektiven und ansteekenden
Krankheiten beschr~nkt, die allgemein als weniger schwer in ihren Wirkungen
betraehtet werden.
Bei dieser Gelegenheit muB ich noch erw~ihnen, dab die Ansicht
Scheubes (44) nicht zul~ssig ist, welcher meint, das in Pestepidemien die
l~iethode der chirurgischen Kur schwerhch anwendbar sei, vielleicht wegen
des Andranges der Kranken, w~ihrend die Ausiibung der Chirurgie besenders in Kriegen bewiesen hat, dab sie auch den grS~ten Ansprfichen
geniigen kann, wie sie in den gef~hrhchsten Pestepidemien nie vorkommen.
Wenn man die in den Statistiken der englischen Kommission so vorzfiglich zusammengestellte Bewegung in allen Hospit~lern Bombays vergleicht, kann man leieht ersehen, dab eine Anzahl yon Chirurgen als Ersatz
oder als Hilfe der ~Lrzte reohtzeitig und angemessen allen Anspriichen
des Dienstes entspreehen kSnnte, auch im H5hepunkt der epidemischen
Periode.
Ich schhe•e diese Arbeit, indem ioh mioh mit einem tiefen Geffihl
der Dankbarkeit des Interesses und der Erleichterungen erinnere, die mir
yon der brasilianisohen Regierung w~hrend meines langen Aufenthaltes in
jenem Lande zuteil geworden sind. Ich danke haupts~chlieh dem Direktor
der 5ffentlichen Gesundheitspflege Profi Dr. l~uno de A n d r a d e die
u
dieser Studien, basiert auf einen yon ihm ausgesproohenen
Wunseh, indem er mir die teehnische Leitung der Kur der Pestkranken
S T U ~ I ~ ff~m~ DI~ PEST.
409
anvertraute: ein praktisehes und sicheres Resultat zu erhalten, indem alle
alten u n d n e u e n ffir die K u r tier K r a n k e n besser a n g e z e i g t e n
M e t h o d e n v e r g l i e h e n wfirden. Ieh denke aul3erdem mit unvergeBlieher Zuneigung an alle meine Mitarbeiter im Isolierhospital yon Juruhuba:
Dr. E. Gomes, Chefi des staatlichen Bakt. Laboratoriums, Dr. Guim a r a e s , jetzt verstorben, Dr. Tavarez Maced o, Direktor des Hospitals,
Dr. Carvalho Leite, u
und die anderen ~lrzte und Internen
[Dr. A. P e d r o P i m e n t e l , Dr. A r a g a % und die Studenten J. ~[ario,
L a n d e l i n o C-omes, Alvaro S a n e h e z ] , welche mir mit ihren Beobaohtungen und mit der grSBten Ergebenheit als Kollegen und Studierende
geholfen haben.
Anhang.
Als Anhang zu der gegenw~rtigen Arbeit lasse ich einige klinisohe Bemerkungen folgen fiber die typischsten F~lle unter den Hunderten, welche
unserer Beobachtung im Seehospital Paula Candido in Rio Janeiro unterstellt wurden, des bei Gelegenheit der Pestepidemien aussohliel31ieh zur
Aufnahme der Pestlrranken bestimmt war.
Das Hospital liegt in einer Krfimmung der Bai yon Jurujuba, der
Stadt Rio Janeiro gegenfiber, und etwa 2 Seemeilen yon ihr entfernt. Es
besteht aus vier dutch eine Veranda verbundene Pavfllons und aus den
ftir die Angestellten n5tigen Geb~uden. Es kann etwa 800 Betten enthalten.
Der Observationspavillon ist auf einer kleinen Insel vet tier Landungsbrficke
des Hospitals errizhtet.
Die Kranken wurden aus den verschiedenen Gegenden der Stadt in
ein nahe bei einem isolierten Kai (Caes da Saude) gelegenes Depot gebracht und yon deft mittels eines szhwimmenden, yon einem kleinen
Dampfer nachgeschleppten Lazaretts ins Hospital fibergeffihrt.
Alle diese Obliegenheiten wurden mit der gr5Bten Sorgfalt und
Sehuelligkeit ausgeffihrt, damit der Beistand der Kranken niehts zu
wtinsohen fibrig liege.
_ .........
In der Klassifikation tier klinischen Formen der Krankheit beabsichtige
ieh den yon tier englisehen Kommission ausgesprochenen Ansichten zu
folgen, sowie dem, was P. Manson (45) in seiner bekannten Abhandlung
fiber die tropisohen Krankheiten darlegt, ausgenommen, was ich schon
in meiner vorhergehenden Arbeit fiber die septik~misehen, yon gastrointestinaler Infektion herrfihrenden Formen gesagt babe.
Unter Pestis minor verstehen wit die leichten Pestfalle, eingeschlossen die abortiven, atypischen und sogen, ambulanten Formen. In
diesem leichtesten Typus yon Pest leidet der Kranke an Kopfweh und
410
C~Lo
TERNI:
Sohlaflosigkeit, an leichter, nur wenige Stunden dauernder Pyrexie, an
Empfindlichkeit und Sehmerz an einer oder mehreren oberfliiehliehen
Drfisen, gewShnlich an den Inguinaldrfisen, die oft geschwollen sind, und
manchmal auoh an Ubelkeit und Erbrechen. Dieser Zustand kann nur
2 bis 3 Tage dauern, aber nieht selten besteht die Schwellung der Drfisen
fort, und deshalb oder weil in den Drfisen Eiterung aufgetreten ist, wird
die Krankheit auf 10. 20 und sogar 30 Tage verlangert. Alle Falle
endigen mit Genesung.
Unter P e s t i s m a j o r verstehen wit alle Falle mit raschem u
in denen die ersten Symptome sieh raseh mit denen einel" schweren allgemeinen Intoxi]ration vermengen (gatliges Erbrechen und DiarrhSe, heftiges
Kopfweh, Schlaflosigkeit, Sehwindel, Palpitationen, Ubelkeit, Stupor,
Lethargie, allgemeine Schw~iehei. Die Temperatur steigt raseh mit kurzen
und unbestandigen periodisehen Schwankungen, Puls und Atem sind
beschleunigt, die Haut ist heil~ und troeken. Der Gang ist unsieher und
taumelnd, und der Kranke unfahig oder unwillig auf Fragen zu antworten,
weil der intellektuelle ProzeB langsam ist, und in dem typisehen Zustande
einer leiehten Trunkenheit. Die Bubonen, die oft multiple sind, sind
stark entwiekelt, sehr sehmerzhaft und widerstehen dem Druek.
In diesen Fiillen ist der Ted das gewShnliche Resultat, verursacht
duroh plStzliches Aussetzen der Herzt~tigkeit, seltener dureh Komplikationen
oder allgemeine Schwache, wean lange fortdauernde Eiterung in einem
oder mehreren Bubonen stattfindet.
In der Besprechung der Serumtherapie der Pest babe ich hervorgehoben, dab die Resultate wenig oder gar nicht zuverlassig sind, weil sie
nut in den leichtesten Fallen (pestis minor) mit fast bestandigem Erfolg
angewandt werden kann, immer jedoch unter Verhaltnissen, we andere
Kurmethoden gleiehes Resultat geben.
In den gewShnlichen und schwersten Fallen schlagt sie vollkommen
fehl, besonders wegen des Mangels an antitoxischer Wirkung; auf alle
Falle ist es hie mSglich, eine bestandige und genaue Beziehung zwischea
den Serumdosen und dem klinischen Verlauf der Infektion festzustellen.
In den folgenden F~llen, unter fast identisehen Krankheitsbedingungen,
bemerkt man die groi3e Veranderliehkeit der Serumdosen mit oft entgegengesetzten Wirkungen und auf alle Falle das Fortbestehen der Taohykardie und der anderen nervSsen StSrungen, aueh wenn man annehmen
konnte, eine gfinstige Krisis erreicht zu haben. Identische Resultate haben
wir in den Fallen Nr. 509, 227, 374, 356, die ausschliel31ieh mit intravenSsen Sublimatinjektionen behandelt, und in den F~llen Nr. 170, 153,
152, 505, die ohne jede Behandlung gelassen wurden.
STUDIEN ~BEI% DIE PEST.
411
In den operierten F~llen dagegen besserte sich der Zustand des
Kranken sofort, oft in fiberraschender Weise angesiohts der Schwere
der Krankheit. Verschiedene ~rzte, auch husl~nder (K a w e 1 b u r g,
W a l t e r W y m a n , Malta), welche unseren Beobachtungen im Hospital
yon Juruhuba beiwohnten, fanden fiir ihr Urteil keinen besseren Ausdruck
als Zu sagen~ es handle sich um wahre Auferstehungen.
Ich besehr~nke reich i einige der schwersten F~lle zu erw~hnen, in
denen die BaziUen sehon im Blute vorhanden waren, und tier Kranke
sich also bereits im pr~agonischen Zustande befand. Ieh bemerke, dab
alle thermischen und Pulskurven der operierten F~lle denen des Falles
Nr. 298 und 472 gleieh sind; d.h. man hat bestiindig sofortiges AufhSren
der thermischeu Elevation und eine ausgesproehene Abnahme aller sohwersten
Symptome der Infektion, ohne Gefahr, dab sie sich in der Folge wiederhelen, es mfi•ten denn andere nioht operierte Bubonen gebheben sein.
Der Fail Nr. 362 ist besonders bemerkenswert, weil es sieh um die
einzige nicht geimpfte Person (eine W~scherin) unter dem ttospitalpersonal
handelt: sie warde in sehr schwerer Form yon der Pest befallen. Unter
dem fibrigen Personal, besonders unter den Krankenw~rtern und iirzten,
die im Hospital yon Jurujuba mehr der Gefahr der Infektion ausgesetzt
waren wegen der Anforderungen der Kurmethode, da sie t~glich 10 bis
20 Kranke operieren und verbinden muBten, hatten wir nicht einmal verd~r
F~lle; und das ist meiner Meinung nach nicht so sehr den allgemeinen strengen Vorsiehtsmal3regeln als dem Faktum der durch die antipestSse Impfung erworbenen Immunit~t zuzusehreiben. Diese auch auf
das Personal des Serumtherapeutischen Laboratoriums in Messina angewandte Prophylaxismethode geniigte, um jede Gefahr yon Infektion auszusehhel~en, obglei~h wit mit sehr virulentem Material arbeiteten und
mit Metho,.ten, die uns mit grSl~erer Leichtigkeit tier Gefahr aussetzten,
uns die Krankheit zuzuziehen.
I. Pestis minor.
F~lle ohne j e d e B e h a n d l u n g .
Beob. ~r. 170. D. Alonzo, Spanier, 40 Jahre. Aufgenommen am
3. Juni, geheilt entlassen am 25. Juli. Rechter Femoralbubo.
Beob..:Nr. 153. D. Figueira da Costa, Portugiese, 26 Jahre. Aufgenommen am 24. Mai, entlassen 30. Juni. Linker Inguinalbubo.
Beob. Nr. 152. D. Estevez, Portugiese, 25 Jahre. Aufgenommen 23. Mai,
entlassen 15. Juni. Reehter Inguinalbubo.
Beob. Nr. 505. J. tIenrique da Couto, Brasilianer~eger. Aufgenommen
12. August, entlassen 22. August. Rechter Femoralbubo.
412
C~_~zz~no TER~:
Mit i n t r a v e n S s e n I n j e k t i o n e n yon S u b l i m a t
(Baecellis 1%rmel: Sublimat 10 % Kochsalz 40 % Aq. dest. sterile 100 ~rm)
b e h a n d e l t e F~ille.
Beob. l~r. 509. A. Ferreira de Soarez, Portugiese, 26 Jahre. Aufgenommen
14. August, entlassen 22. August. Linker Inguinalbubo.
Beob. :Nr. 227. A. de Mello, Brasilianer, 5 Jahre. Aufgenommen
14. Juni, enflassen 3. Juli. Rechter Inguinalbubo.
Beob. Nr. 374. g. A. Camargo, Brasilianer, 18 Jahre, yon brauner
Farbe. Aufgenommen 11. Juli, enflassen 28. Juli. Rechte Femoral- und
Inguinalbubonen.
Beob. :Nr. 356. 1~I. C. Bispo, Brasilianer, 24 Jahre, yon brauner Farbe.
Aufgenommen 25. Juni, entlassen 13. Juli. Linker Femoralbubo.
Mit S e r u m b e h a n d e l t e F~lle.
Das Serum wurde gewShnlieh intravenSs in starken Dosen (30 bis
50 r
injiziert, die in den ersten Tagen wiederholt wurden, dann einen
um den anderen Tag. Man vergleiehe den klinisehen u
dieser in
den Kurven (Taf. X) gezeichneten F~lle mit den vorhergehenden.
Beob. l~r. 139. A. J. Yiera, Brasilianer, 6 Jahre. Aufgenommen
18. April, entlassen 2.1~Iai. Kleiner linker Inguinalbubo, leichter Stupor.
Beob. :Nr. 147. J. Ferreira Royal, Portugiese, 11 Jahre. Aufgenommen
19. April, entlassen 3. Mai. Linker Inguinalbubo.
Beob. l~r. 186. ~I. C. Padilha, Brasilianer 1%ger, 44 Jahre. Aufgenommen
18. Juni, entlassen 19. Juli. Rechter Inguinalbubo.
Beob. l~r. 163. L.R. d'Oliveira Assiz, Brasilianer, 40 Jahre. Aufgenommen
27. ~Iai, entlassen 14. Juni. Linker Femoralbubo.
Beob. l~r. 140. C. de Castro Macedo, Portugiese, 19 Jahre. Auf
genommen 18. April, entlassen 2. ]llai. Linker Inguinalbubo.
Beob. :Nr. 178. A. J. da Silva, Brasilianer, 32 Jahre. Aufgenommen
8. Juni, entlassen 22. Juni. Linker Inguinal-Femoralbubo.
Ih Pestis major.
~[it S e r u m b e h a n d e l t e F~lle.
Ieh erwiihne drei typisehe Beobachtungen, um die Wirkungslosigkeit
der Serumtherapie in den schweren Pestformen zu beweisen. Wegen
anderer Einzelheiten und Kurven beobaehteter F~lle siehe I. Tell meiner
Arbeit in dieser Zeitschrift, Bd. XLIV.
Beob. :Nr. 197. ~. R. ~Ianghera, Portugiese, 23 gahre. Au.fgenommen
27. April, gestorben 6. Mai. Doppelte Femoral-Inguinalbubonen, r. Iliacalbubo.
Beob. :Nr. 190. Amelia Eugenia (Familienname unbekannt), Brasilian.
:Negerin, 60 Jahre. Aufgen0mmen 22. April, gestorben 27. April. Rechte
Femoral-, Inguinal- und Iliacalbubonen und linker Femoralbubo.
Beob. :Nr. 378. J. M. Ribeiro, Brasilianer, 23 Jahre. Aufgenommen
10. Juli, gestorben 17. Juli. Cervikal-Superklavikul~rbubonen und rechte
Inguinal- und Femoralbubonen.
STUDIEN ~-BER DIE PEST.
413
Chirurgische Kur.
Beob. Hr. 350. :N.Pinto do Santos, Brasilianer, 4 lahre. Aufgenommen
6. Juli, entlassen 18. August. Linker Cervikalbubo, doppelter Inguinal-,
rechter Axill~irbubo. Am 7. Juli Exstirpation der Cervikal- und Axill~rbubonen, am 9. Juli Exstirpation tier Inguinalbubonen.
Beob. Nr. 247. Orl. Gomes, Brasilianer, 13 Jahre. Aufgenommen
13. Juni, enflassen 11. August. Rechte Inguinal- und Iliacalbubonen. Operiert
am 17. Juli, well die anderen Kuren wirkungslos blieben.
Fig. 1.
Fall Nr. 159. Doppelte Femoral-Inguinalbubonen und rechter Iliacalbubo.
Beob. Nr. 378. A. dos Santos Reiz, BrasiHaner ~eger, 36 Jahre. ~ufgenommen 29. Juni, entlassen 22. August. Linke Iliaeal- und Inguinalbubonen.
Operier~ am 1. und 4. JuH.
Beob. Hr. 159. ft. u
Spanier, 26 gahre. Aufgenommen 16. Mat,
enflassen 15. Juni. Doppelte Femoral-Inguinalbubonen und reehter Iliacalbubo.
Operiert am 16. Mat auf der rechten Seite, am 18. auf der linkeu. (Fig. 1.)
414
CA~mLO TER~I:
Beob. Nr. 434. BIarg. A. Fragoso, Brasilianer, 43 Jahre. Aufgenommen
25. Juli, entlassen 5. September. Rechte Femoral-Inguinal-und Iliacalbubonen.
Operiert am 28. Juli.
Fig. 2.
Fall Nr. 424. Doppelte Axill~rbubonen und linker Cervikalbubo.
Fig. 3.
Fall Nr. 298. Reehter Inguinal-Iliacalbubo und Pestgeschwtir auf dem FulJ.
STUDIEN UBER DIE PEST.
415
Beob. Nr. 314. R. Muniz Auror% Por~giese, 35 Jahre. Aufgenommen
29. Juni, entlassen 11. August. Linke Femoral-Inguinalbubonen. Operier~
am 30. Juni und 2. Juli.
Fig. 4.
Fall Nr. 472. Linker Axill~rbubo und rech~er Femoralbubo.
Beob. Nr. 424. A. Ferr. de Gouvea, Portugiese~ 9 Jahre. Aufgenommen
23. Jali, entlassen 29. August. Multiple Bubonen, doppelte Axill~r- und
Cervikalbubonen. Operiert am 24. Juli an den Cervikalbubonen, am 27. Juli
an den A~ill~rbubonen. (Fig. 2.)
Fig. 5.
Fall Nr. 459. Rechter Inguinal-Iliaealbubo und linke Axill~r-Epitrochlearbubonen.
416
CA]IIII~LO TEP~NI:
Beob. ~r. 362. M. ft. de Souza, Portugiese, 30 Jahre. Aufgenommen
10. Juli, entlassen 29. August. Doppelte tinke Inguinal- und Femoratbubonen.
Operier~ 10. und 13. Juli.
Beob. :Nr. 298. D. Martins, Portugiese, 19 ffahre. Aufgenommen 26. ffuni,
entlassen 22. August. Reehte Inguinal- und Iliacalbubonen. Operiert am
27. Juni. (Fig. 3.)
Beob. Nr. 472. Ephigenia (Familienname nnbekannt), Brasi]., 9 Jahre.
Aufgenommen 6. August, entlassen 5. September. Linker Axill~r- u. rechter
Femoralbubo. Operiert 7. August. (Fig. 4).
Fig. 6.
Fall Nr. 329. Rechte Inguinal-Crur~fl-Axill~rbubenen. Pestexanthem u. Pestkarbunkel.
~uf dem Ful3.
Beob. Nr. 459. F. Alvez Monteiro, Portugiese, 18 Jahre. Aufgenommen
1. Juni, entlassen 11. August. Reehte Inguinal- und Iliacalbubonen, linke
Axill~ir- und Supertrochlearbubonen. Operiert 2. Juni am Leisten, am 4. Juni
an der Axilla und am Arm. (Fig. 5.)
Beob. Nr. 329. G. Mechino, Ita]., 26 ffahre. Aufgenommen 1. Ju]i,
entlassen 18. August. Rechte Inguinal-, Iliae- und Axill~irbubonen. Allgemeines Pestexanthem. Operiert am 1. ffuli am Leisten, am 3. ffuli an der
Axilla. (Fig. 6.)
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STUDIEN UBBR DIE PEST.
417
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2u
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27
418
CA~IILLO TERNI:
STUDIEN ~BER DIE PEST.
37. T a d i n o , 2~aggua.qllo etc. della gran peste dl Milano del~ anno 1639. Milano
per Filippo Ghisolfi. 1648. - - Siehe auch: P a u l u s A e g i n e t a , OTu, de tge •ediea etc.
Lib. VL Cap. XXXIV. Co]oniae. Opera et imprensa. Jo. Loteris. Anno 1533. -P r o s p e r i A l p i n i , De Medlelna 2iegyT~iorum. Libri quatuor. Venetiis 1591. - B a s s i a n u m L a u d u m , De origin el eau,a Testiae _patavinae. Vene~s. Ann. 1555.
D e r s e l b e , Cura della ~aste. Yen. 1557. - - Th. J o r d a n u s , ~e~Ha _phaenomena etc.
Francofur~i, Wechelus, 1576. - - M a s s a r i a , 2)e /)este. Yenetiis 1597. - - H y e r o n .
M e r c u r i a I i s , JOepestepraeser~im de Feneta et~atavina, Basel 1577. - - P r o s p e r
B o r g a n t i u s , JOe_peste. Venitiis 1565. - - V i c t o r de B o n a g e n t i b u s , JDeeern_problemata de _peste. Venetiis 1556. - - G e o r g i u s A g r i c o l a , De _peste in 1630. Mediolanum 1641.
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41. C a b a n a s , -Bull. gdn. de Thdr. 30. Nov. 1899.
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Jena 1903.
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Erkliirung der Abbildungen.
(Taft X.)
PesUs minor.
Serie 1.
Klinische Kurven der ohne jede kurative Behandlung gelassenen
F~lle. (Kontrolle.)
Serie 2.
Desgl. der mit endoven~sen Injektionen yon Sublimat behandelten
F~lle. ( B a c c e l l i s Methoden.)
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Future Pedagogues’ Attitudes and Knowledge about Inclusive Education in Spain: an exploratory study
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Review of European studies
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1. Introduction We understand Inclusive Education (IE) as a type of education which ensures the exercising of the inalienable
right to a complete, quality education for all. It involves every student’s full personal development and their
maximum integration in society. IE considers students’ diversity an enriching element and, thus, promotes it. Finally, IE supports the idea that everyone should be present and participate and interact at school (EADSNE,
2011; Booth & Aiscow, 2002; López, 2009; López-Torijo & Mengual-Andrés, 2014; López-Torrijo &
Mengual-Andrés, 2015; Opertti & Brady, 2011; UNESCO, 2008). All this has been acknowledged by the international community through declarations which imply an irreversible
commitment to IE. Some significant milestones are the Salamanca Statement and Framework for Action on
Special Needs Education (UNESCO, 1994); the Declaration of Madrid in 2002, where the 50 million European
disabled people were represented by 400 participants; the UN Convention on the Rights of Persons with
Disabilities (UN, 2006), which was signed by 147 countries and ratified by 97; and the 48th Session of the
International Conference on Education “Inclusive Education: the way of the future” (UNESCO, 2008). Together with its European partners, Spain has subscribed to all those international declarations and has even
been at the forefront of some of them (Rao, Cardona & Chiner, 2014). At a regulatory level, it has invested a lot
into IE. Proof of this is the regulatory process started some decades ago and improved in recent years: the
Spanish Constitution (SC, 1978, art. 25 and 49); Law of Social Integration of Disabled People—Ley de
Integración Social de los Minusválidos, LISMI—(LISMI, 1982); Organic Law of General Arrangement of the
Educational System—Ley Orgánica de Ordenación General del Sistema Educativo, LOGSE—(LOGSE, 1990);
Organic Law of Quality of Education—Ley Orgánica de Calidad de la Educación, LOCE—(LOCE, 2002); Law
of Equal Opportunities, Non-discrimination and Universal Accessibility of Disabled People—Ley de Igualdad de
Oportunidades, No Discriminación y Accesibilidad Universal de las Personas con Discapacidad,
LIONDAU—(LIONDAU, 2003). The current Organic Law of Education—Ley Orgánica de Educación,
LOE—(LOE, 2006) establishes IE as the Spanish educational model in its principles (art. 1), its aims (art. 2) and
in Title II, which is dedicated to “Educational equality”. The education provided to students with SEN is conceived from the perspective of IE. Thus, there is no
difference between Special Education and Inclusive Education; actually Special Education is part of Inclusive
Education. Received: March 24, 2015 Accepted: May 4, 2015 Online Published: June 26, 2015
doi:10.5539/res.v7n11p103 URL: http://dx.doi.org/10.5539/res.v7n11p103 Received: March 24, 2015 Accepted: May 4, 2015 Online Published: June 26, 2015
doi:10.5539/res.v7n11p103 URL: http://dx.doi.org/10.5539/res.v7n11p103 Review of European Studies; Vol. 7, No. 11; 2015
ISSN 1918-7173 E-ISSN 1918-7181
Published by Canadian Center of Science and Education Review of European Studies; Vol. 7, No. 11; 2015
ISSN 1918-7173 E-ISSN 1918-7181
Published by Canadian Center of Science and Education Abstract This article analyses the attitudes and knowledge about inclusive education among students reading Pedagogy at
the University of Valencia and how these are influenced by participants’ age, gender, and which academic
programme or year of study they are in. This research comprises a sample of 182 students from the degree’s four
year groups, which guarantee a representativeness of 95%. The principal results indicate that attitudes towards
inclusive education among students reading Pedagogy are highly positive. However, they consider the training
received insufficient. Regarding the participants of the study, the analysis shows that attitudes towards inclusive
education are directly related to age, while gender has no influence over students’ attitudes. Keywords: inclusive education, attitudes, knowledge, pedagogy, students Future Pedagogues’ Attitudes and Knowledge about Inclusive
Education in Spain: An Exploratory Study Santiago Mengual-Andrés1, Manuel López-Torrijo1 & María-Isabel Viana-Orta1
1 Department of Comparative Education & History of Education, University of Valencia, Valencia, Spain
Correspondence: Santiago Mengual-Andrés Avda. Blasco Ibáñez 30, Faculty of Philosophy and Educational
Sciences, Valencia, 46010, Spain. E-mail: Santiago.Mengual@uv.es Received: March 24, 2015 Accepted: May 4, 2015 Online Published: June 26, 2015
doi:10.5539/res.v7n11p103 URL: http://dx.doi.org/10.5539/res.v7n11p103 1. Introduction Apart from the teaching professionals working at ordinary schools, students with SEN receive assistance from
(early, general and specific) psychopedagogical and educational guidance teams (Equipos de Orientación
Educativa y Psicopedagógica), which are allocated on a regional basis. These units are comprised of
psychopedagogists, teachers specialised in hearing and language, teachers specialised in therapeutic pedagogy,
special education teachers and social workers. Psychopedagogists’ specific functions are: (a) to coordinate SEN
students’ psychopedagogical diagnosis and assessments, from which individualised learning programmes and
pedagogical or organisational adaptations are derived; (b) to propose the most suitable schooling modality for
each student; and (c) to provide guidance to schools, professionals and families on anything related to the
prevention, detection and assistance to students with SEN. Specifically, they provide support to school
management teams and teaching staff on the measures, regulations, projects, materials and educational resources
which are necessary for IE. Psychopedagogists are, in short, key specialists in the process of IE and coordinators
of the psychopedagogical units (Servicios Psicopedagógicos). The international declarations quoted above highlight the importance of professionals’ initial and continuous
training, as well as the significance of professionals’ attitudes when they deal with students with SEN (UNESCO,
1994, art. 3 and 4; UN, 2006, art. 8, 24.4 and 26.2). As a result of this, recent studies have tackled those questions. Future professionals’ general attitude towards IE
is positive (Cook, 2002; Haq & Mundia, 2012; Loreman, Earle, Sharman, & Forlin, 2007; Mu, Franck, & Konz,
2007), even in spite of some professionals’ stance denouncing the lack of initial training, time and assistance to
address it correctly (Lambe, 2011). Future professionals show a clearer position regarding IE in the case of
students with learning disabilities than in the case of students with behavioural problems, mental deficiency or
multiple disability (Cook, 2002). Other research shows neutral attitudes towards the social participation of
students with disabilities (de Boer, Pijl, & Minnaert, 2012). And some studies have found a certain rejection
towards IE for disabled people in general (Sharma, Moore, & Sonawane, 2009) and people with sensory, mental,
behavioural problems or multiple disabilities (Haq & Mundia, 2012), and even towards students with low
academic performance or with a shy, withdrawn personality (Sharma et al., 2009). 1. Introduction The principle of individualised attention demands different educational integration degrees adapted to
each student’s abilities, limitations and needs. That means parents can choose among different modalities and 103 Review of European Studies Vol. 7, No. 11; 2015 www.ccsenet.org/res types of schooling: ordinary school with no extra support; ordinary school with extra support (inside and outside
the classroom); combined schooling (in ordinary and specific students’ groups); specific students’ groups in
ordinary schools and, finally, simply specific schools. The type of schooling should always aim at the best
possible modality and level of inclusion for each student and is revised every academic year, sometimes even
during the same year. types of schooling: ordinary school with no extra support; ordinary school with extra support (inside and outside
the classroom); combined schooling (in ordinary and specific students’ groups); specific students’ groups in
ordinary schools and, finally, simply specific schools. The type of schooling should always aim at the best
possible modality and level of inclusion for each student and is revised every academic year, sometimes even
during the same year. According to statistical data, Spain is considered one of the EU countries with the highest levels of IE. Only 0.4 %
of Spanish students with special educational needs (SEN) attend specific educational institutions (EADSNE,
2011; Chiner & Cardona, 2012) in contrast to 4 % in Belgium, Denmark, Germany or Switzerland. According to
the most recent data from the Ministry of Education, Science and Sports—MECD—33.447 students are educated
in 490 specific institutions at non-university level (0.41%), compared to 8.087.347 students who attend ordinary
schools with different modalities and levels of educational inclusion. 1.9% of students have SEN (0.7% in
preschool; 1.9% in primary school; 2.2% in compulsory secondary school; 7.0% in initial qualification
programmes, 0.2% in non-compulsory secondary school—Bachillerato—and 0.4% in vocational training
education) and 755.156 students have foreign origins (9.1%). Spain has the highest student dropout rate in the
EU: 24.9% in contrast to the EU average of 12.8%. In 2012, there were 664.325 professionals teaching at a
non-university level in Spain, which means a student-teacher ratio of 12% (13.2 % in primary education; 10.3%
in compulsory secondary school—ESO—and 9.8% in non-compulsory secondary school—Bachillerato—). In
that same year, the Spanish Government invested 47 million euros in education, which was 4.58 % of the gross
domestic product (MECD, 2012). 1. Introduction As far as methods are concerned, combined programmes—comprising a theoretical part and a part related to
structured experiences—have proved excellent compared to separate programmes (Campbell, Gilmore, &
Cuskelly, 2003; Kim, 2011). However, immersive training courses do not clearly offer better results than short
programmes (Sharma et al., 2008). Students with higher marks or higher qualifications show better attitudes towards people with disabilities than
those with lower marks and qualifications (Forlin et al., 2009; Sharma, Forlin, Loreman, & Earle, 2006). Likewise, slight improvements are found in future professionals’ change of attitude as more training is received
(Costello & Boyle, 2013). Very little research has been carried out for Spain. Gómez and Infante (2004) analysed the attitudes of Spanish
students from various university subjects with regard to intercultural and inclusive education. They discovered
that all of them showed a positive attitude, with final-year Pedagogy students having a better attitude than
Business Engineering students. Sandoval (2009) found shortfalls in training regarding the identification of
learning disabilities, teaching responsibilities and cooperation among teaching staff. However, future education
professionals showed positive attitudes towards IE, with the development of values such as justice, equality and
cooperation. Meanwhile, Llorent and Llorent (2012) studied the semantics of future education professionals from
Brazil, Cabo Verde, Seville and Cordoba (Spain) with regards to IE. Their study was carried out through the
analysis of documentaries, interviews, free comments and surveys, and they confirmed the relation between their
perceptions and the institutional and regulatory context participants were in. However, future Pedagogy specialists’ training and attitudes towards IE have not been researched since the
Bologna Process university reforms and that reform affects the initial training of professionals. In that sense, the
White Paper for the Degree in Pedagogy and Social Education (a document which provides the framework for
the preparation of the new Spanish university degrees since the Bologna process) brought together all the basic
skills those professionals should acquire in order to adequately fulfill the functions required for IE (ANECA,
2005). The University of Valencia is one of the ten Spanish universities included in the Shanghai ranking for 2013. According to the training programme, students on the Pedagogy degree course at this university receive 240
credits (2,400 hours) of training during 4 academic years (of which 200 are theoretical, 32 are practical and 8 are
dedicated to the Final Project). 1. Introduction The importance of future professionals’ attitude (de Boer, Pijl, & Minnaert, 2010) lies in the fact that it can, and
will, affect their ability to apply inclusive criteria in their professional career (Costello & Boyle, 2013). However,
some elements can help develop a positive attitude towards IE. Indeed, all of the aforementioned studies point to
initial training as the main factor (Cook, 2002; Mu et al., 2007), along with professional experience in IE,
knowledge about different disabilities (Brandes, McWhirter, Haring, Crowson, & Millsap, 2012; de Boer et al.,
2010; Sharma, Forlin, & Loreman, 2008), special education training during undergraduate or postgraduate
studies (Forlin, Garcia Cedillo, Romero-Contreras, Fletcher, & Rodriguez Hernandez, 2010; Sharma et al., 2009),
training on inclusion policies and regulation (Forlin, Sharma, & Loreman, 2009), and even personal factors, such
as predisposition or talent (Cook, 2002). However, Cook’s (2002) study also identifies aspects which can be
detrimental to future teachers’ attitude towards IE, such as limited teaching experience, knowledge or aptitude in
the field of education. 104 Vol. 7, No. 11; 2015 Review of European Studies www.ccsenet.org/res Regarding future professionals’ knowledge about IE, almost all studies mention insufficient training and limited
or negative experiences (Forlin & Chambers, 2011; Forlin et al., 2010; Forlin, Loreman, Sharma, & Earle, 2009;
Gokdere, 2012; Sharma et al., 2009). Regarding sociodemographic variables related to IE future professionals’ attitudes and knowledge, (Forlin,
Loreman, et al., 2009) state that female students show a slightly higher positive attitude than male students. However, it is the men who improve their attitude most after training. Similarly, other studies show that younger
future professionals’ attitude is slightly worse than their older peers (Sharma et al., 2008), though this also
improves more than their older classmates after training (Forlin, Loreman, et al., 2009). As regards the level of education acquired, several studies show that specific IE training courses improve future
teachers’ attitudes and self-confidence (Glumbic, Kaljaca, & Brojcin, 2004; Stella, Forlin, & Lan, 2007), as well
as their abilities and self-awareness regarding those abilities (Cologon, 2012). Training in Special Education
(Forlin et al., 2010) and in the field of disabilities (Sharma et al., 2008) has also provided an improvement in
their attitudes towards people with disabilities. Some studies have detected the training areas which are the cause
of future teachers’ greatest concerns: assistance to students with low performance, disaffection, anti-social,
negative behaviour and, especially, bullying (Kyriacou, Avramidis, Stephens, & Werler, 2013). 2.4 Data Analyses The data analyses were carried out with SPSS 22 statistical software. Measures of central tendency and
dispersion were used to analyse participants’ answers about their attitudes and knowledge towards IE. After
checking normal distribution and homogeneity of variances for the various contrasts, different parametric
measures were used. An independent-samples t-test was used to check if there were any differences in attitudes
and knowledge based on gender. A one-way between-groups analysis of variance (ANOVA) was implemented to
explore the differences among attitudes and knowledge based on students’ academic year of study. Last but not
least, post-hoc contrasts were used to find out differences among groups, and effect sizes for parametric tests
were calculated. 2.3 Procedures The AKPEIEQ was distributed in two ways (printed and online) during the second semester of the 2012-2013
academic year. The online version was developed through the Limesurvey software. For the distribution,
permission was given by the School of Education and by the different teachers who provided the students with
the questionnaire. One researcher explained its content and solved the doubts participants had during completion. The on-line version was provided in order to involve a higher number of participants and so that students who
were on their internships could participate if they were interested. Data was collected and recorded during two
weeks and the response rate was high, at 96%. 1. Introduction In theory, they are gaining the qualifications to diagnose, plan and provide
educational guidance in any situation affected by diversity, inequality or discrimination, as well as to promote
interculturality. Quantitatively, however, only 15 credits are dedicated to Inclusive Education in the degree of Pedagogy at
University of Valencia, which means 6.25% of the total. The subjects offered are: a) Intercultural Pedagogy (6
credits, compulsory); b) Diagnosis of students with SEN (4.5 credits, optional) and c) Inclusive Education (4.5
credits, optional). Each credits amounts to 10 learning hours in the Spanish university system. Using the University of Valencia as the Spanish example, this study brings up the following researc Using the University of Valencia as the Spanish example, this study brings up the following research questions:
1) Do future pedagogues have positive attitudes towards IE? 1) Do future pedagogues have positive attitudes towards IE? 2) Do those future professionals consider that they are sufficiently qualified to meet the needs of IE? 2) Do those future professionals consider that they are sufficiently qualified to meet the needs of IE? 105 Vol. 7, No. 11; 2015 Review of European Studies www.ccsenet.org/res 3) To what extent does their age, their gender and the training received at university affect their knowledge and
attitudes towards IE? 2.1 Participamts The sample comprised 182 students—from the degree’s four year groups—reading Pedagogy at the Faculty of
Philosophy and Educational Sciences of the University of Valencia. Nonprobability sampling procedures were
used based on the total sample size, which provided a confidence level of 95.5% ( = .05). Most participants were women, 88.4% (n=161), and only 11.54% (n=21) of them were men. Participants’ age
ranged from 19 to 54, with an average of 21 years old (M=21.6; SD=4.3). The sample was created with students
from the degree’s four year groups: 37.9% were first-year students, 21.4% were second-year, 25.8% were
third-year and 14.8% were fourth-year. 2.2 Instrumentation The Attitudes, Knowledge and Previous Experience about Inclusive Education Questionnaire (AKPEIEQ) was
used to examine future professionals’ attitudes and knowledge about educational inclusion. This instrument
consisted of two parts: (a) the IE Attitude Scale (AS) referred to the dimension “Foundations towards inclusion”
from the Teachers’ Perceptions on Inclusion Questionnaire developed by Cardona, Gómez-Canet and
González-Sánchez (2000) including seven items, and (b) the IE Knowledge Scale (KS), which was designed ad
hoc for this study and which comprised seven items. Both dimensions used a five-level Likert scale (1 =
Strongly disagree, 2 = Disagree, 3 = Undecided, 4= Agree, 5 = Strongly agree). The questionnaire showed high internal consistency ( = .90) and inter-item correlation analyses did not show an
increase of reliability through the elimination or alteration of items. Content validity proved appropriate through
the use of Lawshe’s (1975) content validity index (CVI), showing a global CVI of 0.79. 3.1 Future Pedagogues’ Attitudes and Knowledge towards IE Generally speaking, attitudes towards IE were highly favourable (M=4.35, SD=.8). As Table 1 shows, almost all
participants (96.2%) were in favour of IE and 78.4% declared themselves to be against educational segregation. Similarly, most participants (96.2%) believed IE develops tolerance and respect, also in secondary school for
92.3% of students, pointing out more advantages than disadvantages (85.2%). Lastly, 87% of participants
considered the presence of support professionals in the classroom an indispensable element for an efficient IE. However, participants defined themselves as having very little knowledge (M=2.9, SD=.9) about IE. 42% of
them acknowledged not knowing international or Spanish regulations on IE. Similarly, a high percentage (77%)
considered themselves indecisive or incapable of carrying out an assessment for students with SEN, or admitted
having limited ability when it came to providing guidance to students with SEN about their future professional
opportunities (72.4%). At the same time, 67.6% of participants confirmed they did not know about the different 106 Review of European Studies Vol. 7, No. 11; 2015 www.ccsenet.org/res inclusive schooling modalities. Nevertheless, 56% of participants felt qualified to do their job according to the
requirements of IE. Table 1. Students’ attitudes and knowledge towards IE
Disagree
Unsure
Agree
M
SD
n
%
f
%
n
%
Attitudes towards inclusion
1. It is unfair to separate students with SEN from the rest of
their peers
4.08
1.25
24
13.2
15
8.3
142
78.4
2. Inclusive education develops tolerance and respect among
students
4.64
.57
1
0.5
6
3.3
175
96.2
3. I think that all students, including those with moderate
and severe disabilities, can learn in inclusive settings
3.99
.96
18
10
27
14.9
136
75.2
4. Inclusive education is also possible in secondary
education
4.46
.68
3
1.6
11
6.0
168
92.3
5. Inclusion has more advantages than disadvantages
4.27
.86
8
4.4
19
10.4
155
85.2
6. I am in favour of inclusion
4.64
.55
-
0
7
3.8
175
96.2
7. Inclusion requires the presence in the classroom of
support educators
4.38
.78
4
2.1
19
10.4
159
87.3
Total
4.35
.8
Knowledge about inclusion
8. I know the principles of LISMI, LOE and the UN
convention on the rights of persons with disabilities
2.77
1.11
77
42.6
55
30.4
49
27.1
9. I am able to diagnose a student with SEN
2.65
1.07
78
43.1
63
34.8
40
22.1
10. The relationship between students’ attitudes and knowledge about IE and age was studied using the Pearson
product-moment correlation coefficient. Preliminary analyses were carried out to guarantee the non-violation of
the normality, linearity and homogeneity assumptions. A medium positive correlation was found between
attitudes towards IE and participants’ age (r=.320, n=182, p=.000) and also between knowledge about IE and age
(r=.511, n=182, p=.000). The older participants had greater knowledge and better attitudes. Also, positive
attitudes towards IE went hand in hand with greater knowledge about it, with a medium correlation strength
(r=.313, n=182, p=.000). Similarly, a independent-samples t-test was carried out to evaluate the differences in attitudes towards IE and its 3.2 Relationship between Students’ Attitudes and Knowledge about IE, Gender and Age .3 Differences in Attitudes towards IE as a Function of Students’ Academic Year of Study In order to examine the differences among attitudes towards IE based on the students’ year of study. A one-way
between-groups ANOVA was carried out. The analyses proved that differences were statistically significant
between attitudes and students’ year of study (Table 2). Post-hoc comparisons using the Sheffé test showed that
fourth-year students’ attitudes (M=32.2; SD=2.33) were more positive than those of first-year students (M=28.85;
SD=3.37) and second-year students (M=29.52; SD=3.46). Contrasts also showed that third-year students’
attitudes (M=31.08; SD=2.83) were more favourable than those of first-year students. The differences between
attitudes and year of study were at p <.01 level [F(3,181) = 5.58, p = .001, ηp2 = .08], and the effect size was
medium. It was particularly noticeable that fourth-year students are much more likely to think that IE promotes tolerance
and respect towards difference than first-year students (p =.00). This was also observed between forth and
third-year students and first-year students (p <.01). Similarly, fourth-year students were more in favour of
applying IE in secondary school that those from the first and second year (p <.01). Finally, third-year students
were slightly more likely than first year students to agree that IE has more advantages than disadvantages (p
< .05). Table 2. Students’ attitudes towards IE as a function of their year of study
1st
2nd
3rd
4th
M
SD
M
SD
M
SD
M
SD
F (3,
181)
p
Post
hoca
Attitudes towards
inclusion
1. It is unfair to separate
students with SEN from
the rest of their peers
3.99
1.31
3.82
1.35
4.15
1.22
4.59
.84
2.284
.081
2. Inclusive education
develops tolerance and
respect among students
4.59
.52
4.36
.81
4.77
.42
4.96
.19
7.596
.000**
1<4
2<3,4
3. I think that all students,
including those with
moderate and severe
disabilities, can learn in
inclusive settings
3.93
.94
3.89
.98
4.06
.94
4.15
1.02
.554
.646
4. Inclusive education is
also possible in secondary
education
4.41
.62
4.18
.82
4.53
.68
4.85
.36
5.863
.001**
1,2<4
5. Inclusion has more
advantages than
disadvantages
4.12
.90
4.13
.97
4.51
.71
4.48
.70
2.944
.034*
1<3
6. I am in favour of
inclusion
4.59
.62
4.59
.54
4.68
.47
4.74
.52
.636
.593
7. Inclusion requires the
presence in the classroom
of support educators
4.23
.80
4.51
.64
4.45
.88
4.48
.70
1.470
.224
Total
29.85
3.37
29.52
3.46
3.08
2.83
32.2
2.33
5.58
.001**
1,2<4
1<3
Note. 3.1 Future Pedagogues’ Attitudes and Knowledge towards IE I know the different schooling modalities available for
students with SEN. 2.87
1.11
68
37.4
55
30.2
59
32.4
11. I can provide guidance about the organisational
proposals which should be included in a School Educational
Project in order to develop IE
2.84
1.07
66
36.8
61
34.1
52
29.1
12. I am able to provide guidance about the methodological
adaptations that can be used in class in order to deal with
students’ diversity
3.05
1.02
55
30.2
59
32.4
68
37.3
13. I can provide guidance about future professional
opportunities for students with SEN
2.70
1.18
80
44.2
51
28.2
50
27.6
14. I feel qualified to carry out my work according to the
requirements of IE
3.55
1.09
29
15.9
51
28.0
102
56
Total
2.9
.9 Table 1. Students’ attitudes and knowledge towards IE 3.2 Relationship between Students’ Attitudes and Knowledge about IE, Gender and Age Similarly, a independent-samples t-test was carried out to evaluate the differences in attitudes towards IE and its 107 Vol. 7, No. 11; 2015 Review of European Studies www.ccsenet.org/res specific knowledge based on gender and the results did not prove to be statistically significant at p < .05 level. specific knowledge based on gender and the results did not prove to be statistically significant at p < .05 level. 3.3 Differences in Attitudes towards IE as a Function of Students’ Academic Year of Study specific knowledge based on gender and the results did not prove to be statistically significant
3.3 Differences in Attitudes towards IE as a Function of Students’ Academic Year of Study 3.3 Differences in Attitudes towards IE as a Function of Students’ Academic Year of Study .3 Differences in Attitudes towards IE as a Function of Students’ Academic Year of Study a1 = First year; 2 = Second year; 3 = Third year, 4 = Forth year; * Significant at p < 0.05 level.; **
Significant at p < 0.01 level. Table 2. Students’ attitudes towards IE as a function of their year of study 108 Vol. 7, No. 11; 2015 Review of European Studies www.ccsenet.org/res 3.4 Differences in Knowledge about IE as a Function of Students’ Academic Year of Study The influence on students’ knowledge about IE was examined based on their year of study (Table 3). The results
of the ANOVA showed statistically significant differences with a large effect size at p < .01 level [F(3,181) =
16.37, p = .000, ηp2 = .22]. Post-hoc comparisons revealed that fourth-year students (M=26.0;SD=5.06) and
third-year students (M=19.75;SD=5.31) generally showed greater knowledge about IE than first-year students
(M=17.69;SD=6.05). The biggest differences were observed between fourth-year students and the rest of the
groups, with a noticeably wide range between fourth-year and third-year groups. The contrast analysis showed
that knowledge already improves from first-year students to second-year students. A more detailed analysis showed that students’ knowledge of European and Spanish regulations about IE
improved in the more senior years, especially between third and fourth-year students compared to first-year
students and between fourth-year students and third and second-year students (p = .00). Fourth-year students also
showed a better knowledge of the inclusive schooling modalities than the rest of the groups (p =.00). Similarly, a
recurrent pattern was observed regarding psychopedagogic assessment (p =.01), guidance on organisational
proposals to promote inclusion (p = .00), guidance on socio-professional opportunities to students with SEN (p
=.00) and methodological adaptations (p =.01), with third and fourth-year students having better knowledge than
first-year students. This pattern was also observed with the same significance when comparing fourth-year
students with third-year students. Table 3. Students’ knowledge about IE as a function of their year of study
1st
2nd
3rd
4th
M
SD
M
SD
M
SD
M
SD
F(3,
181)
p
Post
hoca
Knowledge about
inclusion
8. I know the principles
of LISMI, LOE and the
UN convention on the
rights of persons with
disabilities
2.36
1.08
2.61
1.07
2.91
.88
3.81
.87
14.179
.000**
1<3,4
2<4
3<4
9. I am able to diagnose
a student with SEN
2.19
1.10
2.63
.91
2.87
.74
3.48
1.12
12.293
.000**
1<3,4
2<4
10. I know the different
schooling modalities
available for students
with SEN. .3 Differences in Attitudes towards IE as a Function of Students’ Academic Year of Study 2.42
1.11
2.97
.98
2.94
.87
3.74
1.09
11.077
.000**
1<4
2<4
3<4
11. I can provide
guidance about the
organisational proposals
which should be
included in a School
Educational Project in
order to develop IE
2.29
1.12
2.79
.92
3.15
.85
3.70
.72
16.193
.000**
1<3,4
2<4
12. I am able to provide
guidance about the
methodological
adaptations that can be
used in class in order to
deal with students’
diversity
2.68
1.07
2.97
.98
3.30
.83
3.67
.83
8.147
.000**
1<3,4
2<4
13. I can provide
guidance about future
2.23
1.12
2.62
1.11
2.96
1.03
3.59
1.08
11.117
.000**
1<3,4
2<4 109 Vol. 7, No. 11; 2015 Review of European Studies www.ccsenet.org/res professional
opportunities for
students with SEN
14. I feel qualified to
carry out my work
according to the
requirements of IE
3.61
1.16
3.18
1.16
3.51
.88
4.00
1.00
3.207
.024*
2<4
Total
17.69
6.05
19.75
5.31
19.75
5.31
26
5.06
16.37
.000**
1<3,4
2<4
1<2
3<4
Note . a1st = First year; 2nd = Second year; 3rd = Third year, 4th = Forth year * Significant at p < 0.05 level. **
Significant at p < 0.01 level. 4.1 Future Education Professionals’ Attitudes towards IE Echoing the results of other studies, future pedagogues taught at the University of Valencia seem to show highly
positive attitudes towards IE, even to a greater extent than their peers from other countries in similar studies
(Cook, 2002; Gómez & Infante, 2004; Haq & Mundia, 2012; Mu et al., 2007). Specifically, some other countries’
students show doubts or even rejection towards IE for students with sensory deficits, intellectual impairment,
behavioural problems or multiple disabilities (Cook, 2002; Haq & Mundia, 2012), disabilities (Sharma et al.,
2009); or even a more general rejection towards students with low academic performance or with a shy,
withdrawn personality (Sharma et al., 2009). Nevertheless, students in our study are chiefly in favour of IE for
students with moderate and severe disabilities, both in primary and secondary education. This research concludes that, based on the sample participants, IE promotes tolerance and respect in all students. Similar results have been provided by Sandoval (2009), who concludes that IE stimulates justice, equality and
cooperation. Also Llorent and Llorent (2012) show the existing relationship between those values and students’
institutional and social contexts. Similarly, students’ in our study are highly in favour of IE and believe it has more advantages than disadvantages. This data enables us to conclude that they believe in the importance of those attitudes for their professional work,
something which has also been pointed out by de Boer, Pijl and Minnaert (2010) and Costello and Boyle (2013). Some studies have noted, based on future education pedagogues’ opinion, that successful IE depends on
professionals’ initial training (Cook, 2002), professional experience and previous contact with disabled people
(de Boer et al., 2010; Sharma et al., 2008), on special education training (Forlin et al., 2010; Sharma et al., 2009)
or even on teachers’ personal background, aptitudes and qualifications (Cook, 2002). However, our students have
highlighted that successful IE depends on the presence of support teachers in the classroom and on the specific
role played by class tutors. This is partly along the same lines as a study which identifies influential factors for
successful IE such as: (a) lack of personal resources, (b) work volume of ordinary teachers, (c) acceptance of
peers and families and (d) risk of lowering academic level of the class (Sharma et al., 2009). 4. Discussion 4.1 Future Education Professionals’ Attitudes towards IE 4.2 Future Professionals’ Qualification towards IE 4.2 Future Professionals’ Qualification towards IE As for the essential question of future pedagogues’ professional qualification, students from the University of
Valencia consider they are not trained enough to carry out basic IE tasks, although they are willing to complete
their training. These results are along the same lines as those observed in other contexts (Forlin & Chambers,
2011; Forlin et al., 2010; Forlin, Loreman, et al., 2009; Gokdere, 2012; Sharma et al., 2009). 4.3 Influence of Demographic Variables such as Gender, Age and Training Received on Students’ Attitudes and
Knowledge about IE As in most comparable international studies, the sample used in our research had a higher level of women than
men. Forlin et al. (2009) found small differences in attitudes in favour of female students over male students. In
contrast to this, our research did not show such differences regarding attitudes towards IE. Neither were any
significant differences regarding knowledge based on gender observed. 110 Vol. 7, No. 11; 2015 Review of European Studies www.ccsenet.org/res Regarding the variable age, students show a medium bivariate correlation between knowledge about IE and age. That same correlation appears between age and attitudes towards IE. Similarly, Forlin et al. (2009) noted that
younger students showed more negative attitudes towards IE than their older peers, although their attitudes
improved with training, especially among the former group. Regarding the training received, this study shows how future pedagogues consider there are significant
differences in terms of knowledge about IE between third and fourth-year students and first and second-year
students. The same situation is true for their attitudes. This makes us believe that improvements in training affect
attitudes towards IE. Other studies also provide evidence of such improvements in attitudes when there is more
training about IE (Glumbic et al., 2004; Stella et al., 2007), special education (Forlin et al., 2010), politics and
regulations (Forlin & Chambers, 2011), disabled students’ education (Sharma et al., 2008), or postgraduate
training (Forlin et al., 2010; Sharma et al., 2009). Lastly, our research results point out that students consider they have training shortfalls regarding their
professional qualification in: (a) diagnosing students with SEN, (b) providing guidance about work to students
with SEN, (c) knowledge of political and regulatory bases, (d) providing didactic and organisational guidance to
management teams, and (e) knowledge of the different schooling modalities available for students with SEN. 4.2 Future Professionals’ Qualification towards IE Other studies with a similar discourse carried out in different socio-political contexts called for a more complete
basic training to fulfil the needs of students who suffer anti-social behaviour, disaffection and bullying (Kyriacou
et al., 2013). 5. Conclusion Spain is considered one the most inclusive countries at a European level, both in terms of regulation and in terms
of the level of inclusion of students with SEN (Chiner & Cardona, 2012; Chiner, Cardona, & Gomez, 2015). The
results of this study show the highly positive attitudes of future pedagogues towards all groups with SEN at
primary and secondary education. However, the level of training received by these students after the Bologna
university reforms does not equip them sufficiently to carry out their work adequately and meet the requirements
of a quality IE. Therefore, the future of IE in Spain could be seriously affected. We agree with other international
studies (Brandes et al., 2012; Forlin et al., 2010; Forlin, Loreman, et al., 2009; Glumbic et al., 2004; Sharma et
al., 2009) that university curriculums need to be revised in order to guarantee a professional qualification which
meets the requirements of IE. Such training should also include teaching experience, contact with people with
SEN and, in particular, with students with disabilities. Such training should be implemented through specific
postgraduate level courses, since it affects the development of positive attitudes towards IE. This study invites us to carry out further in-depth analysis of its results, paying special attention to the influence
other demographic variables may have on the development of positive attitudes, such as teaching internships,
cooperation with institutions related to IE, political tendencies or religious beliefs. We are also trying to expand our sample to other universities from our socio-political context in order to obtain
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Education. Genova. Copyrights py g
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license (http://creativecommons.org/licenses/by/3.0/). This is an open-access article distributed under the terms and conditions of the Creative Commons Attribution
license (http://creativecommons.org/licenses/by/3.0/). This is an open-access article distributed under the terms and conditions of the Creative Commons Attribution
license (http://creativecommons.org/licenses/by/3.0/). 114
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Disaster management in industrial areas: Perspectives, challenges and future research
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Journal of Industrial Engineering and Management
JIEM, 2019 – 12(1): 133-153 – Online ISSN: 2013-0953 – Print ISSN: 2013-8423
https://doi.org/10.3926/jiem.2663
Disaster Management in Industrial Areas: Perspectives,
Challenges and Future Research
Yousuf A.M.A. Rebeeh
, Shaligram Pokharel*
, Galal M. Abdella
, Abdelmagid S. Hammuda
Department of Mechanical and Industrial Engineering, Qatar University (Qatar)
y.rebeeh@qapco.com.qa, *Corresponding author: shaligram@qu.edu.qa, gmg5005@qu.edu.qa, Hamouda@qu.edu.qa
Received: June 2018
Accepted: January 2019
Abstract:
Purpose: In most countries, development, growth, and sustenance of industrial facilities are given utmost
importance due to the influence in the socio-economic development of the country. Therefore, special
economic zones, or industrial areas or industrial cities are developed in order to provide the required services
for the sustained operation of such facilities. Such facilities not only provide a prolonged economic support
to the country but it also helps in the societal aspects as well by providing livelihood to thousands of people.
Therefore, any disaster in any of the facilities in the industrial area will have a significant impact on the
population, facilities, the economy, and threatens the sustainability of the operations. This paper provides
review of such literature that focus on theory and practice of disaster management in industrial cities.
Design/methodology/approach: In the paper, content analysis method is used in order to elicit the
insights of the literature available. The methodology uses search methods, literature segregation and
developing the current knowledge on different phases of industrial disaster management.
Findings: It is found that the research is done in all phases of disaster management, namely, preventive
phase, reactive phase and corrective phase. The research in each of these areas are focused on four main
aspects, which are facilities, resources, support systems and modeling. Nevertheless, the research in the
industrial cities is insignificant. Moreover, the modeling part does not explicitly consider the nature of
industrial cities, where many of the chemical and chemical processing can be highly flammable thus
creating a very large disaster impact. Some research is focused at an individual plant and scaled up to the
industrial cities. The modeling part is weak in terms of comprehensively analyzing and assisting disaster
management in the industrial cities.
Originality/value: The comprehensive review using content analysis on disaster management is presented
here. The review helps the researchers to understand the gap in the literature in order to extend further
research for disaster management in large scale industrial cities.
Keywords: disaster response, emergency management, disaster situation, decision-making
To cite this article:
Rebeeh, Y.A.M.A., Pokharel, S., Abdella, G.M., & Hammuda, A.S. (2019). Disaster management in industrial
areas: perspectives, challenges and future research. Journal of Industrial Engineering and Management, 12(1), 133-153.
https://doi.org/10.3926/jiem.2663
-133-
Journal of Industrial Engineering and Management – https://doi.org/10.3926/jiem.2663
1. Introduction
Disaster refers to the event of serious interruption that leads to extensive losses of human lives and deterioration
of the environment and physical facilities (Zio & Aven, 2013). The disasters in industrial facilities are classified as
technological disasters as they are essentially man-induced disasters (Granot, 1998). Van Wassenhove (2006)
mentions that disaster disrupts the performance of existing systems, virtually to a standstill on its onset and
threatens the systems' existence itself. Reniers, Khakzad, Cozzani and Khan (2018) mention that disaster in the
industrial areas can happen not only due to the events inside the industry but also outside the industry, for example,
natural disaster leading to flooding and fire in the chemical plants and events in other nearby plants leading to
disaster event in the chemical industry. Therefore, the development of an inclusive disaster management to avoid a
disastrous situation in the industrial cities becomes important (Shaluf, Ahmadun, Mat-Said, Mustapha & Sharif,
2002). There have been guidelines prepared by the international agencies, such as that by the Organisation for
Economic Co-operation and Development (OECD, 2003) and the United Nations Environment Programme
(UNEP, 2010) for chemical disaster management. These guidelines focus on chemical accidents prevention. The
review presented in this paper considers the research on disaster management in industrial cities (an area with many
industrial facilities) as a whole; that means, it considers disaster related to both chemical and non-chemical
processing industrial facilities in industrial cities.
Disasters occur in industrial cities due to the inadequacy of planning of response mechanism or sudden loss of
control and functionality of the risk mitigation process. While many industries focus on internal risk-based
management system to eliminate risks, disaster in industrial cities can be an external threat to a facility within or
outside the industrial city. Inadequate planning, implementation of proper procedures for eliminating, reducing,
reacting and recovering from disasters can create vulnerability of the industrial cities to disaster. As per the
definition by Alexander (1997), vulnerability inherent in industrial cities can be economic (loss of a source of
income to the people and the business owners) and technological (loss of capital assets). Alexander (1997) also
mentioned that disaster management needs a multi-disciplinary approach with the inclusion of available technology
and processes in it.
Chen, Chen and Li (2012) provided concepts on the lifecycle of disaster management system in which responses
are classified into pre-incident, during the incident and post-incident stages. A similar classification is also provided
by Moe and Pathranarakul (2006). Mojtahedi and Oo (2017) adopted this classification to review critical attributes
of stakeholders in disaster management.
In pre-incident phase, the disaster management focuses on prediction, identification, and assessment. During the
incident the focus is on the effective response, coping with the situation and fast recovery, and during post-incident,
the focus is on complete recovery. The response mechanism for these phases are predictive (preventive), reactive
and corrective (Mojtahedi & Oo, 2017; Moe & Pathranarakul, 2006). We follow this classification as well to discuss
disaster management in industrial cities. The assumptions and considerations in each phase are briefly given below.
•
Preventive (prediction) phase is an early stage management in which the focus is on response planning for
avoiding or minimizing the impact of the disaster. This phase requires planners to simulate various disaster
scenarios by considering intensity and spread of the impact in and around the industrial facilities.
Therefore, this type of system needs to support quick data access, communication, selection of locations
to stock the resources for mitigation of disaster impact, and deployment of resources at the quickest
possible time. The main focus here is on the preparedness for all disaster situations.
•
Reactive (response) phase is focused on relief supplies and other processes during or immediately after the
occurrence of the disaster event in order to contain the damages and to minimize the impact of the event.
A good response system gets activated immediately when a trigger for disaster is identified so that the
impact can be contained as quickly as possible. The focus in this phase is to continuously monitor, control
and provide all possible services as needed. Therefore, facilities and support developed for preventive
phase become very important to deploy reactive phase activities.
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Journal of Industrial Engineering and Management – https://doi.org/10.3926/jiem.2663
•
Corrective (recovery) phase is focused on recovery after the occurrence of the disaster event. The main
activities during this phase are recovery centered and support restoring the disaster inflicted situation to the
equilibrium (as-was or better condition) through rehabilitation and reconstruction. Lesson learned from
the disaster becomes a key to update preventive measures so that response system becomes more effective.
The review of literature shows that there is a need to strengthen the knowledge on the available tools for
management of disaster in the industrial areas, particularly those with large facilities and handle highly reactive
materials or compounds, and deal with risky procedures in processing such materials. The review shows that most
of the literature focus on natural disaster although natural disaster may be one of the factors for industrial disaster.
The systematic review and understanding on facilities planning, service system planning, resource planning for
different type of industry and different type of incidents, and the modeling processes that can be adopted for a
comprehensive disaster management is still lacking. Therefore, this review is expected to give a total overview of
the industrial disaster management during different phases of disaster: preparedness, response and recovery.
2. Methodology
In this paper, the review is focused on the publication database such as books and journals available in the
university, the published materials in databases maintained by ScienceDirect, Google Scholar, EmeraldInsight, and
other database websites (such as Taylor and Francis, Wiley online, IEEExplore, Institute of Mechanical Engineers
‘IMECHA’, American Chemical Society ‘ACS’, and EBSCO). It is to note that some of the literature appeared in
more than one database. This type of search of the literature for systematic review is consistent with that proposed
by Benet-Zepf, Marin-Garcia and Küster (2018), Marin-Garcia and Martinez-Tomas (2016), and Marin-Garcia,
Betancour and Giraldo-O’Meara (2018).
In this paper, content analysis method is used to extract the related information presented in the literature as it
helps in developing a meaningful conclusion through the examination of the textual data in a large volume of the
literature. Caunhye, Nie and Pokharel (2012) have also used content analysis to review optimization papers on
emergency logistics and have mentioned that the analysis is helpful in identifying the relevance of literature on the
focus of the study. Beerens and Tehler (2016) used content analysis on the disaster exercise related literature to
obtain various attributes, such as forms, functions, purpose and the methods. Inoue and Havard (2015) mention
that content analysis provides a quick method to analyze the accessible data to identify the forms required for the
review.
The database was searched using keywords such as decision support system (DSS), emergency logistics, disaster
management, industrial accidents, and emergency communication. The search and consequent review included the
following.
•
The framework, mathematical models, and applications of disaster management. The context of reviewed
literature mainly focus on industrial disaster.
•
Published articles (journals and conference proceedings) from 2000 to 2018. One basic paper published in
1997 that provides the emerging knowledge on the disaster is also reviewed.
•
Published papers in English language for the detailed review.
The review excluded the papers before 2000, as preliminary search showed that the focus on disaster management
and those particularly related to oil and gas industry are mainly considered after 2000. The review also excluded the
practitioner papers and books (or book chapters) as they may not have been peer-reviewed.
The analysis of the keywords search revealed that most of the papers on disaster management appeared in
ScienceDirect database followed by the EmeraldInsight database. Of the collected literature, about 60% of the
literature focused on the theoretical aspects while the rest focused on the applications and cases. Altogether, 71
literature were obtained but only 59 literature were found most relevant for this review. The findings from the
analysis are presented in this paper.
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Similar to the techniques used by Benet-Zepf et al. (2018) and Marin-Garcia and Martinez-Tomas (2016), the
literature review methodology used in this paper uses a streamlined and systematic process as shown in Figure 1.
The search started with the keyword search and then examining the literature for their relevance in industrial
disaster.
Figure 1. Methodology used for the literature review
Some of the reviewed literature are directly related to industrial disasters while the others mention of concepts
that can be considered for the application and design of emergency system in industrial areas. The papers
obtained from the search are then classified into different phases of disaster management as mentioned by Moe
and Pathranarakul (2006). The authors mentioned prediction, emergency relief, rehabilitation, and
reconstruction. The activities for these phases are considered as mitigation, preparedness, response, and
recovery. For the purpose of review in this paper, the activities are grouped in terms of phases of disaster
management as preventive, reactive, and corrective phases, as described above. As shown in Figure 1, the phases
of disaster are further divided in to four sub-categories defined as facilities, resources, support systems and
services, and modeling framework. The three sub-categories are further defined in terms of elements and
described next. Modeling framework would encompass mathematical models or frameworks that are used in
different phases of disaster management. This drilling down of the research in this way would help to bring the
value of content analysis on literature review further.
Figure 2. Sub-categories and elements in each phase of disaster response system
Facilities are described by elements: the location of facilities, capacities of these facilities, and their type of
functions such as medical centers, hospitals, shelter camps, and warehouses. Resources are described by elements:
human resources (such as rescue team, firefighting, safety, security, and medical personnel), tools and equipment
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(such as fire trucks, ambulance cars, rescue tools, or any other type of hardware) needed for the response
management, software, and allocation of these resources. Support services or systems are described by elements:
communications, policies and procedures, planning process and skills development. In communication, hardware
and software systems such as GSM network, satellite mobile phones or information exchange system dedicated
within the industrial areas are considered. In policies and procedures, documents in the form of paper or electronic
medium that can be used for control, regulate and standardize the disaster management system are considered. In
planning, the processes to develop clear actions, integrate them and evaluate them to find the fit of the plan with
the response are considered. Finally, in training and skills development, skills enhancement or development for
better utilization of tools, resources coordination as per plans, policies, and procedures are considered.
The rest of the paper is structured in two sections. Research on various types of disaster response mechanisms
proposed and adopted by the researchers and practitioners are given in Section 2. In Section 3, main conclusions
from the review are drawn and directions for research in the area of disaster management in industrial cities have
been proposed.
3. Literature Analysis
The analysis of literature presented here is based on the grouping in terms of the phases of disaster management
and the categories of these phases mentioned above. The frameworks and models used in different phases are also
described in brief in the review. This is to note that some of the literature are cited in more than one phase because
of the applicability of their content presented in different phases of disaster management.
3.1. Preventive Phase
Most of the literature in disaster or emergency management refers to the preventive phase. Planning of safety and
security of industrial facilities is very important. Preventive phase planning can be very important specifically for
the man-made disasters as incubation of the disaster may be imminent. Shaluf et al. (2002) mention that incubation
period can be from a very short period to a very long period. The complacency in assuming everything to be in
working condition, especially in the technological projects, can push away the need to plan for prevention. The
authors give examples of short and long incubation periods causing major disasters in industries. This is to note
that in the review presented here, the focus is on the provisions of measures to avoid or mitigate the disaster. The
review below focuses on the subcategories mentioned in Figure 2 and the modeling framework that considers
preventive phase.
3.1.1. Facilities
Facility location decision for preventive measures should include capacity, type, and accessibility to the disaster sites.
Not only that, the design of industrial facilities and its contents (like materials storage) is also an important
consideration. Various techniques such as analytic network process (ANP) or analytic hierarchical process (AHP)
are used for risk assessment for the storage of hazardous chemicals or installations handling hazardous materials.
The industries are prone to chemical spill and fire and therefore, hazard assessment of such facilities become
important.
Zhao, Huang, Guo and Zhu (2008) used AHP and geographical information system (GIS) to assess the risk of
facilities that use hazardous materials. They defined the facility based risk assessment in terms of intrinsic risk (such
as property of substance and quantity of storage), derivative accident (such as impact on water pollution and traffic
disruption), impact on surrounding environment (such as economic condition and population density) and rescue
force (such as medical and fire forces). Therefore, site accessibility, economic impact, the number of people
affected in a particular area, and readiness, capacity and skills of medical rescue force and fire safety force becomes
important. The authors also use poison index (reflect the level of poisonous chemicals available on site), fire and
explosion index (reflects chemicals substance nature from fire and explosion behavior) and erosion index (reflects
chemicals substance nature from corrosion and erosion behavior) for their assessment.
Shen, Wang, Yan and Wang (2015) studied disaster situations due to a chemical spill. They studied the evacuation of
people, location of shelters and routing of people to the shelters by considering two objectives: individual
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vulnerability (harm caused by the spill on individuals) and social vulnerability (harm caused to the population in an
area and the location of the source of the spill). The three-stage solution methodology provides the best locations
of shelter areas in the first stage, routing based on different factors in the second stage and the improvement in
terms of the solution obtained in the third stage. The factors considered in the second stage are expanded further
for evaluation in the third stage.
Khayal, Pradhananga, Pokharel and Mutlu (2015) mentioned that for large-scale disasters, installing temporary
facilities and sharing resources among themselves are important. Such facilities could be set up for immediate relief
to the affected people.
Davis, Samanlioglu, Qu and Root (2013) considered inventory prepositioning with a two-stage stochastic linear
programming model in which the first stage is used for the prepositioning of supplies based on the forecasted
impact of the disaster event. The authors considered the initial inventory at a location, the capacity of the facility,
penalty cost for damaged supplies in the facility, distance from one supply point to another, the time allowed for
prepositioning and the congestion in the traffic. The authors use scenarios to identify the worst-case demand
(maximum demand) and worst-case supply (with the lowest level of available supply due to supply damage or loss
of supply).
Li, Jin and Zhang (2011) developed a dual-step stochastic disaster preparedness model for relief supplies in the
shelters. The authors used a two-stage optimization model by considering available resources, surplus or shortage
of resources after evacuation in a particular shelter. In the first stage, the model uses the fixed cost of shelters and
inventory cost of supplies in the shelters. In the second stage, the cost of transportation for evacuation and
transportation of supplies, and the cost of surplus or shortage of supplies are minimized.
From the reviewed literature in this subsection, it can be mentioned that for the reduction of disaster impact, the
routes, the location of shelters, impact on traffic due to disaster, the location of industrial facilities, the interaction
of the facilities with the surroundings based on the type of chemical and the environmental factors are important.
Further, it also shows that mathematical models such as single objective (or multi-objective) programming or
analytical hierarchical programming in combination with other models can be used for decisions on facilities.
3.1.2. Resources
Seok, Nof and Filip (2012) proposed a sustainability-based DSS using collaborative control theory (CCT) for
disaster management. The authors used three key factors, called protocols, for consideration in the DSS: disruption
analysis –predict and manage disruption; negotiation management—understand and analyze stakeholders’
objectives for negotiation, and; knowledge management—provide relevant data and information for decisionmaking and to maintain updated databases. The authors mentioned that for disruption analysis, demand
fluctuations should be considered. For negotiation, options to get the supply from different manufacturers (or
supplies) and any additional constraints should be considered. For knowledge management, the information on the
requested amount, available capacity for transportation of the requested quantities, information on the location of
manufacturers and the related costs should be considered. The authors have developed extensive mathematical
models to analyze the decision situation.
Improvement of safety in a cluster of chemical plants is studied by Reniers, Ale, Dullaert and Soudan (2009). The
authors mention that compared to the plant-level safety standards, for cluster level safety, cooperation, joint
workforce planning, joint risks and hazard analysis, and joint planning for disaster response planning are required.
This requires formal information exchanges in terms of documentation and feedback for safety enhancements.
The framework should consider human resources, production, maintenance, and management and safety support.
This framework can also help in defining and monitoring the responsibilities within the plant safety and cluster
safety units. The authors also provide a list of 11 safety items that need to be considered in clusters.
The use of information systems has also been mentioned by Zhao et al. (2008). The authors recommended using a
GIS-based system and AHP to aid in the planning of disaster management. Similarly, Yoon, Velasquez, Partridge
and Nof (2008) mentioned of a computer-based DSS and Shaluf et al. (2002) developed an information-based
framework for disaster management related planning activities.
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3.1.3. Support System and Services
Continuous training and skills development are important elements of disaster management support system. Shaluf
et al. (2002) and Yoon et al. (2008) mentioned communication as one of the important aspects that can help in
running the support system for disaster management smoothly.
Shaluf et al. (2002) observed that the level of emergency preparedness through the understanding of different
causes of hazards are important in disaster management. The authors developed “Ibrahim-Razi” model by
considering different phases of error accumulation leading to disaster. The authors mentioned that longer the
incubation of error, larger is the impact of the disaster.
Yoon et al. (2008) mentioned that communication in disaster situations can be enhanced with a computerized
model for group DSS (GDSS). Their GDSS used real-time information about available resources, communication
contacts, maps, weather, plans and procedures to simulate real-life disaster situation.
The third element considers policies and procedures on disaster management support system. It is relatively a broad
area which covers all important processes and sub-processes in the disaster response system. Generally, these
procedures and communication protocols provide guidelines for groups and individuals on how to react, and
whom to contact in case of disaster.
Calixto and Larouvere (2010) reviewed disaster response plans in Australia, Canada, Japan, United Kingdom, and
the USA and mentioned that disaster plans can be categorized in terms of individual-level or site level (that focus
on serious impact on individuals and the surrounding environment in a specific area), regional level (coordinated
effort of multiple organizations, when an individual plan is not sufficient to cater to the needs for response), and
national level (coordination of response and cooperation from multiple organizations on a larger scale). The
authors developed sensitivity map and index to show criticality of a specific location and the location where critical
resources are needed. The authors view that responses from the local government and the industries can be
different and, therefore, good integration and coordination become necessary for an effective disaster response
plan.
3.1.4. Modeling Framework for Preventive Disaster Response
Table 1 below provides the snapshot of different mathematical models and systems that are used for the
prevention-based disaster management.
The researchers have used various mathematical or procedural frameworks in order to analyze the preventive
aspects of the disaster management system. The authors have mostly considered a single objective programming
based on mixed integer programming in order to arrive at either the location or location-allocation or location
routing decisions. It is also found that decisions are made either through a single stage model or a multi-stage
model. It is also seen that GIS, computer-based system, framework-based system or individual system can be used
in disaster response.
Authors
Context
Objectives used
Single or
multiple
objective
functions
Data types
Deterministic/
Stochastic
Solution
Method
Information
system/
Remarks
Calixto and
Larouvere
(2010)
Chemical
disasters
–
–
Deterministic
Framework
The clusterbased
coordinated
disaster
response plan
Davis et al.
(2013)
Hurricane
Prepositioning
cost
Single
Stochastic
Single objective
programming in
two stages
–
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Authors
Context
Objectives used
Single or
multiple
objective
functions
Data types
Deterministic/
Stochastic
Solution
Method
Information
system/
Remarks
Khayal et al.
(2015)
Generic
disasters
Logistics and
deprivation costs
Single
Stochastic
scenarios
Single objective
programming,
multi-period
solution
–
Li et al.
(2011)
Hurricane
Cost of capacity
and cost of
transportation
Single
Stochastic
scenarios
Stochastic Single
objective
programming in
two stages with
an L-shaped
algorithm
–
Reniers et al.
(2009)
Chemical
industrial areas
–
–
Deterministic
Frameworksafety
management
Development
of clusterbased safety
management
system
Shaluf et al.
(2002)
Fireworks,
chemical, and
hazardous
materials
related
industries
–
–
Deterministic
Framework
Development
of a model for
assessment of
technological
man-made
disaster
Seok et al.
(2012)
Manufacturing
entities
–
–
Deterministic
Collaborative
control theory
Improvement
in DSS with
regard to
sustainability
on delivery
scheduling and
production
planning
Shen et al.
(2015)
Chemical
industrial parks
1. Time
satisfaction
2. The
probability of
using a route
WeightDeterministic
based single
objective
Compromise
programming for
transportation
and location
–
Yoon et al.
(2008)
Generic, but
focus on the
transportation
network
–
–
Deterministic
Framework
Development
of Group DSS
Zhao et al.
(2008)
Chemical
installations
–
–
Deterministic
AHP
Use of GIS
Table 1. Modeling frameworks for preventive measures
It can be seen from Table 1 that most of the models are developed based on deterministic data. Mathematical
models are also developed using the single objective programming method or analytical hierarchical programming.
The reviewed papers focus mainly on routing, the location of facilities, evacuation, and developing a cluster (or
group) based disaster response system.
3.2. Reactive (Response) Phase
Most of the literature that focuses on reactive phase focus on resources, distribution, and changes in demand
situations. Some papers also discuss the temporary location of facilities and inter-facility transfer of supplies.
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3.2.1. Facilities
Location of facilities and distribution of the medical supplies using the rough set theory is illustrated by Wei,
Qiuyan and Jinlong (2012). The authors considered various conditions, expert requirement (or a situation of
disaster), and the feasibility of constructing the distribution facilities in various sites within the disaster area. The
authors developed a decision table by considering different decision-making conditions in order to reduce the
subsets of possibilities for the location of facilities and distribution of supplies. The paper uses different scenarios
for decision-making inference, decision-making conditions, and three levels of possibility to construct a facility in a
chosen location. This decision table and scenario analysis are used to prioritize the location of facilities in industrial
cities.
Celik (2017) presented a method for location of temporary evacuation centers as a part of disaster operations
management. The author developed 14 factors for selection of shelters based on the literature review. The author’s
integrated decision-making trail and evaluation laboratory (DEMATEL) method with interval type-2 fuzzy sets
(IT2FSs) for facility analysis and found that the optimal distribution of relief is the most important factor for
disaster management.
Khayal et al. (2015) show that both preplanning and positioning of resources can be done by anticipating the
disaster. This means that in the industrial cities, a good planning by developing various disaster scenarios can help in
pre-positioning the supplies in different places. However, during the event, not all areas may need all the supplies
positioned at a particular place, therefore, inter-facility transfer of resources becomes important to quickly respond
to the disaster situation.
Davis et al. (2013) also mentioned the need to account for demand changes and supply changes during the disaster.
Although the spatial location of disaster in industrial cities may not be vast as that in the case of natural disasters,
the changes do happen when there are delays in providing the relief services to the affected industrial facility or
shelters. Provisions should be made to meet the demand in order to minimize the deprivation (Holguin-Veras,
Jaller, Van Wassenhove, Pérez & Wachtendorf, 2012).
3.2.2. Resources
Peng, Zhang, Tang and Li (2011) used multi-criteria decision-making system based on the quantity of resource
losses in order to assess and rank the locations in a disaster. Although their three-stage model is developed for the
agricultural situation, this model can be used in the industrial cities as well.
Lee, Wang, Wang and Cheung (2012) proposed an unstructured information management system (UIMS)
framework to manage large databases of the disaster situation. In order to be able to effectively reduce the impact
of disaster during the response phase, information of various forms like data, reports, speeches, images, and video
should be analyzed. The authors proposed a three-tier framework for information input, the first tier is a feed-in
layer for organizational information (emails and internal reports), the second tier is for outside information (like the
websites of its associates and competitors), and the third tier is for a public thesaurus of the domain industry. The
authors used synthesis evaluation to find the precision, usability, comprehensibility, reasonability, and usability of
data and the associative information for an effective disaster response.
Ju, Wang and Liu (2012) mentioned that the emergency response capability of an organization refers to the
processing capability of the department (for example, capability to command, collaboration with urgency),
forecasting capability (for example, monitoring, warning, information system development and use), support
capability (reserve available, communication, and simulating disaster plan), and after disaster processing capability
(for example, social support and reconstruction). The authors mentioned that a scenario-based response system can
help to reduce disaster impact.
Chen et al. (2012) proposed a conceptual framework for incident process assessment during the reactive phase. The
framework considers three factors: possibility related, necessity related and other influence related. Possibility
related factors consider the disaster type, current state and the evolutionary tendency of disaster, affected victims
and property, and ability in terms of resource availability and supply of resources to the required areas. For
necessity related, profit and value are considered. For influence related, the preference and tolerance of the decision
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making, and the emergency situations are considered. The authors divided the during incident assessment strategy
into three measures: mitigability (the ability to reduce the impact of disaster through specific arrangements and
precautions); rescuability (the potential to save lives and properties), and; recoverability (the potential and difficulties
to bring the facilities to the normal or enhanced state of operation). The authors mention that proper assessment
of these measures can provide an appropriate emergency response decision.
Baldini, Oliveri, Braun, Seuschek and Hess (2012) mentioned the use of radio frequency identification technology
(RFID) to increase the efficiency of providing supplies in case of disaster. The authors mention that disaster
incident in chemical installations have low predictability, medium impact, and the medium possibility of extension
to different areas. The authors proposed a cryptographic algorithm to understand the flows correctly so that
supplies can be coordinated for maximum impact. This requires a database with the quantity, type, and usability of
the relief materials.
Immediate identification of disaster situation and mitigation planning has also been the subject of research. Zhong,
Shi, Fu, He and Shi (2010) used Petri net-based disaster response framework to analyze and contain a situation like
SARS (severe acute respiratory syndrome). In this case, tracking and information sharing becomes very important
and the situation can be applicable to industrial disaster as well for meaningfully tracking each victim of disaster and
providing required relief services.
Fancello, Mancini, Pani and Fadda (2017) considered the allocations of emergency vehicles for the response
planning. They segregated emergency vehicles into three types in order to evacuate the victims according to their
injury severity. The consideration of distance between the disaster area and the response facilities such as hospital
and the anticipated congestion also becomes critical in a disaster situation. The choice of the vehicle types and their
number changes when the level of severity changes dynamically during the response period. Therefore, correct
assessment of the type and number of vehicles, dynamic condition of injury and the location and capacity of
facilities becomes important in disaster management.
Yaming, Ming, Weidong and Hongkun (2011) proposed a DSS system that used GIS. The focus is on the chemical
gas dispersion and blowouts that happen in petrochemical industries. The authors have mentioned that there is a
need to develop a gas dispersion simulation model by considering wind speed and direction, production capacity,
the content of the gas, surface roughness, and reference atmospheric pressure. Based on the simulated scenario, a
simple static heuristic is used to identify a route for quick evacuation.
3.2.3. Support System and Services
Laakso and Palomäki (2013) mentioned that different usage of terminologies and languages in a disaster response
can, in fact, increase the damage. In order to minimize this problem, the authors developed a theoretical framework
and mentioned that horizontal level of communication spread across the organization becomes important to
mitigate the impact of the disaster. The authors also mentioned the communication integration on the vertical
levels so that the top level promotes collaboration on information exchange. The authors also mention that
communication flow at the time of disaster and immediately afterward, communication at the event site, and the
cooperation between relief works becomes important. They mention that cooperation is particularly important in
industrial cities to warn the neighboring installation when an installation is exposed to a disaster risk. The use and
integration of technology are important for such cooperation.
Reniers, Audenaert, Pauwels and Soudan (2011) have examined six chemical plants in which there are both fast
shutdowns and slow shutdowns as a means to respond to the fire-induced risks from a nearby installation. The slow
shutdown time in the companies varied from 15 minutes to 36 hours whereas that for fast shutdown time varied
between 15 minutes to about 3 hours. Due to the high cost of shutdown, establishing and following proper policies
and procedures become important.
3.2.4. Modeling Framework for Reactive Disaster Response
Table 2 below shows various modeling framework, from the mathematical model to framework-based models in
order to develop the understanding of the disaster situation, response mechanism system, decision support and
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policy development for a common understanding of the disaster-related situations for tackling the during-incident
situations. Although the coverage of the research is natural disasters to the generic type of disasters, the models
mentioned are applicable to the industrial disasters as well. The review shows that most of the research publications
for the reactive phase are based on the development and implementation of the framework.
Authors
Context
Objectives
used
Single or
multiple
objective
functions
Data types
Deterministic/
Stochastic
Solution Method
Information system/
Remarks
Baldini et al.
(2012)
Generic
–
–
Deterministic
A technology-based
framework for
efficient relief
supplies.
Supports decision
making through a
prior understanding of
the needs and
development of
supply system to meet
the need in the disaster
area.
Celik (2017)
Disasterrelated
–
–
Probabilistic
distribution and
deterministic
A decision model
based on fuzzy
method and
DEMATEL.
Supports decision on
the finding temporary
locations for
evacuation for efficient
disaster management.
Chen et al.
(2012)
Generic
–
–
Deterministic
A framework based
response strategy
Supports decision
making for developing
an appropriate
response plan
Davis et al.
(2013)
Hurricane
Cost of
distribution
–
Stochastic
Single objective
programming. Postincident distribution
is analyzed in the
second stage of the
two-stage model.
Fancello et
al. (2017)
General
Allocation
of
emergency
vehicles for
evacuation
of victims
Single
Stochastic
Two stage model
Supports decision
making for developing
the allocation through
the choice of shortest
route in order to
develop and efficient
response plan.
Ju et al.
(2012)
Generic
–
–
Stochastic
Fuzzy AHP and 2tuple linguistic
approach
Supports the
development of
capability framework
for an organization
Khayal et al.
(2015)
General
Logistics
Single
and
deprivation
costs for
both
prepositioni
ng and postdisaster
distribution
Stochastic
scenarios
Single objective
programming, multiperiod solution
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Authors
Context
Objectives
used
Single or
multiple
objective
functions
Data types
Deterministic/
Stochastic
Solution Method
Information system/
Remarks
Laakso and
Palomäki
(2013)
Generic
–
–
–
Collaborative
framework by
considering various
common issues on
disaster event
Supports policy
development for highlevel collaboration
Lee et al.
(2012)
Data on
various
disaster
response or
planning
related
document
–
–
Deterministic
Development of
framework by
synthesizing the data
and extracting the
knowledge for
disaster response
Supports decision
making through a
case-based knowledge
flow model.
Peng et al.
(2011)
Disasterrelated to
weather
–
–
Deterministic
Data mining and
multiple criteria
method
Interactive
information
management
framework
Reniers et al. Chemical
(2011)
plants
–
–
Deterministic
and scenario
based
Use of real option
theory
Obtain economically
justified shutdown
time for each
installation
Wei et al.
(2012)
Related to
medical
supplies
–
–
Deterministic
but scenario
based
Rough set theory by
considering various
site based
conditions.
–
Zhong et al.
(2010)
Urban areas
related
events
–
–
Stochastic
Petri Net based
system for analysis
of the system
Supports the
integration of
communication
between local and
external emergency
agencies for effective
disaster response
Table 2. Modeling frameworks for reactive measures
3.3. Corrective (Recovery) Phase
Corrective actions are usually taken after the disaster in order to recover it as fast as possible. Chen et al. (2012)
mention that in the post-incident stage, the focus is on complete recovery and bringing the situation to normal or
to an enhanced level of performance. The review here is focused on the post-disaster situation and response
mechanisms. It is to note that some of the literature mentioned in the earlier phases also consider the reactive
phase.
3.3.1. Facilities
For post-disaster situation, the larger the time required to wait for the relief supplies, more the suffering of the
individual people and more impact on the industrial facilities. Therefore, quick analysis and restoration of facilities
become important for disaster management.
Ben-Tal, Do-Chung, Mandala and Yao (2011) considered demand uncertainty in relief supplies and proposed a
multi-period robust optimization (RO) and cell transmission model (CTM). Their method provides dynamic
optimization for assignment of crisis management and control of road traffic movement with time-based on
undefined requirements.
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Tzeng, Cheng and Huang (2007) developed a relief-distribution structure in a multi-objective programming model
for relief supplies. The model minimizes the costs of transfer points and travel time and maximizes satisfaction.
The authors mentioned that compared to the general (or commercial logistics), in relief supplies fairness of
supplies, the temporary installation of facilities, and transfer points for relief supplies should be considered. Relief
supplies decisions are urgent and are based on available information, and therefore, they are very dynamic. The
authors used fuzzy multiple objective models and simulated scenarios to demonstrate the applicability of the model.
Turğut et al. (2011) developed a DSS for the location of disaster logistics center by using analytic hierarchy/fuzzy
analytic hierarchy process. Identification of the principles and sub-parameters needed for the suggested DSS was
done through a survey completed by experts. The authors mentioned that for the choice of such a center, decisionmakers should consider costs related to investment, operation and maintenance, cost of transportation by mode
(air, sea, or land), infrastructure available (IT, energy and water), location (closeness to the city and the disaster
location), and the climatic conditions.
3.3.2. Resources
Nivolianitou and Synodinou (2011) identified factors influencing response to disasters based on feedback from
emergency system documents analysis, interview with experts, official organization, and volunteers. The study
identified 15 factors influencing disaster management, which are: role framing (executing activities of the
organizations and the capabilities of its employees), competence, availability, intervention type, means, guidelines,
cooperation degree, coordination degree, early reaction time, timely information passage, role clearness (exact
information of team’s member accountability), number of people, deeper contextual knowledge (associated
information of disaster situation attributes), organization identity (institute’s identification), and cognitive lock-up
(readiness of the staff to take care of limited kind of incidents). Also, the study identified two main human factors
for consideration: personal factors (the physical fitness, team gender, good memory, stress, fear, perception capacity,
attention, temper, character, and motivation), and labor factors (years of experience, time pressure, and workload).
The study also identified a group of factors for suitable activities and effective involvement with emphasis on
operation and logistic related parameters. One of the major findings of the study is that the importance of human
factors evaluation needs to be seriously considered starting from physical condition till workload and capacity. The
quantification of these factors and the establishment of required minimum levels are very important elements for
disaster response but they are not covered in this study.
In the area of human resources behavior and performance, Subramaniam, Ali and Mohd-Shamsudin (2010)
mentioned that disaster response is a collective team effort; therefore, team level analysis becomes important. Their
research focuses on team member resources like members’ ability, personality, and characteristics of team members
(age, tenure, leadership, roles, and norms).
Zhou, Huang and Zhang (2011) presented a tool to evaluate cause-and-effect relationship in disaster management.
The authors used fuzzy DEMATEL method to obtain five important critical success factors (CSFs) from a list of
20 interdependent factors. The CSFs consist of four cause elements and one effect element which are: i) sensible
organization arrangement and comprehensive knowledge of roles and accountabilities of each individual; ii)
efficient emergency communication system to guarantee information transmitting; iii) unified governmental
management to integrate design and execution; iv) utilization of up-to-date transportation techniques, and v)
continual enhancement of working arrangement of disaster management. The study mentions that continuous
improvement of operational aspects is important for an effective disaster management.
Chiu and Zheng (2007) proposed a cells transformation technique (CTM) based linear programming model for the
mobilization of tools and equipment, determination of best traffic route, and dispatch timing for multi-priority
groups (SMDTS-MPG) for an instantaneous or no-notice disaster. The authors recommended an integrated and
multi-dimension evacuation and relief supplies distribution system in order to deploy the resources. The model
minimizes the travel time of multi-priority groups over the planning horizon. The result of the model provides
travel time, traffic assignment, and departure schedule for different groups.
Vescoukis, Doulamis and Karagiorgou (2012) proposed an environment information management system (EIM)
for crisis management and planning based on 3D modeling software. The model is based on the real-time and
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stored information. The proposed model focuses mainly on systems architecture to support environmental
simulation on the geospatial information for disaster response and decision support.
De Maio, Fenza, Gaeta, Loia and Orciuoli (2011) proposed a DSS framework based on a fuzzy cognitive map
(FCM). The FCM helps in information processing and resources identification based on the disaster situation. The
framework consists of several interactive components that are run by emergency managers. The manager provides
inputs and directives to the framework and receives system outputs and stimuli (such as messages or phone calls).
Stimuli signals either start the DSS session or adapt to a particular disaster plan that exists in the system. In the
fuzzy cognitive mapping process, three parts need to be considered, features of the situation, types of actions that
match the features and the type of resources required. The authors simulated scenarios by analyzing features,
actions, and resources at different time periods. The authors mention that the use of FCM and simulation support
decision-making at a very short period of time.
Chen, Peña-Mora and Ouyang (2011) presented a GIS-based framework to allocate equipment required to respond
to disasters. The proposed framework consists of three components: emergency resource repository portal (E2RP),
mobile resource request client (MR2C), and automated resource management system (ARMS). The authors
mention of the need for a repository model in the form of database in the DSS. The use of GIS in ARMS helped
to provide routes that take minimum time from a set of location to the disaster site.
In terms of resources (tool, equipment, and distribution), de la Torre et al. (2012) provided an analysis on selected
models used in disaster relief routing. The author’s review covered a wide span of research areas that support
disaster management. The review included policies, demand assessment, uncertainty in supply and demand, vehicles
routing, modeling vehicle depot, specialized models, commodities delivery and location, vehicle fleet type and
technology, and finally, the uncertainty in the vehicle fleet.
3.3.3. Support System and Services
The review of literature in corrective phase is mostly related to flow assignments, planning in anticipation of next
disaster, supporting database development, and DSS. The study reviewed here also focus on infrastructure, the
behavior of victims, structure of the team, and the policy formulation as well. Therefore, the holistic view becomes
important in the corrective phase as the lessons learned in the previous incident can help in developing a better
response mechanism for the future.
Park, Hong and Roh (2013) conducted an analysis of Japanese companies that got affected by Tsunami and the
restoration of business. The study mentioned four distinct crisis processes in order to recover from such disasters:
crisis detection, assessment of damages and required responses, preparation to resume duties, and reexamination of
crisis management activities. The authors mentioned two concepts for an efficient disaster management: the
emergency supplies delivery system and the emergency supplies network adaptability. The authors conclude that a
cooperative databank substructure and supply chain information flow makes the management system more
portable and adaptable in disaster situations.
Taber, Plumb and Jolemore (2008) discussed the interaction between the organizational policies and work practices
that happen in industrial installations. The authors conducted an empirical study by interviewing emergency
response workers (such as firefighters and paramedics) and found that organizational support and collaborative
activities with the colleagues in the workplace enhance the learning process beyond the initial training.
There are examples of long-term impact and capabilities analyzed for better disaster response. For example,
Nagarajan, Shaw and Albores (2012) focused on communicating warning messages for evacuation. Parlak, Lambert,
Guterbock and Clements (2012) used multi-criteria analysis to prioritize the initiatives for different behaviors of
disaster victims. Simpson (2008) studied disaster preparedness and response capability for better disaster response
by considering factors like public safety, hazard exposure, and city’s financial state. Kusumasari, Alam and Siddiqui
(2010) mentioned that expertise in assessment of the post-incident situation is important for effective disaster
management. Yang and Hsieh (2013) and Oh, Deshmukh and Hastak (2010) recommended having good
communication and other support infrastructure for monitoring the available stocks and requirements of supplies
for disaster management.
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3.3.4. Modeling Framework for Corrective Disaster Response
Table 3 shows various modeling frameworks and DSS based framework in order to support corrective measures or
to develop future capabilities to manage disaster responses. The research covered not only the disaster events but it
also simulated scenarios and collaborative initiatives among different disciplines related to disaster management.
Although various mathematical models can be used to support decision making, the main focus in the corrective
phase should be related to the development of policies, training, collaborative joint efforts for disaster management
as a culture, a ramped up geospatial database system, and the development of wide range of computer model based
disaster scenarios for active interaction by the emergency response units.
Authors
Context
Single or
multiple
objective
functions
Objectives
used
Data types
Deterministic
/ Stochastic
Solution Method
Information
system/ Remarks
Ben-Tal et al.
(2011)
Generic
Time and
space
dependent
cost for
traffic flow
assignment
Single
Both
Deterministic
and stochastic
Robust
–
optimization, cell
transmission
model, and affinely
adjustable robust
counterpart
Chen et al.
(2011)
Civil
engineering
related
response to
the disaster
–
–
Deterministic
Database analysis
and information
protocol for
request and
dispatch of
equipment
Uses GIS for
developing the
fastest route to the
site (from multiple
locations)
Chiu and
Urban-based
Zheng (2007) no-notice
disaster
Minimization Single
of travel time
Stochastic
Cell transmission
based Linear
programming
Information on
possible routes and
disaster location, but
this is not explicitly
shown.
de Maio et al.
(2011)
Generic
–
–
Deterministic
Framework, fuzzy
cognitive mapping
and simulation
modeling
DSS with GIS-based
data
Subramaniam Generic
et al. (2010)
–
–
Deterministic/
behavioral
A team-based
decision for
disaster response
Team structure is
presented
Taber et al.
(2008)
Paramedics
and fire
fighter
–
–
–
Survey-based study –
for collaborative
work
Turğut et al.
(2011)
Earthquake
–
–
Deterministic
AHP and fuzzy
AHP for location
of the disaster
relief supply center
Tzeng et al.
(2007)
Earthquake
Minimization Multiof fixed cost, objective
minimization
of travel
time,
maximization
of
satisfaction
Deterministic
Scenario-based and Electronic location
fully multiple
map for supply
objective modeling routes. Can be used
in industrial facilities.
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Decision support to
help choice of a
location. Can be
applied for use in
industrial facilities
Journal of Industrial Engineering and Management – https://doi.org/10.3926/jiem.2663
Single or
multiple
objective
functions
Objectives
used
Data types
Deterministic
/ Stochastic
Context
Vescoukis et
al. (2012)
Generic, fire
hazard
discussed
–
–
Deterministic
Systems
architecture for
planning and
simulation
modeling
Expected as a
decision support
system with
integration of
geospatial data
system
Yaming et al.
(2011)
Petrochemica –
l industry
–
Deterministic
Simulation of gas
dispersion and
heuristics for
routing
GDSS based on GIS
Zhou et al.
(2011)
Natural
disasters
–
Stochastic
Fuzzy DEMATEL The focus is on
method
finding the critical
success factors for
disaster management
–
Solution Method
Information
system/ Remarks
Authors
Table 3. Models and framework for corrective measures
4. Conclusions and Research Opportunities
Disaster in industrial cities does not only consider social aspects that involve casualty and displacement of people,
but it also involves economic losses due to the loss of capital assets and loss of revenues. Additionally, it would also
damage the geography around the disaster site. Disaster in the industrial city becomes important because of the
complexity of the processes handled and materials stored in the premises, which may not only impact the disaster
triggering industrial facility, but it may cause more damages in the neighboring facilities as well. Therefore, many
researchers have been working in different phases of disaster management for preplanning, responding or
providing corrective measures. The literature review presented in this paper shows that considerable work has been
done in understanding, planning, and preparing for the disaster situation. However, disaster management in
industrial cities should focus not only on the facility-based process and material characteristics, shutdown, and
evacuation but it should also focus on the industrial city based routing of evacuation, sheltering the evacuated
people for a short-term, protecting the facility and its neighborhood to avoid the resulting disaster. Ramli, Mokhtar
and Aziz (2014) also emphasize the importance of multi-facility safety or cluster-based safety. The combination of
various facility-based and city-based factors for an integrated disaster management is not found in the literature.
We find that modeling for preplanning and localization of response depending on the nature of the area (spatial
and technical), and the capability of the response mechanism are the most important factors to decrease the impact
on people, business, and infrastructure. This type of planning should be done on a cyclic basis by incorporating the
lessons learned, improved mathematical frameworks, and the technology.
4.1. Research Opportunities
In terms of future research, we propose three high-level directions. Each of these directions can be supported by
smaller and focused research mentioned therein. The focus of research should be to increase economic efficiency
in the planning phase and service efficiency during and post-incident phase.
1. Research should be carried out in an industrial area in terms of assessing the impact of no-notice or
known disasters. Various networking models can help to identify the locations for sheltered supplies,
evacuation facilities, evacuation routes and evacuation modes. As disaster response should focus on
response actions rather than on the costs. Mathematical models (exact and heuristics) and simulation
models should be developed for the rigorous planning of disaster response.
2. Research in the industrial cities are usually focused at the individual plant level and is scaled up to the
industrial city area level. This is not necessarily right because of the interaction of one industry with the
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other in an industrial city. For example, if the fire started in one location moves to the next location, there
might be more damages caused in the next location and the first industry can be left with an insignificant
impact. This means that the nature of the environment, the content of the materials handled, stored and
processed in a particular installation also becomes important. This might require developing new indices,
like toxicity index, for disaster management. The response model requires cooperative model (also called
risk pooling) for resources and skills available inside the industrial installations as well. Such a multienterprise collaborative disaster response model, also emphasized by Nof, Morel, Monostori, Molina and
Filip (2006), can lead to an effective disaster management system.
3. Another area to focus on research would be to develop a disaster preparedness maturity model. With
standardized procedures, full integration with the technology, development of maturity model would help
to assess and implement measures for disaster readiness of an organization. This can also help in
developing a common understanding of disaster management as emphasized by Laakso and Palomäki
(2013).
4.2. Limitations
The focus of the review is on highlighting the importance of industrial disaster. The review is limited mainly to the
large-scale disaster management, the modeling process or the decision-making process. The content in the literature
is focussed mainly on the technical aspects as shown in Figure 2. Aspects like leadership and other managerial
aspects, implications of the disaster management in industrial areas, and suitability of the models and frameworks
in all industrial areas are not the focus of the literature review. This is mainly due to the lack of extant literature
available in the printed media. Therefore, the authors believe that this review will open further opportunity to
conduct meaningful research mainly for the preparedness aspects of the disaster management. It should help in
realizing that emergency preparedness in an industry by considering both internal and external factors as the core
value in the sustainability of industrial establishment and economic benefits.
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication
of this article.
Funding
The authors received no financial support for the research, authorship, and/or publication of this article.
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Numerical treatment of the radiated and dissipative power-law nanofluid flow past a nonlinear stretched sheet with non-uniform heat generation
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Scientific reports
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www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports | (2023) 13:22691 Numerical treatment
of the radiated and dissipative
power‑law nanofluid flow
past a nonlinear stretched sheet
with non‑uniform heat generation
OPEN *email:
mmkhader@imamu.edu.sa Abbreviations
A∗, B∗
Coefficients of the space and temperature-depen
c
Constant
C
Concentration of fluid
C∞
Ambient concentration
Cw
The fluid’s concentration adjacent to the sheet
cp
Heat capacity per unit mass
Cfx
Local skin friction coefficient
DB
Coefficient of diffusion
DT
The coefficient associated with thermophoresis
Ec
The factor of viscous dissipation
f
Stream function with no dimensions
k∗
Absorption coefficient
m
Stretching parameter
n
Power-law parameter
Nux
Coefficient of heat transfer 1Department of Mathematics and Statistics, College of Science, Imam Mohammad Ibn Saud Islamic University
(IMSIU), Riyadh 11566, Saudi Arabia. 2Department of Mathematics, Faculty of Science, Benha University, Benha,
Egypt. 3Department of Physics, College of Science, Imam Mohammad Ibn Saud Islamic University (IMSIU),
Riyadh 11566, Saudi Arabia. 4Department of Astronomy, Faculty of Science, Cairo University, Giza, Egypt. *email:
mmkhader@imamu.edu.sa Numerical treatment
of the radiated and dissipative
power‑law nanofluid flow
past a nonlinear stretched sheet
with non‑uniform heat generation
OPEN M. M. Khader 1,2*, Ahmed M. Megahed 2 & A. Eid 3,4 The main aim of this paper is to investigate the effect of non-uniform heat generation and viscous
dissipation on the boundary layer flow of a power-law nanofluid over a nonlinear stretching sheet. Within the thermal domain, the analysis considers both thermal radiation and variable thermal
conductivity. Through the use of similarity transformations, the governing boundary layer equations
are transformed into a system of ODEs. The spectral collocation method (SCM) with shifted Vieta-
Lucas polynomials (VLPs) is implemented to give an approximate expression for the derivatives and
then use it to numerically solve the proposed system of equations. By employing this technique,
the system of ODEs is converted into a system of nonlinear algebraic equations. The dimensionless
temperature, concentration, and velocity are graphically presented and analyzed for various values
of the relevant governing parameters. Through the presented graphical solutions, we can see that the
main outcomes indicate that an increase in the power law index, thermal conductivity parameter, and
radiation parameter leads to a noticeable decrease in the local Nusselt number, with reductions of
around 0.05 percent, 0.23 percent, and 0.11 percent, respectively. In contrast, the Prandtl parameter
demonstrates an opposing effect, elevating the local Nusselt number by about 0.1 percent. We
validated the accuracy of the numerical solutions by comparing them in some special cases with
existing literature. 1
orts | (2023) 13:22691
| https://doi.org/10.1038/s41598-023-49547-7
Abbreviations
A∗, B∗
Coefficients of the space and temperature-dependent heat generation
c
Constant
C
Concentration of fluid
C∞
Ambient concentration
Cw
The fluid’s concentration adjacent to the sheet
cp
Heat capacity per unit mass
Cfx
Local skin friction coefficient
DB
Coefficient of diffusion
DT
The coefficient associated with thermophoresis
Ec
The factor of viscous dissipation
f
Stream function with no dimensions
k∗
Absorption coefficient
m
Stretching parameter
n
Power-law parameter
Nux
Coefficient of heat transfer
1Department of Mathematics and Statistics, College of Science, Imam Mohammad Ibn Saud Islamic University
(IMSIU), Riyadh 11566, Saudi Arabia. 2Department of Mathematics, Faculty of Science, Benha University, Benha,
Egypt. 3Department of Physics, College of Science, Imam Mohammad Ibn Saud Islamic University (IMSIU),
Riyadh 11566, Saudi Arabia. 4Department of Astronomy, Faculty of Science, Cairo University, Giza, Egypt. | https://doi.org/10.1038/s41598-023-49547-7 | https://doi.org/10.1038/s41598-023-49547-7 www.nature.com/scientificreports/ Pr
Prandtl number
qr
Radiative heat flux
q
′′′
Non-uniform heat generation
r, s
Constants related to the temperature and concentration respectively beside the sheet
R
Radiation parameter
Re
Dimensionless Reynolds number
Shx
Coefficient of mass transfer
Sc
Schmidt number
T
Fluid temperature
T∞
Temperature of the fluid at a considerable distance from the surface
Tw
Temperature beside sheet
Uw
Stretching velocity
u, v
Components of the velocity vector in x− and y− directions; respectively
Greek symbols
ρ
Fluid density
ψ
Stream function
η
Similarity variable
t
Thermophoresis parameter
b
Brownian motion parameter
κ, κ∞
Thermal conductivity and Ambient thermal conductivity, respectively
θ
Dimensionless fluid temperature
τ
The ratio of the heat capacity of the nanomaterial to that of the fluid
ε
Conductivity parameter
γ
Temperature-dependent heat generation parameter
µ
Viscosity of the fluid
γ ∗
Space-dependent heat generation parameter
φ
Dimensionless fluid concentration
ν
Kinematic viscosity Greek symbols
ρ
Fluid density
ψ
Stream function
η
Similarity variable
t
Thermophoresis parameter
b
Brownian motion parameter
κ, κ∞
Thermal conductivity and Ambient thermal conductivity, respectively
θ
Dimensionless fluid temperature
τ
The ratio of the heat capacity of the nanomaterial to that of the fluid
ε
Conductivity parameter
γ
Temperature-dependent heat generation parameter
µ
Viscosity of the fluid
γ ∗
Space-dependent heat generation parameter
φ
Dimensionless fluid concentration
ν
Kinematic viscosity Superscripts
w
Sheet condition
′
Derivative with respect to η
∞
Condition away the sheet In recent decades, there has been increasing interest on the part of researchers to study the technological appli-
cations of non-Newtonian power-law fluids due to their diverse potential uses1. These materials are notably
utilized in various fields, including geophysics, cosmetic processes, oil reservoir engineering, paper production,
bioengineering and chemical, polymer solutions, and nuclear industries, among others. Moreover, the response
of all non-Newtonian materials to shear cannot be precisely predicted using a single constitutive relationship. As a result, researchers have introduced multiple models of non-Newtonian fluids to facilitate discussions about
their diverse characteristics and properties. The necessity for employing multiple models stems from the fact that
different non-Newtonian fluids demonstrate distinctive and intricate behaviors under various flow conditions. These diverse models aid in enhancing our comprehension and describing the distinct rheological properties
of these fluids. These endeavors play a significant role in advancing our understanding and application of non-
Newtonian fluids in various industrial and scientific domains. www.nature.com/scientificreports/ www.nature.com/scientificreports/ PL nanofluids are harnessed in these applications to tackle challenges associated with heat transfer, lubrication,
and thermal management across diverse industrial and scientific settings. The findings have confirmed that the
presence of nanoparticles in different types of nanofluids can indeed augment the heat transport mechanism
and the thermal conductivity of the fluid. Further details and related investigations can be found in the listed
references10–15. Enhancing heat transfer in heat exchangers, double-plane windows, electronic cooling, and
similar applications are of utmost importance for energy conservation. Several studies have been conducted to
investigate nanofluids and their potential applications in addressing this concern16–18.f gl
p
pp
g
Indeed, the majority of nonlinear differential equations lack exact solutions, necessitating the use of numerical
and approximate methods as the primary approaches to solving such ordinary differential equations (ODEs)19,20. The SCM is an approximation method utilized to solve numerically ODEs. This technique gives the approxi-
mate solution by summing up basis functions and determining their coefficients by collocating the differential
equation at a limited number of collocation points21–23. Among the variety of base functions available for use
are the orthogonal VLPs. Utilizing the SCM with VLPs comes with the benefit of their remarkable convergence
properties. The accuracy of the solution improves rapidly with an increase in the number of collocation points. Moreover, VLPs exhibit good stability properties, making them suitable for solving differential equations that are
stiff or have rapidly varying solutions. In addition, the VLPs have the added advantage of having a closed-form
expression, which simplifies their computation and manipulation24,25.h p
pi
p
p
The novelty, purpose, and drive behind this study become evident when examining the importance of non-
Newtonian behavior in the industry, and modern technology. No prior research has delved into the amalgamation
of the power-law model with viscous dissipation originating from non-uniform heat generation on a nonlinear
stretched sheet. Therefore, this paper seeks to utilize the Vieta-Lucas spectral collocation approach to numerically
solve the non-Newtonian power-law model and consider elements like viscous dissipation, thermal radiation,
and non-uniform heat generation within the framework of a nonlinear stretching sheet. In light of these advance-
ments, one could ponder the potential future directions that could further explore the applications and behaviors
of PL nanofluid flow. www.nature.com/scientificreports/ To achieve this main aim, we will apply the SCM based on VLPs as a basis to convert the
resulting system of ODEs to a system of algebraic equations. This system is considered a constrained optimization
problem and optimized to get the unknown coefficients of the series of the solution. This connection of the two
well-known techniques will be called “the shifted Vieta-Lucas collocation optimization method (SVLCOM)”. | https://doi.org/10.1038/s41598-023-49547-7 Among these models, the power-law model stands
out as the simplest one, representing the commonly observed behaviors of fluids, such as shear thinning and shear
thickening. Schowalter2 was the one who applied the concept of the boundary layer to power-law fluids (PLFs). Fluids with power-law flow characteristics are commonly encountered in various practical applications, includ-
ing blood, polymers, molten plastics, foodstuffs, and more. PL non-Newtonian fluids present numerous benefits
in diverse industrial and practical applications when contrasted with Newtonian fluids. These advantages arise
primarily from the presence of the flow behavior index in the PLFs, which enables a broad spectrum of viscosity
adjustments depending on the shear rate. Through careful selection of the flow behavior index, engineers can
customize the fluid’s viscosity to suit particular application needs, making it an invaluable asset in processes that
demand precise control over flow characteristics3. Several studies focusing on flows described by the power-law
model have been referenced in works4–6.li Lately, nanotechnology’s exploration involving nanofluids has garnered significant interest due to its exten-
sive applications across various engineering and technological fields. A nanofluid consists of particles with sizes
smaller than 100 nanometers that are dispersed within the base fluid7. Nanofluids represent the next evolution-
ary step in heat transfer liquids, presenting intriguing prospects for improving heat transfer efficiency when
compared to pure liquids. Nanofluids find diverse uses in hybrid-powered engines, chemical catalytic reactors,
and other applications. This is because traditional fluids like water and oil are generally considered inefficient
heat transfer fluids due to their low thermal conductivity8. One highly dependable method to improve the
thermal conductivity of such fluids involves incorporating nanoparticles with relatively higher conductivities
into the base fluid9. The non-Newtonian behavior exhibited by fluids containing nanoparticles, known as power
law nanofluid flow, presents numerous real-life applications. These applications span biomedical uses, cooling
for electronic devices, solar thermal systems, and food processing. The distinctive rheological properties of the Scientific Reports | (2023) 13:22691 | https://doi.org/10.1038/s41598-023-49547-7 Mathematical formulation of the problem p
We are examining the continuous 2-dimensional movement of a non-compressible nanofluid with power-law
properties, flowing over a stretching sheet with a nonlinear shape. The components of velocity u and v are
respectively aligned parallel and ⊥ to the surface. The flow is induced by two opposite and equal forces along the
x-axis, causing the sheet to stretch at a velocity Uw = cxm , where c and m are positive real numbers, while the
origin remains stationary. The origin is positioned at the slit, where the sheet is pulled through the fluid medium. The stretching sheet is held at different temperatures Tw = T∞+ A xr , and concentrations Cw = C∞+ B xs for
some constants A and B; where T∞ and C∞ , are constants and uniforms. Furthermore, the constants m, r, and s
are fixed and can be determined to ensure the satisfaction of the similarity solution. Additionally, it is postulated
that the power-law nanofluid is influenced by the thermal radiation phenomenon, aligning with the presence
of non-uniform heat generation. Further, the nanofluid’s thermal conductivity κ is considered to vary during
its motion, and this variation follows a linear temperature dependence. The relationship with temperature can
be expressed as follows26: (1)
κ = κ∞
1 + ε
T −T∞
Tw −T∞
. (1) In the given context, κ∞ represents the thermal conductivity at a distance from the sheet surface. In the energy
equation, we take into account the viscous dissipation based on the power-law model, thermal radiation effects,
Brownian motion of nanoparticles, and thermophoresis phenomena. Figure 1 provides a visual representation
depicting the flow of the nanofluid induced by a nonlinear stretching sheet. The following equations represent Figure 1. Description of the proposed power law nanofluid model. 3
Vol.:(0123
Scientific Reports | (2023) 13:22691 |
https://doi.org/10.1038/s41598-023-49547-7
Figure 1. Description of the proposed power law nanofluid model. Figure 1. Description of the proposed power law nanofluid model. Mathematical formulation of the problem Scientific Reports | (2023) 13:22691 | https://doi.org/10.1038/s41598-023-49547-7 www.nature.com/scientificreports/ www.nature.com/scientificreports/ the two-dimensional flow of the PL nanofluid, taking into account all the previously mentioned assumptions
∇=
∂
∂x , ∂
∂y
27,28: the two-dimensional flow of the PL nanofluid, taking into account all the previously mentioned assumptions
∇=
∂
∂x , ∂
∂y
27,28: (2)
∇.(u, v) = 0, (2)
∇.(u, v) = 0, ∇.(u, v) = 0, (2) (3)
ρ((u, v).∇u) = µ ∂
∂y
−∂u
∂y
n−1 ∂u
∂y
, (3) (4)
ρcp((u, v).∇T) = ∂
∂y
κ ∂T
∂y
+ ρcpτ
DB
∂T
∂y
∂C
∂y + DT
T∞
∂T
∂y
2
+ µ
−∂u
∂y
n+1
+ q
′′′ −∂qr
∂y , (4) (5)
(u, v).∇C = DB
∂2C
∂y2 + DT
T∞
∂2T
∂y2 . (5) When the power-law index n < 1 , the fluid is categorized as a pseudo-plastic material, exhibiting shear-thinning
properties. If n > 1 , it is referred to as a dilatant substance, showing shear-thickening characteristics. Finally,
when n = 1 , the fluid behaves as a Newtonian fluid. Likewise, the radiative heat flux, denoted by qr , plays a crucial
role in our model. It is governed by the following relationship29: When the power-law index n < 1 , the fluid is categorized as a pseudo-plastic material, exhibiting shear-thinning
properties. If n > 1 , it is referred to as a dilatant substance, showing shear-thickening characteristics. Finally,
when n = 1 , the fluid behaves as a Newtonian fluid. Likewise, the radiative heat flux, denoted by qr , plays a crucial
role in our model. It is governed by the following relationship29: (6)
qr = −4σ ∗
3k∗
∂T4
∂y . qr = −4σ ∗
3k∗
∂T4
∂y . (6) The Stefan-Boltzmann constant, denoted by σ ∗ , and k∗ , are constants in this formula of qr . These characteristics
have a significant impact on the radiative heat flux and its interactions with the system. Also, we suppose that
the temperature difference within the nanofluid flow allows expressing the term T4 as a linear combination of
temperatures. By utilizing Taylor’s series and considering only terms of low order, we arrive at the following
formulation30: (7)
T4 ∼= −3T4
∞
1 −4T
3T∞
. Mathematical formulation of the problem (3, 4) are transformed into the following expressions: (14)
nf ′′′
−f ′′ n−1 −mf ′2 + ϒm,n ff′′ = 0, nf ′′′
−f ′′ n−1 −mf ′2 + ϒm,n ff′′ = 0, (14) https://doi.org/10.1038/s41598-023-49547-7 https://doi.org/10.1038/s41598-023-49547-7 Scientific Reports | (2023) 13:22691 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ (15)
1
Pr
(1 + R + ε θ)θ′′ + ε θ′2
−r θf ′ + ϒm,n f θ′ + Ec
−f ′′ n+1
+ (1 + ε θ)
γ ∗e−η + γ θ
+ t θ′2 + b θ′φ′ = 0, (15) (16)
φ′′ + t
b
θ′′ −s φ f ′ + Sc ϒm,n f φ′ = 0, (16) where ϒm,n = m(2n−1)+1
n+1
. The modified boundary conditions are defined in the following form: m(2n−1)+1
n+1
. The modified boundary conditions are defined in the following form: f (0) = 0,
f ′(0) = θ(0) = φ(0) (17)
f (0) = 0,
f ′(0) = θ(0) = φ(0) = 1, f (0) = 0, (17) (18)
f ′ →0,
θ →0,
φ →0,
as
η →∞. (18) Below is a concise overview of the explanations for each parameter governing the aforementioned system of
momentum, energy, and concentration: (19)
R =16σ ∗T3
∞
3κ∞k∗,
Ec =
U2
w
cp(Tw −T∞),
Pr = ρ cp
κ
x(3m−1)(n−1)c3(n−1)(K/ρ)2
1
n+1 ,
Sc = ν
DB
,
(20)
b =τDB(Cw −C∞)
ν
,
t = τDT(Tw −T∞)
νT∞
,
γ ∗= κ∞A∗
µcp
,
γ = κ∞B∗
µcp
. (19)
R =16σ ∗T3
∞
3κ∞k∗,
Ec =
U2
w
cp(Tw −T∞),
Pr = ρ cp
κ
x(3m−1)(n−1)c3(n−1)(K/ρ)2
1
n+1 ,
Sc = ν
DB
, (19) (20) Further, based on the definitions of the preceding parameters, it can be deduced that a similarity solution exists
when m = 1
3 and r = s = 2
3. Engineering and industrial quantities
h
l
f
d Engineering and industrial quantities
Using the similarity transformation, we can derive the local Sherwood number Shx , local Nusselt number Nux ,
and local skin-friction coefficient Cfx as follows: g
g
q
Using the similarity transformation, we can derive the local Sherwood number Shx , local Nusselt number Nux ,
and local skin-friction coefficient Cfx as follows: (21)
Shx = −Re
1
n+1
x
φ′(0),
Cfx = 2(−1)n Re
−1
n+1
x
f ′′(0)
n,
Nux = −Re
1
n+1
x
(1 + R)θ′(0). (21) where Rex = U2−n
w
xn
ν
is the local non-Newtonian Reynolds number. where Rex = U2−n
w
xn
ν
is the local non-Newtonian Reynolds number. U2−n
w
xn
ν
is the local non-Newtonian Reynolds number. Basic concepts on the shifted VLPs Basic concepts on the shifted VLPs p
We are going to research a class of orthogonal polynomials, which lies at the heart of our work and they are
necessary to reach our goal. With the help of their recurrence relations and analytical formulae, we can generate
and construct a new family of these polynomials that will be known as VLPs.h and construct a new family of these polynomials that will be known as VLPs. The VLPs; VLm(x) of degree m ∈N0 is obtained through the following formula32: VLm(x) = 2 cos(mχ),
χ = cos−1 (0.5 x),
χ ∈[0, π],
−2 ≤x ≤2. These VLPs satisfy the following recurrence formula: These VLPs satisfy the following recurrence formula: VLm(x) = xVLm−1(x) −VLm−2(x),
m = 2, 3, . . . ,
VL0(x) = 2,
VL1(x) = x. Now, by utilizing the linear transformation x = 4η −2 , we can generate a new class of orthogonal polynomi-
als of VLPs but on the interval [0, 1], and it will be denoted by VLs
m(η) and can be obtained from the formula
VLs
m(η) = VLm(4η −2).hi m(η)
( η
)
The polynomials VLs
m(η) satisfy the recurrence relation defined as: VLs
m+1(η) = (4 η −2)VLs
m(η) −VLs
m−1(η),
m = 1, 2, . . . , where, VLs
0(η) = 2, VLs
1(η) = 4η −2. Also, we find VLs
m(0) = 2(−1)m and VLs
m(1) = 2, m = 0, 1, 2, .... The analytical formula for VLs
m(η) is given by: where, VLs
0(η) = 2, VLs
1(η) = 4η −2. Also, we find VLs
m(0) = 2(−1)m and VLs
m(1) = 2, m = 0, 1, 2, .... The analytical formula for VLs
m(η) is given by: VLs
m(η) = 2m
m
j=0
(−1)j
4m−jŴ(2m −j)
Ŵ(j + 1)Ŵ(2m −2j + 1) ηm−j,
m = 2, 3, . . . . Mathematical formulation of the problem (7) Below are the explanations for the dimensionless boundary conditions: (8)
v = 0,
u = Uw = c xm,
T = Tw = T∞+ A xr,
C = Cw = C∞+ B xs,
at
y = 0, (8) (9)
u →0,
C →C∞,
T →T∞
as ,
y →∞. (9) The similarity transformations, employed in terms of θ , f and φ as well as the similarity variable η to solve the
governing equations, are outlined as follows27: The similarity transformations, employed in terms of θ , f and φ as well as the similarity variable η to solve the
governing equations, are outlined as follows27: (10)
η =
µ
ρ
−1
n+1
x
m(2−n)−1
n+1
c
2−n
n+1
y,
ψ(η) =
µ
ρ
1
n+1
x
m(2n−1)+1
n+1
c
2n−1
n+1 f (η), (10) (11)
φ(η) = C −C∞
Cw −C∞
,
θ(η) = T −T∞
Tw −T∞
. (11) In the last relations, ψ fulfills the continuity equation (2) and is characterized by: In the last relations, ψ fulfills the continuity equation (2) and is characterized by: (12)
u = ∂ψ
∂y ,
v = −∂ψ
∂x . (12) Additionally, we have considered the particular form of non-uniform heat generation q
′′′ , which was introduced
earlier by Abo-Eldahab and El Aziz31, in our analysis. This form is represented as follows: Additionally, we have considered the particular form of non-uniform heat generation q
′′′ , which was introduced
earlier by Abo-Eldahab and El Aziz31, in our analysis. This form is represented as follows: (13)
q
′′′ = κUw
νx
A∗(Tw −T∞)e−η + B∗(T −T∞)
. (13) Here, we must mention that when the coefficients A∗ and B∗ are both positive, it indicates internal heat genera-
tion. Conversely, if both A∗ and B∗ are negative, it suggests internal heat absorption.ii Here, we must mention that when the coefficients A∗ and B∗ are both positive, it indicates internal heat genera-
tion. Conversely, if both A∗ and B∗ are negative, it suggests internal heat absorption.ii y
g
gg
p
By utilizing similarity transformations defined in Eqs. (10, 11), the continuity equation (2) is satisfied exactly
nd Eqs. An approximate of g(n)
m (η)
h pp
gm (η)
In this section, we present an approximate formula of g(n)
m (η) , and state some notes and formulas concerning
the convergence analysis by computing the error estimate of that approximation. Theorem 1 The n-order derivative for the function gm(η) which is defined in Eq. (22) can be estimated by the fol-
lowing formula33: (23)
g(n)
m (η) =
m
j=n
j−n
k=0
αj
(−1)k 4j−k(2 j) (2j −k −1)! (j −k)! k! (2j −2k)! (j −k −n)! ηj−k−n. (23) Theorem 2 34 Assume that g(η) ∈L2
˜w [0, 1] w.r.t. the weight function ˜w(η) =
1
√
η−η2 , and |g′′(η)| ≤ , for some
constant . Then the series (22) converges uniformly to the function g(η) as m →∞ . In addition, we have the fol-
lowing facts: Theorem 2 34 Assume that g(η) ∈L2
˜w [0, 1] w.r.t. the weight function ˜w(η) =
1
√
η−η2 , and |g′′(η)| ≤ , for some
constant . Then the series (22) converges uniformly to the function g(η) as m →∞ . In addition, we have the fol-
lowing facts: 1. The coefficients’s series in Eq. (22) are bounded, i.e. 1. The coefficients’s series in Eq. (22) are bounded, i.e. αj
≤
4j
j2 −1
,
j > 2. 2. The error estimate norm
L2
˜w [0, 1] −norm
can be defined as follows: 2. The error estimate norm
L2
˜w [0, 1] −norm
can be defined as follows:
g(η) −gm(η)
˜w <
L
12
√
m3 . g(η) −gm(η)
˜w <
L
12
√
m3 . g(η) −gm(η)
˜w <
L
12
√
m3 . 3. If g(m)(η) ∈C[0, 1] , then the absolute error bound holds: 3. If g(m)(η) ∈C[0, 1] , then the absolute error bound holds:
g(η) −gm(η)
≤ m+1
(m + 1)! √π,
= max
η∈[0,1] g(m+1)(η), and = max {1 −η0, η0}. 3. If g(m)(η) ∈C[0, 1] , then the absolute error bound holds:
g(η) −gm(η)
≤ m+1
(m + 1)! √π,
= max
η∈[0,1] g(m+1)(η), and = max {1 −η0, η0}. 3. If g(m)(η) ∈C[0, 1] , then the absolute error bound holds:
g(η) −gm(η)
≤ m+1
(m + 1)! √π,
= max
η∈[0,1] g(m+1)(η), and = max {1 −η0, η0}. g(η) −gm(η)
≤ m+1
(m + 1)! √π,
= max
η∈[0,1] g(m+1)(η), and = max {1 −η0, η0}. Basic concepts on the shifted VLPs Let g(η) be a function in the space L2[0, 1] , then by using the shifted VLPs, this function can be expressed and
approximated in terms of the first (m + 1)-terms of VLs
m(η) as follows: Let g(η) be a function in the space L2[0, 1] , then by using the shifted VLPs, this function can be expressed and
approximated in terms of the first (m + 1)-terms of VLs
m(η) as follows: (22)
g(η) ≈gm(η) =
m
j=0
αjVLs
j(η), (22) where αj are constants that we should evaluate with the help of the orthogonality condition of these polynomials. https://doi.org/10.1038/s41598-023-49547-7 https://doi.org/10.1038/s41598-023-49547-7 https://doi.org/10.1038/s41598-023-49547-7 Scientific Reports | (2023) 13:22691 | www.nature.com/scientificreports/ Procedure solution using SVLCOM Procedure solution using SVLCOM g
In this section, we are going to solve numerically the given problem (14–16) by implementing the shifted Vieta-
Lucas collocation optimization method, through the following steps24: . We approximate the solution of the problem (14–16) in the following form as a finite series of shifted VLPs (24)
fN(η) =
N
j=0
αj VLs
j(η),
θN(η) =
N
j=0
βj VLs
j(η),
φN(η) =
N
j=0
δj VLs
j(η). (24) 2. We connect between (24) and the approximation (23) in the given model (14–16) to get: (25)
n
f (3)
N (η)
−f (2)
N (η)
n−1
−m
f (1)
N (η)
2
+ ϒm,n
fN(η)
f (2)
N (η)
= 0, (25) (26)
1
Pr
(1 + R + ε θN(η))
θ(2)
N (η)
+ ε
θ(1)
N (η)
2
−r (θN(η))
f (1)
N (η)
+ ϒm,n
fN(η)
θ(1)
N (η)
+ Ec
−f (2)
N (η)
n+1
+ (1 + ε θN(η)). γ ∗e−η + γ θN(η)
+ t
θ(1)
N (η)
2
+ b
θ(1)
N (η)
φ(1)
N (η)
= 0, (26) (27)
φ(2)
N (η) + t
b
θ(2)
N (η)
−s (φN(η))
f (1)
N (η)
+ Sc ϒm,n
fN(η)
φ(1)
N (η)
= 0. (2
φ(2)
N (η) + t
b
θ(2)
N (η)
−s (φN(η))
f (1)
N (η)
+ Sc ϒm,n
fN(η)
φ(1)
N (η)
= 0. (27) 3. We collocate the system (25–27) at N −2 of points ηk, k = 0, 1, 2, . . . , N −3 as follows:
(28)
n
f (3)
N (ηk)
−f (2)
N (ηk)
n−1
−m
f (1)
N (ηk)
2
+ ϒm,n
fN(ηk)
f (2)
N (ηk)
= 0, 3. We collocate the system (25–27) at N −2 of points ηk, k = 0, 1, 2, . . . Validation of the code’s accuracy In this section, to evaluate the precision of the SCM combined with the shifted VLPs, a comparison is presented
in Table 1 with the results obtained previously through a literature review. This table presents the results obtained
in a particular scenario for different values of the power law index n. The outcomes closely align with the find-
ings of previous research conducted by Andersson and Kumaran36 in a similar situation, thereby validating the
accuracy of the solution. Procedure solution using SVLCOM We solve numerically the constrained optimization problem (33–36) by implementing the Penalty Leap Frog
procedure35 for the unknowns αj, βj, δj, j = 0, 1, 2, . . . , N . This led us to construct the approximate solution
as given in the formula (24). Procedure solution using SVLCOM , N −3 as follows:
(28)
n
f (3)
N (ηk)
−f (2)
N (ηk)
n−1
−m
f (1)
N (ηk)
2
+ ϒm,n
fN(ηk)
f (2)
N (ηk)
= 0, (28) https://doi.org/10.1038/s41598-023-49547-7 Scientific Reports | (2023) 13:22691 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ 1
Pr
(1 + R + ε θN(ηk))
θ(2)
N (ηk)
+ ε
θ(1)
N (ηk)
2
−r (θN(ηk))
f (1)
N (ηk) (29)
1
Pr
(1 + R + ε θN(ηk))
θ(2)
N (ηk)
+ ε
θ(1)
N (ηk)
2
−r (θN(ηk))
f (1)
N (ηk)
+ ϒm,n
fN(ηk)
θ(1)
N (ηk)
+ Ec
−f (2)
N (ηk)
n+1
+ (1 + ε θN(ηk)). γ ∗e−ηk + γ θN(ηk)
+ t
θ(1)
N (ηk)
2
+ b
θ(1)
N (ηk)
φ(1)
N (ηk)
= 0, (29)
+ ϒm,n
fN(ηk)
θ(1)
N (ηk)
+ Ec
−f (2)
N (ηk)
n+1
+ (1 + ε θN(ηk)). (29) γ ∗e−ηk + γ θN(ηk)
+ t
θ(1)
N (ηk)
2
+ b
θ(1)
N (ηk)
φ(1)
N (ηk)
= 0, (30)
φ(2)
N (ηk) + t
b
θ(2)
N (ηk)
−s (φN(ηk))
f (1)
N (ηk)
+ Sc ϒm,n
fN(ηk)
φ(1)
N (ηk)
= 0. (30)
ηk) + t
b
θ(2)
N (ηk)
−s (φN(ηk))
f (1)
N (ηk)
+ Sc ϒm,n
fN(ηk)
φ(1)
N (ηk)
= 0. (30) Also, we approximate the boundary conditions (17)-(18) at the finite interval (0, η∞) in the following form: (31)
N
j=0
2(−1)j αj = 0,
N
j=0
αj VLs′
j (0) = 1,
N
j=0
2(−1)j βj = 1,
N
j=0
2(−1)j δj = 1,
(32)
N
j=0
aj VL∗′
j (η∞) = 0,
N
j=0
2βj = 0,
N
j=0
2δj = 0. (31)
N
j=0
2(−1)j αj = 0,
N
j=0
αj VLs′
j (0) = 1,
N
j=0
2(−1)j βj = 1,
N
j=0
2(−1)j δj = 1, (31) (32)
N
j=0
aj VL∗′
j (η∞) = 0,
N
j=0
2βj = 0,
N
j=0
2δj = 0. (32)
N
j=0
aj VL∗′
j (η∞) = 0,
N
j=0
2βj = 0,
N
j=0
2δj = 0. (32) 4. Procedure solution using SVLCOM We recognize the obtained system (28)-(32) as a constrained optimization problem by introducing the fol-
lowing cost functions (CFs): (33)
CF1 =
N
k=0
n
f (3)
N (ηk)
−f (2)
N (ηk)
n−1
−m
f (1)
N (ηk)
2
+ ϒm,n
fN(ηk)
f (2)
N (ηk)
, (33) (34)
CF2 =
N
k=0
1
Pr
(1 + R + ε θN(ηk))
θ(2)
N (ηk)
+ ε
θ(1)
N (ηk)
2
−r (θN(ηk))
f (1)
N (ηk)
+ ϒm,n
fN(ηk)
θ(1)
N (ηk)
+ Ec
−f (2)
N (ηk)
n+1
+ (1 + ε θN(ηk)).
γ ∗e−ηk + γ θN(ηk)
+ t
θ(1)
N (ηk)
2
+ b
θ(1)
N (ηk)
φ(1)
N (ηk)
, CF2 =
N
k=0
1
Pr
(1 + R + ε θN(ηk))
θ(2)
N (ηk)
+ ε
θ(1)
N (ηk)
2
−r (θN(ηk))
f (1)
N (ηk) (34)
γ ∗e−ηk + γ θN(ηk)
+ t
θ(1)
N (ηk)
2
+ b
θ(1)
N (ηk)
φ(1)
N (ηk)
, CF3 =
N
k=0
φ(2)
N (ηk) + t
b
θ(2)
N (ηk)
−s (φN(ηk))
f (1)
N (ηk)
+ Sc ϒm,n
fN(ηk)
φ(1)
N (ηk)
, (35)
CF3 =
N
k=0
φ(2)
N (ηk) + t
b
θ(2)
N (ηk)
−s (φN(ηk))
f (1)
N (ηk)
+ Sc ϒm,n
fN(ηk)
φ(1)
N (ηk)
, (35) and the constraints (Cons.) (36)
Cons. =
N
j=0
2(−1)j αj
+
N
j=0
αj VLs′
j (0) −1
+
N
j=0
2(−1)j βj −1
+
N
j=0
2(−1)j δj −1
+
N
j=0
αj VLs′
j (η∞)
+
N
j=0
2βj
+
N
j=0
2δj
. (36) 5. We solve numerically the constrained optimization problem (33–36) by implementing the Penalty Leap Frog
procedure35 for the unknowns αj, βj, δj, j = 0, 1, 2, . . . , N . This led us to construct the approximate solution
as given in the formula (24). 5. We solve numerically the constrained optimization problem (33–36) by implementing the Penalty Leap Frog
procedure35 for the unknowns αj, βj, δj, j = 0, 1, 2, . . . , N . This led us to construct the approximate solution
as given in the formula (24). 5. Discussion of numerical results Our study primarily focuses on examining the transportation of thermal energy and mass within a power-law
nanofluid flow over a nonlinear stretching surface. We also consider the effect of non-uniform heat generation,
viscous dissipation, and thermal radiation as integral aspects of our analysis. In this section, we showcase the
numerical outcomes utilizing the SCM employing shifted VLPs. We give a comprehensive portrayal of the flow
and heat mass transportation, considering the impact of various physical factors. These factors include n, R, ε ,
t , b , γ , γ ∗ , and Ec. During the numerical calculation, we kept the relevant parameters constant at a value of https://doi.org/10.1038/s41598-023-49547-7 Scientific Reports | (2023) 13:22691 | www.nature.com/scientificreports/ n
Work in36
Current work
0.8
1.028400
1.028399854
0.9
1.011300
1.011298709
1.1
0.992400
0.992399014
1.3
0.984000
0.983899953
1.7
0.979501
0.979500892
1.8
0.979468
0.979467789 Table 1. Comparison of values of skin friction coefficients −f ′′(0) in connection with the prior work
conducted by Andersson and Kumaran36 for different values of n when m = 1. Table 1. Comparison of values of skin friction coefficients −f ′′(0) in connection with the prior work
conducted by Andersson and Kumaran36 for different values of n when m = 1. Table 1. Comparison of values of skin friction coefficients −f ′′(0) in connection with the prior work
conducted by Andersson and Kumaran36 for different values of n when m = 1. n = 1.2, R = 0.5, ε = t = 0.1, γ = γ ∗= Ec = 0.2, Pr = 3.0, Sc = 2.0, and b = 0.8 for the analysis. Figure 2
illustrates how variations in n influence the dimensionless concentration φ(η) , dimensionless velocity f ′(η) ,
and dimensionless temperature θ(η) . The graphs in Figure 2 indicate that as n increases, the velocity flow field
diminishes, resulting in flow occurring primarily near the surface. Conversely, the concentration and thermal
fields exhibit an opposite trend. Physically, an elevation in the power-law index corresponds to an amplification
of the viscous force experienced within the fluid flow. This heightened viscous force functions as a resisting fac-
tor opposing the motion of the fluid, leading to a decrease in the flow field across all orientations. However, it is
evident that the power law index has a clear effect on the temperature distribution, as it directly affects the
energy equation. Discussion of numerical results In contrast, the power law index has an indirect effect on the concentration field, which
leads to its minimal and insignificant effect on the temperature distribution.l n = 1.2, R = 0.5, ε = t = 0.1, γ = γ ∗= Ec = 0.2, Pr = 3.0, Sc = 2.0, and b = 0.8 for the analysis. Figure 2
illustrates how variations in n influence the dimensionless concentration φ(η) , dimensionless velocity f ′(η) ,
and dimensionless temperature θ(η) . The graphs in Figure 2 indicate that as n increases, the velocity flow field
diminishes, resulting in flow occurring primarily near the surface. Conversely, the concentration and thermal
fields exhibit an opposite trend. Physically, an elevation in the power-law index corresponds to an amplification
of the viscous force experienced within the fluid flow. This heightened viscous force functions as a resisting fac-
tor opposing the motion of the fluid, leading to a decrease in the flow field across all orientations. However, it is
evident that the power law index has a clear effect on the temperature distribution, as it directly affects the
energy equation. In contrast, the power law index has an indirect effect on the concentration field, which
leads to its minimal and insignificant effect on the temperature distribution.l gif
p
Figure 3 depicts how θ(η) is influenced by changes in the radiation R and thermal conductivity ε param-
eters. It’s evident that when both R, and ε are raised, both the thickness of the temperature boundary layer and
the temperature distribution experience an increase. Physically, elevated values of both radiation and thermal
conductivity parameters serve to improve the ability of the fluid to handle heat, ultimately resulting in a con-
spicuous rise in its temperature. This means that increased efficiency in both radiation and thermal conductivity Figure 2. (a) f ′(η) for some values of n. (b) φ(η) and θ(η) for some values of n. Figure 3. (a) θ(η) for some values of R. (b) θ(η) for some values of ε. Figure 2. (a) f ′(η) for some values of n. (b) φ(η) and θ(η) for some values of n. Figure 2. (a) f ′(η) for some values of n. (b) φ(η) and θ(η) for some values of n. Figure 2. (a) f ′(η) for some values of n. (b) φ(η) and θ(η) for some values of n. Figure 3. (a) θ(η) for some values of R. www.nature.com/scientificreports/ contributes significantly to the fluid’s enhanced heat management, leading to a discernible escalation in its overall
temperature. Moreover, the influence of thermal radiation on the mechanisms of heat and mass transfer can be
substantiated by referring to pertinent and significant previously published papers outlined in references (37–39).f contributes significantly to the fluid’s enhanced heat management, leading to a discernible escalation in its overall
temperature. Moreover, the influence of thermal radiation on the mechanisms of heat and mass transfer can be
substantiated by referring to pertinent and significant previously published papers outlined in references (37–39).f i
Figure 4 illustrates the effect of γ , and γ ∗ on the dimensionless temperature θ(η) . It is evident that when
both γ , and γ ∗ are positive, the heat generation results in a rise in temperature across the entire boundary layer. Additionally, it is evident that the influence of the temperature-dependent heat generation parameter on θ(η)
is considerably greater than that of the spatially varying heat generation parameter. Abel et al.40 have reported
similar outcomes concerning γ and γ ∗ , affirming the accuracy of our findings. gfi
g
yi
g
Figure 5a gives θ(η) in the boundary layer region for a range of Ec. In contrast to the scenario without viscous
dissipation, it’s noticeable that the dimensionless temperature rises with an increase in Eckert number Ec. The
enhancement in fluid temperature caused by frictional heating is noted to be more prominent for elevated values
of Ec, as anticipated. Figure 5b shows the effect of Pr on θ(η) . This figure displays a declining trend in tempera-
ture distribution and the thickness of the thermal boundary layer as the Prandtl number enhances. Physically,
in the PLF, a higher Prandtl number indicates a lower thermal conductivity. Wall heat transfer increases as a
result of this decrease in thermal conductivity, which also lessens conduction. The power-law fluid’s ability to
conduct heat is essentially reduced when the Prandtl number is raised, which amplifies the heat transfer at the
fluid-solid interface.h l
The depictions of various thermophoresis parameters, denoted as t , regarding θ(η) and φ(η) distributions
can be observed in Fig. 6. This figure suggests that as the thermophoresis parameter rises, there is an expected
increase in both the concentration and temperature distributions. Physically, the heat transfer coefficient linked to
the fluid is directly linked to the thermophoresis parameter in a proportionally manner. Discussion of numerical results (b) θ(η) for some values of ε. Figure 3. (a) θ(η) for some values of R. (b) θ(η) for some values of ε. https://doi.org/10.1038/s41598-023-49547-7 Scientific Reports | (2023) 13:22691 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ scientificreports/
Figure 6. (a) θ(η) for some values of t . (b) φ(η) for some values of t. Figure 7. (a) θ(η) for some values of b . (b) φ(η) for some values of b. Figure 6. (a) θ(η) for some values of t . (b) φ(η) for some values of t. Figure 6. (a) θ(η) for some values of t . (b) φ(η) for some values of t. Figure 6. (a) θ(η) for some values of t . (b) φ(η) for some values of t. Figure 7. (a) θ(η) for some values of b . (b) φ(η) for some values of b. Figure 7. (a) θ(η) for some values of b . (b) φ(η) for some values of b. of the parameter function to impede the diffusion of nanoparticles in the fluid at distances from the surface,
which lowers the dispersion or spread of concentration.fi
1 p
p
Table 2 displays the numerical data for the local skin friction coefficient (LSFC) 1
2Re n+1
x
Cfx , local Sherwood
number (LSN) Re
−1
n+1
x
Shx , and local Nusselt number (LNN) Re
−1
n+1
x
Nux . These values are presented for various
combinations of parameters including n, R, ε, γ , γ ∗, Ec, Pr, t , and b . Observing the data in the table, it becomes
apparent that increasing n leads to an overall decrease in the values of the LNN, LSN, and LSFC. Furthermore,
the same table indicates that the local heat and mass transfer rates decrease when the thermophoresis param-
eter takes on larger values. It can be observed that higher values of both the heat generation parameter and the
spatially varying heat generation parameter result in a reduction of the LNN, while they lead to an improvement
in the LSN. This phenomenon arises because the heat generation mechanism elevates the fluid temperature in
proximity to the surface. Consequently, the temperature gradient at the surface diminishes, ultimately causing a
decrease in heat transfer at the sheet. Additionally, it can be determined that variations in the thermal conductiv-
ity parameter and Eckert number indices lead to only slight decreases in the Nusselt number. www.nature.com/scientificreports/ y p
y
g
In Table 3, we evaluated the residual error function (REF)41 of the present technique with the values of param-
eters n = 1, m = 1/3, r = s = 2/3, R = 0.5, ε = t = 0.1, Pr = 3.0, Sc = 2.0, γ = Ec = γ ∗= 0.2, b = 0.8,
and N = 7 . These values show the thoroughness of the proposed technique in this paper and confirm that this
technique gives better accuracy. 1. To reduce the energy distribution of the power-law nanofluid flow, it is necessary to utilize a fluid with a high
Pr and decrease n. www.nature.com/scientificreports/ Hence, in the presence
of a temperature gradient within the particle system’s flow area, smaller particles tend to spread more rapidly in
hotter zones and at a slower pace in colder sections. This differential dispersion leads to an overall movement of
particles from warmer to cooler areas. This migration outcome leads to the buildup of particles, causing higher
particle concentrations within the colder portions of the particle mixture. p
p
p
Figure 7 illustrates the impacts of b on the distributions of both concentration φ(η) and temperature θ(η) . The information depicted in this figure clearly indicates that as the Brownian motion parameter increases, the
concentration distribution of the fluid decreases. Conversely, the temperature field exhibits the opposite pattern. Physically, this phenomenon results from the fact that the collective effects of fluid molecules colliding with the
particle surfaces are what fundamentally cause the Brownian motion of the particles. Furthermore, high values 9
(2023) 13:22691 |
https://doi.org/10.1038/s41598-023-49547-7
Figure 4. (a) θ(η) for some values of γ . (b) θ(η) for some values of γ ∗. Figure 5. (a) θ(η) for some values of Ec. (b) θ(η) for some values of Pr. Figure 4. (a) θ(η) for some values of γ . (b) θ(η) for some values of γ ∗. Figure 4. (a) θ(η) for some values of γ . (b) θ(η) for some values of γ ∗. Figure 4. (a) θ(η) for some values of γ . (b) θ(η) for some values of γ ∗. Figure 5. (a) θ(η) for some values of Ec. (b) θ(η) for some values of Pr. Figure 5. (a) θ(η) for some values of Ec. (b) θ(η) for some values of Pr. Figure 5. (a) θ(η) for some values of Ec. (b) θ(η) for some values of Pr. Scientific Reports | (2023) 13:22691 | https://doi.org/10.1038/s41598-023-49547-7 Conclusions A study was conducted to investigate the heat and mass transfer characteristics of power-law nanofluid flow over
a nonlinear stretching sheet, with various parameters being analyzed. The non-Newtonian nanofluid flow over
a nonlinear stretching sheet was influenced by factors such as non-uniform heat generation, viscous dissipa-
tion, thermal radiation, and thermal conductivity effects. By employing similar transformations, the governing
equations for energy, momentum, and concentration are converted into ODEs. The numerical solution to the
transformed equations is obtained using the SCM with shifted VLPs, and a comprehensive analysis of the results
is presented, considering various parameters related to the power-law fluid. Several conclusions can be drawn
from the analysis, which are summarized as follows: 1. To reduce the energy distribution of the power-law nanofluid flow, it is necessary to utilize a fluid with a high
Pr and decrease n. . To reduce the energy distribution of the power-law nanofluid flow, it is necessary to utilize a fluid with a high
Pr and decrease n. Table 2. Values of Re
−1
n+1
x
Shx , Re
−1
n+1
x
Nux and 1
2Re
1
n+1
x
Cfx for various values of n, R, ε, γ , γ ∗, Ec, Pr, t , and b
with Sc = 2.0, m = 1
3 , and r = s = 2
3. Conclusions https://doi.org/10.1038/s41598-023-49547-7 Scientific Reports | (2023) 13:22691 | www.nature.com/scientificreports/ n
R
ε
γ
γ ∗
Ec
Pr
t
b
1
2 Re
1
n+1
x
Cfx
Re
−1
n+1
x
Nux
(1+R)
Re
−1
n+1
x
Shx
0.7
0.5
0.1
0.2
0.2
0.2
3.0
0.1
0.8
0.768059
0.279378
1.04090
1.0
0.5
0.1
0.2
0.2
0.2
3.0
0.1
0.8
0.677696
0.274849
1.03038
1.2
0.5
0.1
0.2
0.2
0.2
3.0
0.1
0.8
0.636029
0.261189
1.02017
1.2
0.0
0.1
0.2
0.2
0.2
3.0
0.1
0.8
0.636029
0.265060
1.02330
1.2
0.5
0.1
0.2
0.2
0.2
3.0
0.1
0.8
0.636029
0.261189
1.02017
1.2
1.0
0.1
0.2
0.2
0.2
3.0
0.1
0.8
0.636029
0.239985
1.01924
1.2
0.5
0.0
0.2
0.2
0.2
3.0
0.1
0.8
0.636029
0.295616
1.01744
1.2
0.5
0.2
0.2
0.2
0.2
3.0
0.1
0.8
0.636029
0.227158
1.02280
1.2
0.5
0.5
0.2
0.2
0.2
3.0
0.1
0.8
0.636029
0.122618
1.03035
1.2
0.5
0.1
0.0
0.2
0.2
3.0
0.1
0.8
0.636029
0.473281
1.00359
1.2
0.5
0.1
0.2
0.2
0.2
3.0
0.1
0.8
0.636029
0.261189
1.02017
1.2
0.5
0.1
0.3
0.2
0.2
3.0
0.1
0.8
0.636029
0.063584
1.03099
1.2
0.5
0.1
0.2
0.0
0.2
3.0
0.1
0.8
0.636029
0.440750
1.00557
1.2
0.5
0.1
0.2
0.2
0.2
3.0
0.1
0.8
0.636029
0.261189
1.02017
1.2
0.5
0.1
0.2
0.4
0.2
3.0
0.1
0.8
0.636029
0.076553
1.03516
1.2
0.5
0.1
0.2
0.2
0.0
3.0
0.1
0.8
0.636029
0.324640
1.01448
1.2
0.5
0.1
0.2
0.2
0.5
3.0
0.1
0.8
0.636029
0.165594
1.02875
1.2
0.5
0.1
0.2
0.2
1.0
3.0
0.1
0.8
0.636029
0.005134
1.04316
1.2
0.5
0.1
0.2
0.2
0.2
2.0
0.1
0.8
0.636029
0.221093
1.01940
1.2
0.5
0.1
0.2
0.2
0.2
3.0
0.1
0.8
0.636029
0.231189
1.02017
1.2
0.5
0.1
0.2
0.2
0.2
6.2
0.1
0.8
0.636029
0.244238
1.02710
1.2
0.5
0.1
0.2
0.2
0.2
3.0
0.0
0.8
0.636029
0.275598
1.02764
1.2
0.5
0.1
0.2
0.2
0.2
3.0
0.2
0.8
0.636029
0.247806
1.01401
1.2
0.5
0.1
0.2
0.2
0.2
3.0
0.4
0.8
0.636029
0.223747
1.00438
1.2
0.5
0.1
0.2
0.2
0.2
3.0
0.1
0.5
0.636029
0.356354
1.00249
1.2
0.5
0.1
0.2
0.2
0.2
3.0
0.1
1.0
0.636029
0.212541
1.02480
1.2
0.5
0.1
0.2
0.2
0.2
3.0
0.1
1.5
0.636029
0.129750
1.02886 Table 2. Conclusions Values of Re
−1
n+1
x
Shx , Re
−1
n+1
x
Nux and 1
2Re
1
n+1
x
Cfx for various values of n, R, ε, γ , γ ∗, Ec, Pr, t , and
with Sc = 2.0, m = 1
3 , and r = s = 2
3. Table 2. Values of Re
−1
n+1
x
Shx , Re
−1
n+1
x
Nux and 1
2Re
1
n+1
x
Cfx for various values of n, R, ε, γ , γ ∗, Ec, Pr, t , and b
with Sc = 2.0, m = 1
3 , and r = s = 2
3. Table 3. Values for the REF in the present technique. η
REF of f (η)
REF of θ(η)
REF of φ(η)
0.0
3.951753E−07
4.756542E−08
8.014712E−06
1.0
4.753951E−08
3.852035E−07
5.985214E−08
2.0
9.014740E−08
2.852014E−07
2.014782E−08
3.0
2.963258E−06
9.015975E−08
0.963258E−08
4.0
7.023987E−08
3.321987E−08
1.654123E−07
5.0
4.753654E−08
7.456852E−06
4.014736E−06
6.0
5.741933E−06
0.854560E−08
7.654258E−07
7.0
0.321047E−08
9.014785E−07
3.011563E−08
8.0
9.852012E−08
7.852014E−08
5.456852E−07
9.0
3.741230E−08
5.951023E−07
9.759512E−08
10.0
1.756542E−06
0.987123E−08
6.550086E−06 Table 3. Values for the REF in the present techniq
η
REF of f (η)
REF of θ(η)
REF of φ(η)
0.0
3.951753E−07
4.756542E−08
8.014712E−06
1.0
4.753951E−08
3.852035E−07
5.985214E−08
2.0
9.014740E−08
2.852014E−07
2.014782E−08
3.0
2.963258E−06
9.015975E−08
0.963258E−08
4.0
7.023987E−08
3.321987E−08
1.654123E−07
5.0
4.753654E−08
7.456852E−06
4.014736E−06
6.0
5.741933E−06
0.854560E−08
7.654258E−07
7.0
0.321047E−08
9.014785E−07
3.011563E−08
8.0
9.852012E−08
7.852014E−08
5.456852E−07
9.0
3.741230E−08
5.951023E−07
9.759512E−08
10.0
1.756542E−06
0.987123E−08
6.550086E−06 2. Increasing the thermophoresis parameter and the power-law index, while simultaneously reducing the
Brownian motion parameter, leads to an elevation in the concentration of the power-law nanofluid flow. 3. Elevating the temperature-dependent heat generation, Eckert number, space-dependent heat generation,
and radiation parameter result in an increase in temperature. Conversely, an increase in the power-law index
leads to a decrease in velocity. 4. The LNN, and LSN exhibit a declining trend as the power-law index, radiation parameter, and thermopho-
resis parameter increase. 2. Increasing the thermophoresis parameter and the power-law index, while simultaneously reducing the
Brownian motion parameter, leads to an elevation in the concentration of the power-law nanofluid flow. 2. Increasing the thermophoresis parameter and the power-law index, while simultaneously reducing the
Brownian motion parameter, leads to an elevation in the concentration of the power-law nanofluid flow. 3. Conclusions Elevating the temperature-dependent heat generation, Eckert number, space-dependent heat generation,
and radiation parameter result in an increase in temperature Conversely, an increase in the power-law index 2. Increasing the thermophoresis parameter and the power-law index, while simultaneously reducing the
Brownian motion parameter, leads to an elevation in the concentration of the power-law nanofluid flow. ll
. Elevating the temperature-dependent heat generation, Eckert number, space-dependent heat generation
and radiation parameter result in an increase in temperature. Conversely, an increase in the power-law index
leads to a decrease in velocity.h ll
3. Elevating the temperature-dependent heat generation, Eckert number, space-dependent heat generation,
and radiation parameter result in an increase in temperature. Conversely, an increase in the power-law index
leads to a decrease in velocity.h y
4. The LNN, and LSN exhibit a declining trend as the power-law index, radiation parameter, and thermopho-
resis parameter increase. y
4. The LNN, and LSN exhibit a declining trend as the power-law index, radiation parameter, and thermopho-
resis parameter increase. https://doi.org/10.1038/s41598-023-49547-7 Scientific Reports | (2023) 13:22691 | www.nature.com/scientificreports/ 5. The concentration profile responds oppositely to changes in b compared to the thermophoresis parameter,
while both parameters yield a similar impact on the temperature field.h 5. The concentration profile responds oppositely to changes in b compared to the thermophoresis parameter,
while both parameters yield a similar impact on the temperature field.h p
y
p
pi
6. The future research direction, building upon the foundation laid by this paper, involves investigating the
flow of a hybrid power-law nanofluid subjected to the influences of Ohmic heating and variable density. i
6. The future research direction, building upon the foundation laid by this paper, involves investigating the
flow of a hybrid power-law nanofluid subjected to the influences of Ohmic heating and variable density. References Unsteady mixed convective radiative nanofluid flow in the stagnation point region of a revolving sphere consider-
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ing the influence of nanoparticles diameter and nanolayer. J. Indian Chem. Soc. 99(10), 100716 (2022).il gl
p
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14. Naz, R. et al. Investigation of entropy generation in stratified MHD Carreau nanofluid with gyrotactic microorganisms Von
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using spectral collocation methods. Chaos 29, 1–5 (2019). using spectral collocation methods. Chaos 29, 1–5 (2019). 21. Khader, M. M. et al. A numerical approach for solving the problem of biological invasion (fractional Fisher equation) using the
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41. Khader, M. M. et al. © The Author(s) 2023 Additional information Correspondence and requests for materials should be addressed to M.M.K. Correspondence and requests for materials should be addressed to M.M.K Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
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English
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High-performance ECRH at W7-X: experience and perspectives
|
Nuclear fusion
| 2,021
|
cc-by
| 3,989
|
High-performance ECRH at W7-X: experience and
perspectives Measurements of the parameter
dependencies of the bootstrap current in
the W7-X stellarator
U. Neuner, K. Rahbarnia, C.D. Beidler et
al. - Measurements of the parameter
dependencies of the bootstrap current in
the W7-X stellarator
U. Neuner, K. Rahbarnia, C.D. Beidler et
al. - (Expected difficulties with) density-profile
control in W7-X high-performance plasmas
C D Beidler, Y Feng, J Geiger et al. - To cite this article: H.P. Laqua et al 2021 Nucl. Fusion 61 106005 To cite this article: H.P. Laqua et al 2021 Nucl. Fusion 61 106005 View the article online for updates and enhancements. View the article online for updates and enhancements. This content was downloaded from IP address 129.13.72.195 on 17/11/2021 at 08:39 You may also like You may also like
Measurements of the parameter
dependencies of the bootstrap current in
the W7-X stellarator
U. Neuner, K. Rahbarnia, C.D. Beidler et
al. -
(Expected difficulties with) density-profile
control in W7-X high-performance plasmas
C D Beidler, Y Feng, J Geiger et al. -
Turbulent transport in the scrape-off layer
of Wendelstein 7-X
Carsten Killer, Yann Narbutt, Olaf Grulke
et al. - ∗Author to whom any correspondence should be addressed.
a See Klinger et al 2019 (https://doi.org/10.1088/1741-4326/ab03a7) for the
W7-X Team.
Original content from this work may be used under the terms
of the Creative Commons Attribution 4.0 licence. Any further
distribution of this work must maintain attribution to the author(s) and the title
of the work, journal citation and DOI. This content was downloaded from IP address 129.13.72.195 on 17/11/2021 at 08:39 This content was downloaded from IP address 129.13.72.195 on 17/11/2021 at 08:39 International Atomic Energy Agency Nuclear Fusion https://doi.org/10.1088/1741-4326/ac1a1b Nucl. Fusion 61 (2021) 106005 (6pp) Abstract The second operation phase of W7-X (OP1.2) showed the potential of exclusively electron
cyclotron resonance heating (ECRH)-sustained plasma operations in stellarators. Employing
multi-pass ECRH scenario in the second harmonic O-mode (O2-ECRH), stationary densities
of up to 1.4 × 1020 m−3 could be achieved. This scenario also made stationary divertor
detachment possible, which is a reactor-relevant scenario for power and particle exhaust. At
high densities and with sufficiently high density gradients for an improved ion confinement,
the coupling between the electrons and ions was strong enough to bring the ion temperature to
values above 3 keV and to the neoclassical limit for some magnetic configurations, thus
enabling to test the W7-X neoclassical optimization. The planned enhancement of the ECRH
performance will enable to advance towards reactor-relevant beta values and to investigate
their stability and confinement of fast particles, which is a priority goal of W7-X. Keywords: ECRH, W7-X, stellarator, high performance, high density, detachment (Some figures may appear in colour only in the online journal) similar to that expected in a fusion reactor, where in a reac-
tor it is the fusion alpha particles that transfer their energy to
the electrons by slowing down collisions. This scenario can be
simulated in W7-X with microwave heating of the electrons. However, it should be noted that although the fast alpha par-
ticles do not significantly interact with the thermic ions, they
can have an influence on the ion confinement [1]. High-performance ecrh at w7-x: experience
and perspectives H.P. Laqua1,∗J. Baldzuhn1
, H. Braune1, S. Bozhenkov1, K. Brunner1,
M. Hirsch1, U. Hoefel1, J. Knauer1, A. Langenberg1, S. Marsen1, D. Moseev1,
E. Pasch1, K. Rahbarnia1, T. Stange1, R.C. Wolf1, N. Pablant2
, O. Grulke1,3
and the W7-X Team1,a 1 Max-Planck-Institute for Plasma Physics, Greifswald, Germany
2 Princeton Plasma Physics Laboratory, 08543 Princeton, United States of America
3 Technical University of Denmark, Kongens Lyngby, Denmark 1 Max-Planck-Institute for Plasma Physics, Greifswald, Germany
2 Princeton Plasma Physics Laboratory, 08543 Princeton, United States of America
3 Technical University of Denmark, Kongens Lyngby, Denmark E-mail: laqua@ipp.mpg.de Received 31 May 2021
Accepted for publication 3 August 2021
Published 30 August 2021 1. Introduction W7-X is an optimized stellarator in which the otherwise strong
neoclassical transport is reduced so that high confinement time
values can be achieved.In particular this can be done with elec-
tron cyclotron resonance heating (ECRH) only, whereby only
the electrons are heated directly, while the ion temperature is
increased by electron ion collisions. Although it is expected
that high ion temperatures will also be achieved by direct ion
heating with NBI or ICRH, pure electron heating is a situation The superconductingcoil system of W7-X generates a mag-
netic field strength of 2.5 T. This requires an ECRH frequency
of 140 GHz, which corresponds to the second harmonic res-
onance of the electrons. Unfortunately, only the X2 mode
is completely absorbed in the plasma at the resonance sur-
face. However, the X2-mode can only propagate up to 1.2 ×
1020 m−3. The second harmonic O-mode, which has a cut-
off density of 2.4 × 1020 m−3, is incompletely absorbed in
the single pass. On the other hand, the highest performance
is expected at densities above 1 × 1020 m−3, because of the © EURATOM 2021
Printed in the UK 1 1741-4326/21/106005+6$33.00 Nucl. Fusion 61 (2021) 106005 H.P. Laqua et al Figure 1. W7-X ECRH system. Here, one out of two models with five gyrotrons/beams is shown. The individual gyrotron beams, marked by
different colours, are combined into a bundle and sent over a long distance with large common mirrors, the so called multi-beam section. They are divided into individual beams close to the vacuum vessel. Inside the vacuum vessel the beams are launched into the plasma by
steerable mirrors. Figure 1. W7-X ECRH system. Here, one out of two models with five gyrotrons/beams is shown. The individual gyrotron beams, marked by
different colours, are combined into a bundle and sent over a long distance with large common mirrors, the so called multi-beam section. They are divided into individual beams close to the vacuum vessel. Inside the vacuum vessel the beams are launched into the plasma by
steerable mirrors. an efficiency of 95% as shown in figure 1. The transmission
takes place with cooled mirrors and at atmospheric pressure. A diamond disc forms the interface to the plasma vessel. Inside
the plasma vessel, the individual rays are radiated flexibly in
both toroidal and poloidal direction into the plasma with the
help of movable mirrors. 1. Introduction The 140 GHz EC-wave with ordinary
(O2) polarization gives access to densities beyond the cut-off
limit (1.2 × 1020 m−3) of the commonly used extra-ordinary
(X2) polarized waves, but to the expense of incomplete single
pass absorption for the W7-X plasma parameters. This dis-
advantage could be compensated by a special multi-pass sce-
nario, where the partially (60%–70%) absorbed ECRH-beams
were reflected by specially shaped tiles and passed three times
through the plasma core with an overall absorption of up to
90%. The O2-operation scenario has been also routinely used
for plasma operation below the X2 cutoff at densities above
0.8 × 1020 m−3 prohibiting the otherwise high risk of uncon-
trolled beam deflection in the X2-modeECRH case. In order to
keep the absorption high, the density was feed-back controlled
in such a way that a central Te was always kept above 2 keV. It should be noted, however, that the O2 process comes at the
price of flexibility. Ray trajectories are fixed by the reflector
tile positions. positive confinement scaling and the increasing coupling
between the electrons and ions with density. Therefore, a
multi-pass ECRH scenario was developed so that an absorp-
tion of the O2 mode of more than 90% could be achieved. It
should be noted that in a stellarator reactor a magnetic field
strength of above 5T is expected. In that case the well absorbed
first harmonic O-mode (O1) will be used. Here the full flexibil-
ity of the ECRH with respect of current drive and off-axis heat-
ing will be achieved and further optimization of the electron
temperature profile as well as the iota profile will be possible. The high performance high density scenario must also be
compatible with a technically reasonable particle and energy
exhaust. For this purpose, W7-X is equipped with a so-called
island divertor, which separates the core plasma from the
plasma wall interaction. 3. Results An outstanding result is the stationary operation with pure
O2-ECRH at plasma densities of 1.4 × 1020 m−3, which is
clearly above the X2 cutoff density as shown in figure 2. Here
the electron-ion coupling was so high that the ion tempera-
ture approached the values of the electrons. Unfortunately, the
error bars of the x-ray spectroscopy were too high to carry
out a precise profile analysis. The high density operation with
the multi-pass O2-ECRH scenario showed an excellent plasma
performance. However the maximum achieved temperature
was limited the available heating power (6 MW for 15 s) and
the respective transport parameters of the plasma scenarios. This scenario could be kept stationary and was only limited
in time by of the maximum heating energy applicable to this
W7-X operation phase with uncooled first wall elements.i The achievable large density range enabled a combined
operation with pellet injection without the risk of approach-
ing the cut-off condition for the here pellet-induced peaked
density profiles. After the pellet injection phase, the trans-
port properties were improved for both ions and electrons and
thus high plasma performance with high triple product values
has been achieved [5]. Here the ion power flux approached
the neoclassical value enabling to test the neoclassical trans-
port optimization of W7-X [6]. In figure 3, the time traces of
the pellet-fueled plasma discharge is shown. First of all, the
plasma start-up had to be performed with the well-absorbed
X2 mode. While the plasma density was then built up with
gas fuelling, the polarization of the ECRH beams was changed
from X-mode to O-mode. It should be noted that the polar-
ization change can be performed during gyrotron operation. As long as the plasma density is below the X2 cutoff den-
sity both ECRH modes will be absorbed. Then the density
was further increased with the aid of pellet injection and fur-
ther ECRH beams were switched on. After the pellet injection,
a peaked density profile was established which improved the
ion confinement and increased both the ion temperature and
the plasma energy (diamagnetic energy). There reason for the
confinement improvement is the simultaneous stabilization of
the ion temperature gradient (ITG) turbulence and the trapped
electron mode (TEM) turbulence. This is a unique feature of A further benefit of the O2-ECRH scenario was its com-
patibility with high neutral pressure at the plasma edge. 2. Experimetal set-up W7-X has the world’s largest microwave heating system for
plasma generation at the moment [2]. The ECRH works at
a frequency of 140 GHz. That is twice the resonance fre-
quency of the electrons at a magnetic field strength of 2.5 T,
the nominal magnetic field of the superconducting coil sys-
tem. The ECRH consists of 10 microwave sources, the so-
called gyrotrons, with a unit power of up to 1 MW and an
operating time of 30 min, which is also the targeted W7-
X plasma duration of 30 min in the final completion phase. The maximum total port through power was about 8 MW,
but for higher reliability in longer discharges the gyrotrons
were detuned to a total port through power of 6 MW. The
main reason for power limitation was arcing in the transmis-
sion line due to a high air humidity during the experimental
campaign in summer 2018.The 10 microwavebeams are trans-
mitted with a purely quasi-optical transmission line from the
gyrotrons into the plasma over a distance of about 40 m with Three high performance scenarios are reported here. In
the first scenario, a stationary plasma at a density of 1.4 ×
1020 m−3 with only 6 MW O2-ECRH was achieved. The elec-
tron and ion temperatures almost equalized each other. In the
second scenario, the density was built up with the help of pel-
let injection, which lead to a temporarily peaked density profile
with a strongly improved ion heat confinement. This scenario
could not be explored towards steady state due to the limited
number of available hydrogen ice pellets of the existing blower
gun pellet injector. In the third scenario, the so-called detach-
ment was demonstrated,in which the power flux to the divertor
is strongly reduced. 2 H.P. Laqua et al Nucl. Fusion 61 (2021) 106005 Figure 2. Left: time traces of a gas fuelled high density plasma with O2-ECRH only. Right: density and temperature profiles of the high
density discharge. Figure 2. Left: time traces of a gas fuelled high density plasma with O2-ECRH only. Right: density and temperature profiles of the high
density discharge. neoclassical confinement times of 2–10 s [4]. Even more in
cases, where impurity accumulation has been found, like in the
exclusively NBI-heated high density plasmas, additional O2-
ECRH significantly flattened the otherwise peaked impurity
profiles and pushed the impurities towards the plasma edge. 4. Outlook For the next operation campaign an upgrade of the available
ECRH power to 10 MW and more efficient multi-pass reflector
tiles are planned. In particular a new more powerful (1.5 MW)
gyrotron is being developed now and an increase of the num-
ber of gyrotrons and beamlines from 10 to 12 is envisaged. The development strategy for the new gyrotron is to largely
retain the successful basic W7-X gyrotron design and only
to increase the operating mode from TE28.8 to TE28.10 in
order to increase the cavity diameter and thus to keep the
power load density at the cavity surface below the critical
level of 20 MW m−2. Keeping the azimuthal mode unchanged
allowed to use the same electron optics as for the 1 MW class
W7-X gyrotrons. Therefore the new 1.5 MW class gyrotron
is fully compatible with the W7-X installation, in particular
with the gyrotron magnet, and outdated gyrotrons can easily
be replaced by new more powerful gyrotrons. Inside the new
gyrotron only cavity and the microwave optics are adjusted
accordingly. The beam current is increased from 40 to 60 A. In experiments at KIT with an uncooled short-pulse gyrotron
with the same TE28.10 microwave system, 1.5 MW output
power was achieved and thus the gyrotron concept has been
successfully confirmed [10]. The 1.5 MW power cw gyrotron
is currently being built by the Thales company and expected
for November 2021 [11]. A general challenge of nuclear fusion with magnetic con-
finement is the power and particle exhaust. The advantage of
the particle motion guided by field lines in magnetic confine-
ment turns into a challenge when the power and the particles
come into contact with the walls. Here the flux concentrated
on the magnetic field lines leads to power densities that are
technically difficult to control. The low shear concept of the
W7-X has an advantage here, because it has long (>100 m)
connection lengths between the last closed flux surface and the
divertor target. Due to the different magnetic geometry and the
much longer connection length compared to tokamak diver-
tors, the W7-X stellerator detachment differs fundamentally
from that in tokamaks. A detailed description can be found in
[8]. Nevertheless, the detachment at the W7-X is also charac-
terized by a strong reduction of the power flux on the divertor
surface. 3. Results Even
though after boronization of the W7-X wall no glow discharge
cleaning has been performed, the density control was never
lost and the high density operation was very robust. But on
the other hand in the presence of high neutral fluxes, plasma
radiation and charge exchange losses pushed down the edge
temperatures as shown in figure 2. The neoclassical impu-
rity transport in stellarators predicts an inward pinch and thus
an impurity accumulation. Temperature gradient driven turbu-
lence is counteracting here and thus in ECRH-plasmas with
gas fuelling no accumulation has been found [3]. In particu-
lar the laser blow off experiments estimated impurity confine-
ment time of the order of 70–80 ms which is far below the 3 H.P. Laqua et al Nucl. Fusion 61 (2021) 106005 Figure 3. O2 ECRH scenario in combination with pellet injection (#20170904.015). For the plasma start-up three ECRH beams are
polarized in X2-mode. During the density ramp their polarization is changed into O2-mode. The gray shaded area is the over-dense phase. Figure 3. O2 ECRH scenario in combination with pellet injection (#20170904.015). For the plasma start-up three ECRH beams are
polarized in X2-mode. During the density ramp their polarization is changed into O2-mode. The gray shaded area is the over-dense phase. plasma was terminated regularly and no components had been
at their power load limit. the W7-X configuration with a ‘stability valley’ for both ITGs
and TEMs [7]. Unfortunately, in this experimental campaign W7-X was
insufficiently equipped to control the density gradient. This
requires a controlled pellet injection for the central fuelling
and a high neutral gas pumping capability for a low neutral
gas pressure at the edge. 5. Summary Figure 5. Tungsten covered TZM reflector tile with prolarization
grating on top. In the W7-X operation phase 1.2, a routine high-density opera-
tion with O2 ECRH could be established. The O2-ECRH made
it possible to achieve high performance plasma scenarios in
various aspects. Stationary gas puff fuelled discharges of high
densities were achieved, where the electron and ion tempera-
tures were equalized due to the high collision frequency. The
good heating efficiency at high densities also made it possi-
ble to use hydrogen ice pellet injection with which the density
gradients were increased with a strongly beneficial effect on
the confinement. Finally, the stationary high-density operation
also enabled to reach the detachmentat the plasma edge, which
strongly reduced the thermal load on the divertor plates and
thus represents a possible scenario for safe long-pulse oper-
ation. For the next phase of operation for W7-X (OP2), in
addition to the installation of water-cooled first wall and diver-
tor components, further upgrades of the active components
are also planned. The ECRH power will be increased by two
more gyrotron positions and new, more powerful gyrotrons. The continuous-wave pellet injector and the high-performance
cryo-pumps should, among other things, enable the density
profile control for scenario optimization. humidity below 20% at 20◦c. Here a high microwave power
transmission test has been performed in 2020 where the suc-
cessful transmission of a 0.9 MW beam for 2 min through the
whole in air section of the transmission line has been demon-
strated. Since here the maximum power was limited by the
maximum gyrotron out-put power only, we expect a further
margin enabling the transmission of the 1.5 MW beam. A detailed analyses of the absorption and losses with the
O2-mode for all 10 ECRH beams in the experiments enabled
further optimization of the reflector tiles. The new reflec-
tor will also correct the polarization of the reflected beam,
such that the O-mode polarization is maintained as shown in
figure 5. Thus in the next campaign an overall absorption of
95% is envisaged. This is at first glance a small improvement. 5. Summary But it will reduce the none-absorbed ECRH stray radiation
by 50%, which is a remarkable step forward for prospective
future steady state operation, since the microwave stray radia-
tion gives an additional load to all W7-X components even if
they are outside a line of sight to the plasma [12].li The multi-reflection system can also be used for efficient
plasma heating with the third harmonic X-mode at the mag-
netic field strength of 1.7T, thus opening a new operation point
for W7-X. In particular with reduced magnetic field a higher
relative plasma pressure (beta) can be achieved with same 4. Outlook With its the help, steady state conditions could also be
achieved for the uncooled test divertor of W7-X. Although the
peak densities in the detachment experiments were just below
the X2 cutoff density, the plasma had to be heated with the
O2 mode in order to avoid the risk of uncontrolled ECRH X2
beam deflection so close to the X2-cutoff. In the experiment
shown in figure 4 the transition to detachment was reached at
3 s and the radiated power approached the heating power. The
power flux on the divertor was reduced from 3.5 MW m−2 to
0.4 MW m−2. This state could be kept stable for 26 s [9]. The In addition the maximum power capability of the in-
air quasi-optical transmission line is being enhanced with a
powerful air drying system which brings down the relative 4 H.P. Laqua et al Nucl. Fusion 61 (2021) 106005 Figure 4. Left: time traces of the maximum heat flux at the divertor tile. Right: IR-picture of the divertor for attachment and detachment. Reproduced from [9]. Figure 4. Left: time traces of the maximum heat flux at the divertor tile. Right: IR-picture of the divertor for attachment and detachment. Reproduced from [9] Figure 4. Left: time traces of the maximum heat flux at the divertor tile. Right: IR-picture of the divertor for attachment and detachment. Reproduced from [9]. Figure 5. Tungsten covered TZM reflector tile with prolarization
grating on top. heating power compared to the nominal operation at 2.5 T. Plasma performance enhancement is also expected by an
improved edge neutral density control with powerful cryo
pumps in the divertor pumping gap and gas valves in the diver-
tor region. In addition a steady state pellet injector will be
installed as a US contribution to W7-X. It will enable contin-
uous core fuelling at low neutral edge density. This injector is
a prototype of the US ITER pellet injector. Four microwave pickup holes are connected to waveguides
the back of the tile, which measure the position, power and
polarization of the incoming beam. ORCID iDs y
[8] Reinbold F. et al 2021 IAEA-CN-123/45 (private communica-
tion) J. Baldzuhn
https://orcid.org/0000-0001-5667-351X
N. Pablant
https://orcid.org/0000-0001-6617-8459 [9] Jakubowski M. et al 2021 Overview of the results from the diver-
tor experiments at Wendelstein 7-X and their implications for
steady state operation Preprint: 2020 IAEA Fusion Energy
Conf. [EX/7-4] Acknowledgments This work has been carried out within the framework of
the EUROfusion Consortium and has received funding from 5 H.P. Laqua et al Nucl. Fusion 61 (2021) 106005 the Euratom research and training program 2014–2018 and
2019–2020 under Grant Agreement No. 633053. The views
and opinions expressed herein do not necessarily reflect those
of the European Commission. the Euratom research and training program 2014–2018 and
2019–2020 under Grant Agreement No. 633053. The views
and opinions expressed herein do not necessarily reflect those
of the European Commission. [4] Wegner T. et al 2018 Rev. Sci. Instrum. 89 073505 g
[5] Sunn Pederson T. et al 2019 Plasma Phys. Control. Fusion 61
014035 [6] Beidler C. et al 2021 Nature 596 221–6 [7] Alcus´on J.A., Xanthopoulos P., Plunk G.G., Helander P., Wilms
F., Turkin Y., von Stechow A. and Grulke O. 2020 Plasma
Phys. Control. Fusion 62 035005 [1] Di Siena A., Bañ´on Navarro A. and Jenko F. 2020 Phys. Rev.
Lett. 125 105002
[2] Erckmann V. et al 2007 Fusion Sci. Technol. 52 291–312
[3] Langenberg A. et al 2019 Plasma Phys. Control. Fusion 61
014030 References 0] Avramidis K.A. et al 2021 Fusion Eng. Des. 164 112173 [11] Gantenbein G. et al 2021 High power gyrotron development for
advanced fusion devices Preprint: 2020 IAEA Fusion Energy
Conf. [TECH/P7-19] [1] Di Siena A., Bañ´on Navarro A. and Jenko F. 2020 Phys. Rev. Lett. 125 105002
[2] Erckmann V. et al 2007 Fusion Sci. Technol. 52 291–312
[3] Langenberg A. et al 2019 Plasma Phys. Control. Fusion 61
014030 [1] Di Siena A., Bañ´on Navarro A. and Jenko F. 2020 Phys. Rev. Lett. 125 105002 [1] Di Siena A., Bañ´on Navarro A. and Jenko F. 2020 Phys. Rev. Lett. 125 105002
[2] Erckmann V. et al 2007 Fusion Sci. Technol. 52 291–312
[3] Langenberg A. et al 2019 Plasma Phys. Control. Fusion 61
014030 [12] Laqua H.P. et al 2001 Proc. of the 28th EPS Conf. Control. Fusion and Plasma Phys., Funchal 2001, ECA 25A (Geneva:
European Physical Society) pp 1277–80 6
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Beam test performance of prototype silicon detectors for the Outer Tracker for the Phase-2 Upgrade of CMS
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Journal of Instrumentation To cite this article: W. Adam et al 2020 JINST 15 P03014 To cite this article: W. Adam et al 2020 JINST 15 P03014 View the article online for updates and enhancements. This content was downloaded from IP address 129.13.72.196 on 11/05/2020 at 06:38 c⃝2020 CERN for the benefit of the CMS collaboration. Published by
IOP Publishing Ltd on behalf of Sissa Medialab. Original content from
this work may be used under the terms of the Creative Commons Attribution 3.0 licence.
Any further distribution of this work must maintain attribution to the author(s) and the
title of the work, journal citation and DOI. https://doi.org/10.1088/1748-0221/15/03/P03014 This content was downloaded from IP address 129.13.72.196 on 11/05/2020 at 06:38 This content was downloaded from IP address 129.13.72.196 on 11/05/2020 at 06:38 Published by IOP Publishing for Sissa Medialab Received: November 20, 2019
Accepted: February 12, 2020
Published: March 17, 2020 Received: November 20, 2019
Accepted: February 12, 2020
Published: March 17, 2020 Contents 1
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20 2020 JINST 15 P030 0 JINST 15 P03014 7
Summary Tracker group of the CMS collaboration Beam test performance of prototype silicon detectors for
the Outer Tracker for the Phase-2 Upgrade of CMS Beam test performance of prototype silicon detectors for
the Outer Tracker for the Phase-2 Upgrade of CMS 2020 JINST 15 P030 0 JINST 15 P03014 Abstract: A new CMS tracker detector will be installed for operation at the High Luminosity LHC
(HL-LHC). This detector comprises modules with two closely spaced parallel sensor plates and
front-end ASICs capable of transmitting tracking information to the CMS Level-1 (L1) trigger at the
40 MHz beam crossing rate. The inclusion of tracking information in the L1 trigger decision will
be essential for selecting events of interest efficiently at the HL-LHC. The CMS Binary Chip (CBC)
has been designed to read out and correlate hits from pairs of tracker sensors, forming so-called
track stubs. For the first time, a prototype irradiated module and a full-sized module, both equipped
with the version 2 of the CBC, have been operated in test beam facilities. The efficiency of the stub
finding logic of the modules for various angles of incidence has been studied. The ability of the
modules to reject tracks with transverse momentum less than 2 GeV has been demonstrated. For
modules built with irradiated sensors, no significant drop in the stub finding performance has been
observed. Results from the beam tests are described in this paper. Keywords: Front-end electronics for detector readout; Particle tracking detectors; Performance of
High Energy Physics Detectors c⃝2020 CERN for the benefit of the CMS collaboration. Published by
IOP Publishing Ltd on behalf of Sissa Medialab. Original content from
this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the
title of the work, journal citation and DOI. Beam test performance of prototype silicon detectors for
the Outer Tracker for the Phase-2 Upgrade of CMS https://doi.org/10.1088/1748-0221/15/03/P03014 https://doi.org/10.1088/1748-0221/15/03/P03014 Contents
1
Introduction
1
2
CMS tracker for HL-LHC
2
2.1
The concept of pT discrimination
3
2.2
Prototype detectors
4
3
Beam test infrastructure
6
3.1
Beam test setup
6
3.2
Data acquisition system
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4
Preparations for data-taking
9
4.1
Pedestal and noise
9
4.2
Latency scans
9
5
Reconstruction
10
5.1
DUT reconstruction
11
5.2
Tracking
13
5.3
DUT alignment
14
6
Results
14
6.1
Performance of mini-modules
15
6.2
Performance of the full-size module
20
7
Summary
22
Tracker group of the CMS collaboration
26 Contents
1
Introduction
1
2
CMS tracker for HL-LHC
2
2.1
The concept of pT discrimination
3
2.2
Prototype detectors
4
3
Beam test infrastructure
6
3.1
Beam test setup
6
3.2
Data acquisition system
8
4
Preparations for data-taking
9
4.1
Pedestal and noise
9
4.2
Latency scans
9
5
Reconstruction
10
5.1
DUT reconstruction
11
5.2
Tracking
13
5.3
DUT alignment
14
6
Results
14
6.1
Performance of mini-modules
15
6.2
Performance of the full-size module
20
7
Summary
22
Tracker group of the CMS collaboration
26 Contents
1
Introduction
2
CMS tracker for HL-LHC
2.1
The concept of pT discrimination
2.2
Prototype detectors
3
Beam test infrastructure
3.1
Beam test setup
3.2
Data acquisition system
4
Preparations for data-taking
4.1
Pedestal and noise
4.2
Latency scans
5
Reconstruction
5.1
DUT reconstruction
5.2
Tracking
5.3
DUT alignment
6
Results
6.1
Performance of mini-modules
6.2
Performance of the full-size module
7
Summary
Tracker group of the CMS collaboration 1
Introduction The Large Hadron Collider (LHC) at CERN will undergo major upgrades by 2025 to be able to
deliver peak instantaneous luminosities of 5−7.5×1034cm−2s−1. This High Luminosity upgrade of
the LHC (HL-LHC) will allow the CMS (Compact Muon Solenoid) [1] experiment to collect data
corresponding to integrated luminosities of the order of 300 fb−1 per year. Eventually, a total of
3000 fb−1 will be collected during ten years of operation. At the nominal instantaneous luminosity
of the HL-LHC, a single bunch crossing will produce 140-200 proton-proton collisions. The vast
majority of these collisions are “pileup” interactions with low momentum transfer that are of little
physics interest. In order to fully exploit the increased luminosity and to cope with the very high pileup
environment, the detector and the trigger system of the CMS experiment need to be upgraded – 1 – significantly [2]. The present CMS tracker was designed to operate up to an integrated luminosity
of 500 fb−1 [2, 3], beyond which radiation damage will lead to degradation of its performance. The
CMS experiment will replace the current tracker with a new silicon tracker. The upgraded tracker [3]
will feature increased radiation hardness, higher granularity, compatibility with higher data rates,
and a longer trigger latency. In addition, the tracker will provide tracking information to the Level-1
trigger, allowing trigger rates to be kept at a sustainable level without sacrificing physics potential [3]. The CMS tracker for the HL-LHC period will consist of modules with two “stacked“ silicon
sensors, read out by front-end ASICs with the capability to discriminate tracks based on their
transverse momentum (pT). The concept of pT discrimination by means of very short track
segments called stubs, in so-called pT modules, will be discussed in the following section. 2020 JINST 15 P03 A number of module prototypes described in the following section, each with two stacked strip
sensors, also known as 2S modules, were subjected to particle beams at CERN, Fermilab, and DESY
beam test facilities to measure the performance of the stub finding mechanism, the uniformity of
the stub finding efficiency in the entire detector, the potential to reject low pT tracks (< 2 GeV),
and the ability to work efficiently up to the expected overall HL-LHC radiation level. In this paper,
results from beam tests carried out at CERN are reported and, where possible, compared to those
obtained at Fermilab and DESY. 1
Introduction The results from previous beam test are reported in ref. [4]. 0 JINST 15 P03014 2
CMS tracker for HL-LHC The layout of the new tracker is shown in figure 1. The new tracker will consist of two parts: an Inner
Tracker (IT) and an Outer Tracker (OT). Both the IT and the OT will have a barrel section, made
out of coaxial cylindrical layers, and two endcaps, one on each side of the barrel, made out of discs. The IT barrel will feature four layers of pixel detectors, providing three-dimensional hit coordinates,
resulting in excellent vertex resolution. Each IT endcap will consist of 12 pixel discs on each side
of the barrel. The OT barrel will comprise six layers of detector modules each having two silicon
sensors separated by a small distance and read out by the same front-end electronics. The separation
between the sensors of a module, defined by the distance between the sensor mid planes, will vary
between 1.6 mm and 4 mm [3]. Of the six layers of the OT barrel, the three inner layers will be
equipped with modules made of one macro-pixel sensor and one strip sensor (PS pT module). The
three outer layers will be equipped with modules with two strip sensors (2S pT module). The OT
endcaps will feature six discs and will be equipped with PS and 2S modules, as shown in figure 1. The main specifications of the PS and 2S modules for the OT are listed in table 1. Table 1. Main parameters of the 2S and PS modules of the proposed CMS Phase-2 tracker [3]. 2S module
PS module
∼2 × 90 cm2 active area
∼2 × 45 cm2 active area
No. of strips/sensor plane Strip length
Pitch
No. of strips/macro-pixels Strip/macro-pixel length
Pitch
2 × 1016
∼5 cm
90 µm
2 × 960/32 × 960
∼2.4 cm/∼1.5 mm
100 µm Table 1. Main parameters of the 2S and PS modules of the proposed CMS Phase-2 tracker [3]. – 2 – 0
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referred to as Inner Tracker and will be instrumented with pixel modules. 2
CMS tracker for HL-LHC In the Outer Tracker, the radial
region between 200 and 600 mm is equipped with PS modules (blue lines), while the region beyond 600 mm
will be populated with 2S modules (red lines). The CMS coordinate system is defined in ref. [1]. 0
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η 2020 JINST 15 P030 Figure 1. Sketch of one quarter of the tracker layout in r −z view. The radial region below 200 mm is
referred to as Inner Tracker and will be instrumented with pixel modules. In the Outer Tracker, the radial
region between 200 and 600 mm is equipped with PS modules (blue lines), while the region beyond 600 mm
will be populated with 2S modules (red lines). The CMS coordinate system is defined in ref. [1]. 0 JINST 15 P03014 Figure 2. Illustration of the pT module concept [3]. Correlation of signals in closely spaced sensors enables
rejection of low-pT particles. The channels shown in green represent the selection window to define an
accepted stub; a low-pT rejected track is shown in red. ST 15 P03014 Figure 2. Illustration of the pT module concept [3]. Correlation of signals in closely spaced sensors enables
rejection of low-pT particles. The channels shown in green represent the selection window to define an
accepted stub; a low-pT rejected track is shown in red. 2.1
The concept of pT discrimination In the presence of the 3.8 T solenoidal magnetic field inside the CMS detector, the trajectories of
charged particles produced in a collision will bend in a plane transverse to the direction of the beam. The radius of the curvature of the trajectory of these particles depends on the particle pT. The
concept of pT discrimination is shown in figure 2. As a charged particle passes through the module,
it generates signals (hits) in the bottom and top sensors of the module. A hit in the bottom sensor
is then matched to the one in the top sensor and if they are within a predefined window, these two
hits are combined to form a short track segment or stub. These stubs will be used in the Level-1
(L1) track trigger. The readout chips will provide the pT discrimination logic described above. The window for
hit matching can be set within the readout chip according to the pT threshold to be used. For the 2S
module, the readout chip is called the CMS Binary Chip (CBC) [5–9]. Each CBC has 254 readout – 3 – Figure 3. Left: sketch of the full-size 2S module. Right: cross section of the 2S module. The connection
of the front-end chips to strips of both the top and the bottom sensor via routing lines in the flexible hybrid
(flex kapton circuit), which is bent around a stiffener/spacer sandwich [3], is visible. 2020 JINST 15 P03 Figure 3. Left: sketch of the full-size 2S module. Right: cross section of the 2S module. The connection
of the front-end chips to strips of both the top and the bottom sensor via routing lines in the flexible hybrid
(flex kapton circuit), which is bent around a stiffener/spacer sandwich [3], is visible. channels with alternate channels connected to the top and bottom sensors in a module, as shown in
figure 3 (right), so that coincidences between channels of the two sensors can be obtained. 0 JINST 15 P03014 The 2S module, shown in figure 3, consists of two sensors (n-type strips in p-type silicon
substrate), support structures made from Al-CF (carbon fibre reinforced aluminium), two front-end
hybrids [10], each with eight CBCs and one concentrator integrated circuit (CIC) that aggregates
data from the CBCs, and a service hybrid for powering and output data serialization followed by
opto-electrical conversion. 2.1
The concept of pT discrimination All prototype modules discussed in this paper use the second prototype of the CMS Binary
Chip, the CBC2 [7–9]. The block diagram of the analogue front-end (FE) of the CBC2 ASIC is
shown in figure 4. Three I2C registers are used to control the main settings of the analogue FE :
Vplus, which controls the global DC baseline of the post-amplifier output, Voffset (labelled “Offset”
in figure 4) for fine control of the baseline of the post-amplifier output for individual channels on
the CBC2, and VCTH, which controls the comparator threshold. The readout for the CBC2 chip is
binary, thus it does not measure the amount of charge induced on each strip. If the charge on a strip
exceeds the comparator threshold, a hit is registered. Offset
Figure 4. Block diagram of the analogue front-end (FE) of the CBC2 ASIC [7–9]. Three registers are used
to control the analogue FE. Figure 4. Block diagram of the analogue front-end (FE) of the CBC2 ASIC [7–9]. Three registers are used
to control the analogue FE. 2.2
Prototype detectors Prototypes of the 2S module have been investigated at different test beam facilities (table 2). For
the beam tests described in section 3, two small prototype modules and one full-size module have – 4 – – 4 – Table 2. Details of modules used in various beam tests. Module type
No. of CBC2s Sensor active thickness Sensor separation Bias voltage
Beam Test facility
Non-irradiated mini-module
2
270 µm
2.75 mm
250 V
CERN, DESY, Fermilab
Irradiated mini-module
2
240 µm
3.05 mm
600 V
CERN
Full-size module
16
240 µm
1.80 mm
240 V
CERN, Fermilab
Figure 5. Left: the irradiated 2S mini-module assembled from a small prototype hybrid comprising two
CBC2 readout chips and two silicon sensors with 254 strips of 5 cm length. Right: the full-size 2S module
comprising two hybrids with eight CBC2 readout chips each and two full-size 2S sensors. Table 2. Details of modules used in various beam tests. No. of CBC2s Sensor active thickness Sensor separation Bias voltage
Beam Test facility 2020 JINST 15 P030 0 JINST 15 P03014 Figure 5. Left: the irradiated 2S mini-module assembled from a small prototype hybrid comprising two
CBC2 readout chips and two silicon sensors with 254 strips of 5 cm length. Right: the full-size 2S module
comprising two hybrids with eight CBC2 readout chips each and two full-size 2S sensors. been studied. The strip sensors of the modules have 5 cm long n-type strips at 90 µm pitch on about
300 µm thick silicon sensors with p-type bulk. A negative voltage is applied to bias the sensors at
the sensor backplane but in the following the absolute values of the bias voltage applied are quoted. been studied. The strip sensors of the modules have 5 cm long n-type strips at 90 µm pitch on about
300 µm thick silicon sensors with p-type bulk. A negative voltage is applied to bias the sensors at
the sensor backplane but in the following the absolute values of the bias voltage applied are quoted. The small prototype modules, called mini-modules, consist of a version of the front-end hybrid
housing two CBC2s. The hybrid is made of a rigid material with bond-pads on both sides and
the sensors are wire-bonded to the top and bottom sides of it. 2.2
Prototype detectors 0
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/cm
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After irradiation to 6
Before irradiation 2020 JINST 15 P03 JINST 15 P03014 Figure 6. The current-voltage characteristic of a sensor of the mini-module before (red) and after (black)
irradiation to 6 × 1014 neq/cm2, showing the increased current after irradiation. The measurements were
taken at −20◦C and 20◦C for the irradiated and non-irradiated sensors, respectively. with a rotation angle between the strips of both sensors of below 400 µrad. This module is shown
in figure 5 (right). 3
Beam test infrastructure The prototype modules have been studied at beam test facilities at CERN, Fermilab and DESY. In
all of the facilities, the detector under test (DUT) is placed within a tracking detector, referred to
as ‘telescope’ in the following. The telescope provides a reference to reconstruct the tracks of the
incident particles. The beam test facility at CERN is described in detail in the following section, and
the key features of the DESY and Fermilab test beam facilities are highlighted. The data acquisition
systems (DAQ) of the three facilities are also described. 2.2
Prototype detectors This contrasts with the flex-kapton
design used for full-sized modules that folds over the CF spacer to provide bond-pads for the bottom
sensor [3, 11]. The sensors have been glued on a small frame made of aluminium. One mini-module
was left unirradiated. The sensors of this module have an active thickness of 270 µm and their
separation is 2.75 mm. The second mini-module, shown in figure 5 (left), with an active sensor
thickness of 240 µm and a sensor separation of 3.05 mm, was irradiated with 23 MeV protons at
Irradiation Center Karlsruhe [12] to a fluence of 6×1014 neq/cm2 with an annealing of approximately
two weeks at room temperature. The maximum expected fluence for the innermost layer of the 2S
modules of the OT is 3 × 1014 neq/cm2 [3]. This value corresponds to 3000 fb−1 of proton-proton
(pp) collisions at √s = 14 TeV assuming a total inelastic cross section, σpp, of 80 mb. The current-voltage characteristic of the sensors before and after irradiation can be seen in
figure 6. The effect of irradiation is reflected by an increase of the leakage current by three orders
of magnitude. The full-size module consists of two sensors of about 10 cm × 10 cm, with two columns of
1016 strips each. The active thickness of each sensor is 240 µm and the sensors are separated by
1.8 mm. Each of the front-end hybrids on both ends of the module houses eight CBC2s. A flex
hybrid is used to provide bond-pads for the top and bottom sensors (figure 3). The module is built – 5 – 0
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Figure 6. The current-voltage characteristic of a sensor of the mini-module before (red) and after (black)
irradiation to 6 × 1014 neq/cm2, showing the increased current after irradiation. The measurements were
taken at −20◦C and 20◦C for the irradiated and non-irradiated sensors, respectively. 3.1
Beam test setup Schematic drawing of the beam test setup at CERN showing the three detector systems used to char-
acterize the performance of the 2S prototype module: the 6 MIMOSA planes, the ATLAS FE–I4 plane and the
four scintillators used to generate the NIM trigger. The DUT is placed within the telescope system as shown. The synchronization of the data streams from the three detector systems (the 2S prototype, the
MIMOSA-26 sensor planes, and the FE–I4 plane) is performed by an FPGA-based Trigger Logic
Unit (TLU) [17, 18]. During the beam tests, dedicated NIM logic is used to generate a trigger signal
using the output signals from the two pairs of crossed scintillators at either extremity of the EUDET
telescope. This trigger signal is provided as input to the TLU, which distributes this signal to the
DUT and to the telescope’s sensor planes. A simple handshake protocol is used by the DAQ system to maintain synchronization among the
different detector systems. The detector systems assert busy signals on separate lines, which inhibit
triggers from the TLU until all of the lines are cleared. No new triggers are sent by the TLU until all
detectors drop their busy-lines. This ensures that detectors with different dead-times can be triggered
and read out synchronously. In addition, the TLU can send a timestamp for each trigger via a dedi-
cated clock-data line, or it can receive a back-pressure (veto) signal from the DUTs on the same line. The additional ATLAS FE–I4 plane is used to improve the timing resolution of the telescope
by associating the FE-I4 hits with the individual hits in the 115.2 µs rolling-shutter frame of the
telescope during event building. This allows the multiple tracks in a telescope frame to be correlated
to individual triggers. Because the FE-I4 readout has no dead-time and runs on an internal 40 MHz
clock, the required time resolution of 25 ns for the CBC DAQ is achieved. The data streams from the
telescope and the FE-I4 are sent to the EUDAQ [13] online software via the TCP/IP protocol [19]. The two streams are stored together in the same format in a file, which makes the reconstruction
easier in the EUDET [20] framework. The beam test at DESY uses the EUDET based telescope called DURANTA [13], similar to the
one used during the CERN beam test. 3.1
Beam test setup A schematic diagram of the setup at CERN is shown in figure 7. Data were collected using a
120 GeV pion beam. The EUDET telescope [13] used in the CERN beam test of the 2S prototype
modules is a tabletop tracking detector composed of six planes of MIMOSA-26 [14] silicon pixel
sensors for accurate track reconstruction, a fast-timing reference plane (FE–I4) [15] for accurate
timing resolution, and a pair of crossed scintillators with photomultiplier tubes (PMTs) located at
either end of the telescope for trigger generation. The six MIMOSA-26 sensor planes, each covering
an active area of 10.6×21.1 mm2, consist of 50 µm thick 18.4 µm×18.4 µm square pixels arranged
in 576 rows and 1152 columns. The fast-timing plane covers an active area of 16.8 × 20.0 mm2
and consists of 200 µm thick pixels arranged in 336 rows and 80 columns read out by the FE–I4
chip, which was designed for the innermost layer of the upgraded pixel detector of the ATLAS – 6 – experiment. Each sensor plane is mounted inside a 20 mm thick aluminium jig, and two sets of
three jigs are attached via rail systems to the upstream/downstream arms of the telescope. The
minimum distance between sensor planes is defined by the thickness of the aluminium jig (and is
therefore 20 mm), and the maximum distance between sensor planes is defined by the length of each
arm (150 mm for equidistant spacing between the sensor planes). The resolution of the telescope
system over the six sensors used is 3.24 µm [16]. The jigs are cooled to a constant temperature of
16◦C to increase the stability of operation. X
Y
Z
2S prototype
254 Strips
FeI4
USBPIXII
1152 columns
576 rows
80 columns
336 rows
(Downstream)
Scintillators
(Upstream)
Scintillators
(Downstream)
Mimosa Planes
(Upstream)
Mimosa Planes
Figure 7. Schematic drawing of the beam test setup at CERN showing the three detector systems used to char-
acterize the performance of the 2S prototype module: the 6 MIMOSA planes, the ATLAS FE–I4 plane and the
four scintillators used to generate the NIM trigger. The DUT is placed within the telescope system as shown. (Upstream)
Mimosa Planes JINST 15 P03014 Figure 7. 3.1
Beam test setup It also uses six MIMOSA-26 pixel sensors with four crossed
scintillators for triggering and a TLU, however it was equipped with a CMS Phase-1 pixel [21] – 7 – module as a timing reference plane instead of the FE–I4. The data were collected with a positron
beam of 5 GeV energy. The Fermilab Test Beam Facility, or FTBF [22], is equipped with two silicon telescopes aligned
along the beam line and configured to operate synchronously. It has a pixel telescope assembled
from eight planes and a telescope with strip modules made up of 14 detector planes. The strip
telescope increases the coverage of the pixel telescope and improves its tracking performance. The
trigger is generated by a coincidence signal of three scintillation counters, one placed in front and
two placed behind the telescopes. The synchronization of the data streams from the two telescopes
and the 2S module is performed by a Fermilab-designed FPGA-based trigger board. The data are
taken with a 120 GeV proton beam. 2020 JINST 15 P030 4.1
Pedestal and noise The pedestal and noise values of an individual channel in a system with binary readout can be
inferred from a channel’s S-curve. An S-curve is obtained by measuring the noise occupancy as
a function of the comparator threshold (VCTH in figure 4). The comparator threshold has been
measured in VCTH DAC units. One VCTH DAC unit corresponds to 375 electrons, as measured using
an X-ray source. The noise occupancy is given by the fraction of triggers for which a given channel
registers a hit. Higher numerical values of VCTH correspond to lower thresholds in the CBC2. Figure 4 also shows the per-channel 8-bit DAC used to control the offset of the output voltage of
the second amplification stage to compensate for any channel-to-channel variations. 2020 JINST 15 P03 0 JINST 15 P03014 The pedestal value and the channel noise are extracted directly from the S-curve either by
fitting the curve with a sigmoid of the form f (x, µ, σ) = 1
2
1 + er f
x −µ
√
2σ
,
(4.1) (4.1) or by numerically differentiating it. The mean parameter, µ, in eq. (4.1) (or the mean of a Gaussian
fitted to the differential histogram) then corresponds to the pedestal and σ (or the RMS of a Gaussian
fitted to the differential histogram) corresponds to the noise. An example of an S-curve recorded
for a CBC2 on a non-irradiated prototype module and the corresponding differential histogram are
shown in figure 9. Both methods return similar (i.e. consistent within 3σ) values for the pedestal and
noise. The pedestal value, obtained from fitting the left plot of figure 9 with a sigmoid function, is
120.0 ± 0.1 VCTH DAC units. A pedestal value of 119.3 ± 0.2 VCTH DAC units has been obtained by
fitting the distribution shown in figure 9, right, with a Gaussian function. For the noise, 2.12 ± 0.06
VCTH DAC units and 2.14 ± 0.15 VCTH DAC units are obtained, respectively. Figure 10 shows the uniformity of the front-end response after adjustment of the individual
channels’ offsets. The pedestal and noise values were extracted from the fits to the individual
channels’ S-curves using eq. (4.1). The channel-to-channel variation in the pedestal, defined as the
RMS of the measured distribution, is measured to be 0.30 ± 0.01 and 0.37 ± 0.02VCTH DAC units for
the first and second CBC2, respectively. 3.2
Data acquisition system The DAQ system for the CBC2 modules at CERN and Fermilab test beams is based on the CERN
Gigabit Link Interface Board (GLIB) [23] µTCA Advanced Mezzanine Card (AMC). Different
firmware versions are used to read data from the 2 and 16 CBC2s on the tested modules. Control
signals and readout data are exchanged between the GLIB and the control PC via the IPBus [24]
protocol, whereas trigger, busy and veto signals are interfaced to the TLU/Fermilab equivalent via a
dedicated five-channel I/O FPGA Mezzanine Card (FMC). A simple block diagram of the different
components of the DAQ system is shown in figure 8. 0 JINST 15 P03014 2S Prototype
40 MHz
External Clock
MIMOSA Planes
80 MHz
Internal Clock
FEI4
40 MHz
Internal Clock
Trigger Logic Unit
NIM Logic
Discriminator + Coincidence
GLIB
( DIO5 + CBC FMC )
Scintillators
Trigger/Busy/Veto
EUDAQ Run Control
CMS Run Control
USB
TCP/IP
TCP/IP
IPBUS
Trigger Input
Figure 8. Block diagram of the DAQ system used in the CERN and Fermilab beam test setups. The
correlation of the data from the two DAQ chains is described later in section 5. Figure 8. Block diagram of the DAQ system used in the CERN and Fermilab beam test setups. The
correlation of the data from the two DAQ chains is described later in section 5. An external high-precision clock generator was used to provide the clock signals to the GLIB via
the same FMC that connects to the TLU. The CBC2 data are processed and formatted by the firmware
and then sent to a XDAQ [25] application that formats events in a CMS compatible format and
stores the data for later processing within the standard CMS reconstruction software, CMSSW [26]. The binary raw data stream is also stored and can be used for online data quality monitoring. The beam test at DESY used a novel DAQ system, based on the FC7 [27] card. The FC7 hosts a
Kintex-7 FPGA and comes with a system firmware allowing for communication with other devices – 8 – on the FC7 card and IPBus communication. A DIO5 FMC is used to send trigger and busy signals
via LEMO connectors. These are fed into a custom-built LVDS converter box and sent to the TLU
via a standard RJ45 connector. on the FC7 card and IPBus communication. A DIO5 FMC is used to send trigger and busy signals
via LEMO connectors. 3.2
Data acquisition system These are fed into a custom-built LVDS converter box and sent to the TLU
via a standard RJ45 connector. 4.1
Pedestal and noise The mean noise was found to be 1.36 ± 0.06 and 2.38 ± 0.60
VCTH DAC units for the first and second CBC2, respectively. The same figure also clearly shows that
11 of the strips connected to the second CBC2 on the hybrid are significantly noisier than the rest. These 11 strips are included in the noise figure quoted for the second CBC2. The strips exhibiting
a value of noise larger than 3 VCTH DAC units (1125 electrons) are not considered for analysis. 4.2
Latency scans After the pedestal and noise scans, two latency scans, one for data and one for stubs, were carried out. The data latency, measured in units of 40 MHz clock cycles and set using an on-chip configuration – 9 – 105
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125
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135
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/dV
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dN
Figure 9. S-curve measured for a single input channel on one of the two CBC2s on a non-irradiated prototype
module at room temperature with the sensor biased at 250 V. On the left the measured data are shown along
with a fit to the measured data performed using eq. (4.1), while on the right the differential histogram is
shown with the corresponding Gaussian fit. 105
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dN 2020 JINST 15 P03 2020 Figure 9. S-curve measured for a single input channel on one of the two CBC2s on a non-irradiated prototype
module at room temperature with the sensor biased at 250 V. On the left the measured data are shown along
with a fit to the measured data performed using eq. (4.1), while on the right the differential histogram is
shown with the corresponding Gaussian fit. JINST 15 P03014 register, defines the position in the on-chip RAM from which the data are read upon reception of a
trigger. The stub latency, also measured in units of 40 MHz clock cycles and set by a configuration
register in the back-end FPGA, defines the delay between hit and stub data arriving at the back-end
of the data acquisition system and is required to assemble the data at the back-end. The resolution of the data latency measurement was improved using a high-resolution time-to-
digital converter (TDC) in the back-end FPGA. The TDC measures the time of arrival of the trigger
signal at the back-end with respect to the 40 MHz clock edge in time slices of 3.125 ns, using a 3 bit
counter operating at 320 MHz. The results of the latency scans performed in the CERN beam test
are shown in figure 11. 4.2
Latency scans These scans were used to identify the stub and data latencies to use during
data taking by counting the number of stubs and hits contained in the data stream for a fixed number
of triggers and selecting values for the data and stub latency that maximize the fraction of events
containing stubs and hits, respectively. Both scans were performed at a threshold of 113 DAC units
(3σ away from the pedestal). For further data taking, the data latency and stub latency were fixed
at 13 and 4, respectively, as shown by the dashed lines in figure 11. 5
Reconstruction Dedicated software is used to reconstruct the data collected from the telescope system and the DUT. Initially, the reconstruction of tracks of the incident particle is carried out using the hits in the
telescope system. The reconstruction of data from the DUT involves the formation of clusters and
stubs using the hits from the individual channels. The reconstructed tracks are then extrapolated to
the DUT, and the estimated position of the track on the DUT is computed. Using this information,
an alignment is performed to correct for the relative offset in position of the DUT with respect to
the telescope system. The reconstruction of tracks from the telescope data, clusters and stubs from
the DUT data, and alignment procedures are described in the following sections. – 10 – 110
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Second CBC
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Figure 10. Pedestal (top) and noise (bottom) for both CBC2s on a prototype module. 110
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140
160
Number of Channels
First CBC
Second CBC 2020 JINST 15 P030 0 JINST 15 P03014 1
Number of Channels 0
1
2
3
4
5
6
7
Noise in DAC units
1
10
2
10
Number of Channels
First CBC
Second CBC Figure 10. Pedestal (top) and noise (bottom) for both CBC2s on a prototype module. 5.1
DUT reconstruction The schematic diagram for the processing of the DUT data is shown in figure 12. The raw data
received from the 2S modules by the FPGA are converted to the CBC2 event format by the DAQ
software and served to the online Data Quality Monitoring (DQM) system. The raw data are also
sent to the CMS event builder (EVB) [28] which provides data in the Event Data Model (EDM)
format [29, 30]. The EDM data are then processed by the CMS offline software, CMSSW [26],
to produce clusters and stubs used in the offline analysis. Hits in adjacent strips of the DUT are
combined to form a cluster. The number of strips included in a cluster is called the cluster width. The cluster position is defined by the center of the cluster rounded down to an integer strip number. – 11 – 0
2
4
6
8
10
12
14
16
18
20
Data Latency in 40 MHz clock cycles
0
0.2
0.4
0.6
0.8
1
Fraction of events with hits
1
2
3
4
5
6
7
8
9
10
Stub Latency in 40 MHz clock cycles
0
0.2
0.4
0.6
0.8
1
Fraction of events with stubs
Figure 11. Results from data (top) and stub (bottom) latency scans. The TDC phase gives fine resolution
within a 40 MHz clock cycle (1/8). The dashed lines indicate the chosen values. 0
2
4
6
8
10
12
14
16
18
20
Data Latency in 40 MHz clock cycles
0
0.2
0.4
0.6
0.8
1
Fraction of events with hits 2020 JINST 15 P030 0 JINST 15 P03014 1
2
3
4
5
6
7
8
9
10
Stub Latency in 40 MHz clock cycles
0
0.2
0.4
0.6
0.8
1
Fraction of events with stubs Figure 11. Results from data (top) and stub (bottom) latency scans. The TDC phase gives fine resolution
within a 40 MHz clock cycle (1/8). The dashed lines indicate the chosen values. The CBC2 reconstructs stubs, by calculating the cluster positions in integer strip numbers. However,
it outputs only the information that a stub was present, not its position (in contrast to later versions
of the chip, which include this functionality). Therefore the stub reconstruction is done offline, by
emulating the logic in the CBC2. Clusters with cluster width greater than 3 are excluded from stub
formation. 5.1
DUT reconstruction The difference in position (in number of strips) of the clusters in the bottom sensor is cal-
culated with respect to clusters in the top sensor. If this difference is less than the predefined window,
an offline stub is formed. The position of the stub is defined as the position of the cluster in the bot-
tom sensor seeding the stub. As the DAQ systems for the telescope and for the DUT are different, an – 12 – additional processing step is needed to synchronize the events coming from telescope and DUT data
streams by matching the individual trigger numbers using the ROOT data analysis framework [31]. Telescope
DUT
Raw Data
Raw Data
Telescope Event
CBC Event
DUT Event
Final Event
Telescope
Framework
DAQ software/ DQM
CMS EVB
CMSSW
Figure 12. A schematic representation of the data processing for the beam tests at CERN. The Telescope
event contains information about the incident track parameters. The DUT event contains the information of
the hits as read from the DUT and also the clusters and stubs reconstructed using offline software. The CBC
event contains the information about CBC errors. Data from all three sources are merged and stored into a
single file for offline analysis. Raw Data Raw Data 2020 JINST 15 P030 DAQ software/ DQM CMS EVB
CMSSW Telescope
Framework 0 JINST 15 P03014 Final Event Figure 12. A schematic representation of the data processing for the beam tests at CERN. The Telescope
event contains information about the incident track parameters. The DUT event contains the information of
the hits as read from the DUT and also the clusters and stubs reconstructed using offline software. The CBC
event contains the information about CBC errors. Data from all three sources are merged and stored into a
single file for offline analysis. 5.2
Tracking Tracks from the EUDET telescope are reconstructed in the EUTelescope [13] framework using
MIMOSA-26 planes. A database of noisy pixels (pixels with exceptionally high occupancy), is
built and used to exclude such pixels from subsequent steps of the analysis. Clusters are built
according to the nearest neighbour search algorithm, which iteratively joins adjacent pixels with
hits to form a cluster. A “pre-alignment” is performed in the telescope global frame, correcting only
for the misalignment in X and Y directions (as shown in figure 7). The output of this step is used
to constrain the alignment step itself, based on solving exact matrix equations with the Millipede
II framework [32]. Shifts in X, Y and Z coordinates and 3 Euler rotation angles for each Mimosa
plane are corrected for. Tracks are then reconstructed with a Deterministic Annealing Filter (DAF)
algorithm [33, 34], where all hits within a given radius are used for the track reconstruction. Tracks
reconstructed with the DAF are further cleaned to remove any duplicates, defined as two or more
tracks with X and Y coordinates at the FE–I4 plane less than 1 µm apart. While Mimosa planes are read out with a rolling shutter having a window of 115 µs, the
maximum acquisition rate for the DUT and the FE–I4 plane is 40 MHz. The presence of a hit in the
FE–I4 plane that can be matched to the track is used as a timestamp, which largely reduces track
combinatorics. Residuals at the FE–I4 plane are used to determine a nominal distance between – 13 – the track impact point and the FE–I4 hit. The residuals are fitted with a step rectangular function
convolved with a Gaussian smearing. The maximum distance to accept a track is set to half the width
of the step function, compatible with the FE–I4 pitch, plus two times the width of the Gaussian,
compatible with the track pointing resolution. Track reconstruction and telescope alignment at the Fermilab test beam facility are performed
using a single dedicated software package [22] that provides a graphical interface to execute the
various steps. An iterative algorithm implements a least-squares minimization to compute 1st-
order roto-translational corrections using tracks reconstructed with a preliminary description of the
geometry. 2020 JINST 15 P03 The reconstruction of beam test data at DESY follows a similar procedure to that used for
beam tests at CERN. 5.3
DUT alignment The DUT alignment procedure consists of minimizing the residuals at the DUT plane to constrain
the degrees of freedom of the system: χ2 = 1
N
N
Õ
i=0
xDUT −xTkAtDUT
σtkres
2
, χ2 = 1
N
N
Õ
i=0
xDUT −xTkAtDUT
σtkres
2
, where xDUT is the hit position in X and xTkAtDUT the position of the hit as derived from the track
extrapolation to the DUT location, while σtkres is the telescope pointing resolution. The sum runs
over all events in which at least one cluster in the DUT and one track are reconstructed. For each
event the closest pair is selected. To remove outliers, the sum is further restricted to events where
the residual |xDUT −xTkAtDUT| is less than 3σtkres away from the mean value of a Gaussian fit of the
residual distribution. The track impact point on the FE–I4 plane is propagated to the first sensor plane of the DUT,
which corresponds to the plane of the sensor facing the beam direction, including degrees of freedom
for the X position of the first plane, Z position of the first plane, θ angle around the Y-axis, and the
distance between the two sensor planes of the DUT. This procedure eliminates the sign degeneracy
of the θ angle. For efficiency studies reported in section 6, a track is matched to a hit, cluster or
stub on the DUT if the residual, |xDUT −xTkAtDUT|, is less than 3σtkres. 5.2
Tracking The main difference is that the entire reconstruction is performed within the
EUTelescope framework and that the General Broken Lines (GBL) [35] algorithm for alignment
is used. The GBL algorithm is required to account for the increased multiple scattering of the
comparatively low-energy particles available at the DESY beam test facility. 0 JINST 15 P03014 6
Results After calibration, the threshold (VCTH) and the angle of rotation of the DUT with respect to the
beam were varied in suitable step sizes and the properties of hits, clusters and stubs were studied. The axis of rotation of the DUT was the Y axis, as shown in figure 7. A scan of VCTH was performed at vertical beam incidence and measurements of the cluster
and stub efficiencies were carried out as a function of a number of functional parameters to fully
characterize the mini-modules (section 6.1) and the full-size module (section 6.2). – 14 – 50
60
70
80
90
100
110
120
CTH
V
1
−
10
1
10
Mean number of hits per event
Top sensor (non-irradiated)
Bottom sensor (non-irradiated)
Top sensor (irradiated)
Bottom sensor (irradiated)
Figure 13. Average number of hits per event on non-irradiated and irradiated sensors as a function of VCTH. A bias voltage of 250 V (600 V) was applied to the non-irradiated (irradiated) mini-module. 2020 JINST 15 P03 JINST 15 P03014 Figure 13. Average number of hits per event on non-irradiated and irradiated sensors as a function of VCTH. A bias voltage of 250 V (600 V) was applied to the non-irradiated (irradiated) mini-module. 6.1
Performance of mini-modules Figures 13 and 14 show the average number of hits and clusters on the non-irradiated and irradiated
mini-modules, respectively. Lower numerical values of VCTH mean a higher signal threshold, as
mentioned in section 4.1. For the non-irradiated mini-module, the average number of hits/clusters
increases as VCTH is increased and a plateau with a value close to 1 is visible, up to VCTH values
of about 110. However, for the irradiated module, the average number of hits/clusters is mostly
less than 1 as VCTH is increased. This indicates that, for a given value of VCTH, we see a lower
number of hits/clusters in the irradiated mini-module as compared to the non-irradiated one. As
the VCTH setting is increased further (⪆110 ), the noise increases in both mini-modules, leading to
a sharp rise in the average number of hits/clusters. Differential histograms of cluster occupancy as
a function of VCTH, derived by numerically differentiating the distributions of the cluster occupancy
as shown in figure 14, are shown in figure 15. The differential distributions show an inverted
Landau distribution, caused by the actual signal generated from the incident particle, and a noise
peak. Comparing the differential distributions, it can again be seen that the total number of clusters
is lower in the irradiated mini-module. The loss in the number of clusters for the irradiated mini-
module as seen in figure 14 and figure 15 indicates that the charge collection in the irradiated
mini-module is worsened due to radiation induced effects. Along with radiation induced effects,
the lower sensor active thickness of 240 µm for the irradiated module, compared to 270 µm of the
non-irradiated module, also leads to lower charge collection. By choosing appropriate VCTH values
of 106 and 110 DAC units for the non-irradiated and irradiated module, respectively, signals from
incident particles can be collected preferentially. The cluster efficiency, defined as the ratio between the number of events with a cluster matched
to a track in a single track event and the total number of events with a single track, is then measured as – 15 – 50
60
70
80
90
100
110
120
CTH
V
1
−
10
1
10
Mean number of clusters per event
Top sensor (non-irradiated)
Bottom sensor (non-irradiated)
Top Sensor (irradiated)
Bottom sensor (irradiated)
Figure 14. Average number of clusters per event for non-irradiated and irradiated sensors as a function of
VCTH. 6.1
Performance of mini-modules A bias voltage of 250 V (600 V) was applied to the non-irradiated (irradiated) mini-module. 2020 JINST 15 P030 0 JINST 15 P03014 Figure 14. Average number of clusters per event for non-irradiated and irradiated sensors as a function of
VCTH. A bias voltage of 250 V (600 V) was applied to the non-irradiated (irradiated) mini-module. 50
60
70
80
90
100
110
120
CTH
V
4
−
10
3
−
10
2
−
10
1
−
10
1
Differential cluster occupancy
Top sensor (non-irradiated)
Bottom sensor (non-irradiated)
Top sensor (irradiated)
Bottom sensor (irradiated)
Figure 15. Differential cluster occupancy for non-irradiated and irradiated sensors as a function of VCTH. A
bias voltage of 250 V (600 V) was applied to the non-irradiated (irradiated) mini-module. Figure 15. Differential cluster occupancy for non-irradiated and irradiated sensors as a function of VCTH. A
bias voltage of 250 V (600 V) was applied to the non-irradiated (irradiated) mini-module. – 16 – a function ofVCTH for different values of the trigger phase (TDC), to check for a potential dependency. The cluster efficiencies for one of the sensors of the non-irradiated and irradiated mini-modules
are shown in figure 16. The lower charge collection in the irradiated module results in a smaller
effciency plateau (figure 16, bottom) compared to the non-irradiated module (figure 16, top). As
the VCTH increases further (> 110), the efficiency starts to degrade for both the mini-modules due to
increase of noise. Due to the higher noise occupancy, the probability of a neighbouring strip to fire
increases, resulting in larger cluster width. This shifts the position of the actual cluster away from
the track causing the track matching to fail. A small dependency on the trigger phase is present for
both sensors and is more evident at lower VCTH. A trigger phase is present in the CERN beam test
because the trigger signal is asynchronous with respect to the 40 MHz clock that drives the readout
electronics. 2020 JINST 15 P030 Because there is no magnetic field, the dependence of the mini-module performance on the
transverse momentum of tracks is emulated by rotating the DUT with respect to the beam direction. As the incident angle (referred to as α) of the particles increases, the charge deposited is shared
by multiple strips and hence the cluster width is expected to increase, which is shown in figure 17. 6.1
Performance of mini-modules This effect is less evident on the irradiated module due to the radiation induced defects both in
the sensor bulk and on the surface, that change the electric field inside the sensor. This leads to a
modification of the charge sharing and further to a higher average cluster size at normal incidence. The same effect is also evident from the distribution of the fraction of clusters with different strip
multiplicities, as shown in figure 18. The non-irradiated module shows a correlation between the
cluster fractions and the angle. This dependence is much less significant for the irradiated module. 0 JINST 15 P03014 In figure 19 the cluster efficiencies for different TDC values as a function of the DUT rotation
angle for the two modules are shown. The dependency on the trigger phase is negligible and the
mean cluster efficiency for the full range of the angular scan is 99.56 ± 0.01% and 98.21 ± 0.02%
for the non-irradiated and irradiated module, respectively. For the CMS field strength of B = 3.8 T, the relationship between the beam incident angle
(α) and the emulated transverse momentum pT of the traversing particle for a radial position of the
module (R) is given by pT [GeV] ≈0.57·R[m]
sin (α) . The stub efficiency, defined as the ratio of the number
of events with stubs matched to a track in single track events to the number of events with a single
track, was measured for each incident angle. Tracks and stubs must match within 4σ of the spatial
resolution. The stub efficiency of the two mini-modules as a function of effective pT (beam-incident
angle) is shown in figure 20. For larger angles of incidence the relative shift in cluster position in
the two sensors of a module is larger, which leads to lower probability of correlating them as stubs. The stub efficiency drops for larger angles for this reason. A stub correlation window of 5 strips
is used. A radius of 60 cm was used for the calculation of the effective pT from the beam incident
angle. The turn-on curve is different for the two modules due to different sensor spacing. 6.1
Performance of mini-modules The turn-on curve was fitted with an error function of the form f (pT) = 0.5A
1 + er f
pT −pTµ
σpT
, where A is the efficiency at the plateau, pTµ is the turn-on threshold for which the efficiency is 50%,
and σpT is the width of the Gaussian in the error function. The pT resolution is defined as the ratio
of the width of the Gaussian to the pT value at 50% of the plateau height, or σpT/pTµ. For the – 17 – 50
60
70
80
90
100
110
120
CTH
V
0.2
0.4
0.6
0.8
1
Cluster efficiency
TDC 4
TDC 5
TDC 6
TDC 7
TDC 8
TDC 9
TDC 10
TDC 11
TDC 12
50
60
70
80
90
100
110
120
CTH
V
0.2
0.4
0.6
0.8
1
Cluster efficiency
TDC 4
TDC 5
TDC 6
TDC 7
TDC 8
TDC 9
TDC 10
TDC 11
TDC 12
Figure 16. Cluster efficiency of the non-irradiated (top) and irradiated (bottom) 2S mini-modules presented
as a function of VCTH for different phase differences between trigger and readout clocks. A bias voltage of
250 V (600 V) was applied to the non-irradiated (irradiated) mini-module. 50
60
70
80
90
100
110
120
CTH
V
0.2
0.4
0.6
0.8
1
Cluster efficiency
TDC 4
TDC 5
TDC 6
TDC 7
TDC 8
TDC 9
TDC 10
TDC 11
TDC 12 2020 JINST 15 P030 0 JINST 15 P03014 50
60
70
80
90
100
110
120
CTH
V
0.2
0.4
0.6
0.8
1
Cluster efficiency
TDC 4
TDC 5
TDC 6
TDC 7
TDC 8
TDC 9
TDC 10
TDC 11
TDC 12 Figure 16. Cluster efficiency of the non-irradiated (top) and irradiated (bottom) 2S mini-modules presented
as a function of VCTH for different phase differences between trigger and readout clocks. A bias voltage of
250 V (600 V) was applied to the non-irradiated (irradiated) mini-module. non-irradiated module, the turn-on threshold is 1.88 GeV with a pT resolution of 5%, whereas the
expected turn-on threshold is 2 GeV. The plateau efficiency for the non-irradiated module is 99%. The high plateau efficiency with sharp turn-on demonstrates that the module can reject tracks with
pT < 2 GeV efficiently. For the irradiated mini-module, the plateau efficiency reaches 97% with
a pT resolution of 6%. 6.1
Performance of mini-modules This shows that the stub finding logic of the 2S modules will work even
after being irradiated to a fluence of 6 × 1014 neq/cm2, which is twice the expected fluence for the
first layer of 2S modules. The stub efficiency measured using data collected at the DESY test beam
facility with the non-irradiated mini module is found to be 99%. – 18 – 2
4
6
8
10
12
14
16
Angle (deg)
1.1
1.15
1.2
1.25
1.3
1.35
1.4
1.45
1.5
1.55
Cluster width (number of strips)
Irradiated module
Non-irradiated module
Figure 17. Mean cluster width of non-irradiated and irradiated 2S mini-modules as a function of the beam
incident angle. Due to radiation induced defects, charge sharing is higher in the irradiated module, leading
to a larger mean cluster size. A bias voltage of 250 V (600 V) was applied to the non-irradiated (irradiated)
mini-module. A VCTH value of 106 (110) DAC units was used for the non-irradiated (irradiated) mini-module. 2
4
6
8
10
12
14
16
Angle (deg)
1.1
1.15
1.2
1.25
1.3
1.35
1.4
1.45
1.5
1.55
Cluster width (number of strips)
Irradiated module
Non-irradiated module 2020 JINST 15 P030 0 JINST 15 P03014 Figure 17. Mean cluster width of non-irradiated and irradiated 2S mini-modules as a function of the beam
incident angle. Due to radiation induced defects, charge sharing is higher in the irradiated module, leading
to a larger mean cluster size. A bias voltage of 250 V (600 V) was applied to the non-irradiated (irradiated)
mini-module. A VCTH value of 106 (110) DAC units was used for the non-irradiated (irradiated) mini-module. 0
2
4
6
8
10
12
14
16
Angle (deg)
3
−
10
2
−
10
1
−
10
1
Fraction of clusters
1 strip cluster
2 strips cluster
>2 strips cluster
0
2
4
6
8
10
12
14
16
Angle (deg)
3
−
10
2
−
10
1
−
10
1
Fraction of clusters
1 strip cluster
2 strips cluster
>2 strips cluster
Figure 18. Fraction of clusters with different strip multiplicity; (left) non-irradiated module; (right) irradiated
module. A bias voltage of 250 V (600 V) was applied to the non-irradiated (irradiated) mini-module. A
VCTH value of 106 (110) DAC units was used for the non-irradiated (irradiated) mini-module. 6.1
Performance of mini-modules For the irradiated module three angular scans were performed, each with different stub corre-
lation windows. As shown in figure 21, the turn-on curve of the efficiency depends on the selected
correlation window, while the efficiency plateau does not. For the irradiated module three angular scans were performed, each with different stub corre-
lation windows. As shown in figure 21, the turn-on curve of the efficiency depends on the selected
correlation window, while the efficiency plateau does not. 6.1
Performance of mini-modules 0
2
4
6
8
10
12
14
16
Angle (deg)
3
−
10
2
−
10
1
−
10
1
Fraction of clusters
1 strip cluster
2 strips cluster
>2 strips cluster 0
2
4
6
8
10
12
14
16
Angle (deg)
3
−
10
2
−
10
1
−
10
1
Fraction of clusters
1 strip cluster
2 strips cluster
>2 strips cluster Figure 18. Fraction of clusters with different strip multiplicity; (left) non-irradiated module; (right) irradiated
module. A bias voltage of 250 V (600 V) was applied to the non-irradiated (irradiated) mini-module. A
VCTH value of 106 (110) DAC units was used for the non-irradiated (irradiated) mini-module. – 19 – 0
2
4
6
8
10
12
14
16
Angle (deg)
0.92
0.93
0.94
0.95
0.96
0.97
0.98
0.99
1
1.01
Cluster efficiency
TDC 4
TDC 5
TDC 6
TDC 7
TDC 8
TDC 9
TDC 10
TDC 11
TDC 12
4
6
8
10
12
14
16
Angle (deg)
0.92
0.93
0.94
0.95
0.96
0.97
0.98
0.99
1
1.01
Cluster efficiency
TDC 4
TDC 5
TDC 6
TDC 7
TDC 8
TDC 9
TDC 10
TDC 11
TDC 12
gure 19. Cluster efficiency of the non-irradiated (top) and irradiated (bottom) 2S mini-modules as a
nction of the beam incident angle for different TDC phases. A bias voltage of 250 V (600 V) was applied
the non-irradiated (irradiated) mini-module. A VCTH value of 106 (110) DAC units was used for the
n-irradiated (irradiated) mini-module. 0
2
4
6
8
10
12
14
16
Angle (deg)
0.92
0.93
0.94
0.95
0.96
0.97
0.98
0.99
1
1.01
Cluster efficiency
TDC 4
TDC 5
TDC 6
TDC 7
TDC 8
TDC 9
TDC 10
TDC 11
TDC 12 2020 JINST 15 P030 0 JINST 15 P03014 4
6
8
10
12
14
16
Angle (deg)
0.92
0.93
0.94
0.95
0.96
0.97
0.98
0.99
1
1.01
Cluster efficiency
TDC 4
TDC 5
TDC 6
TDC 7
TDC 8
TDC 9
TDC 10
TDC 11
TDC 12 Figure 19. Cluster efficiency of the non-irradiated (top) and irradiated (bottom) 2S mini-modules as a
function of the beam incident angle for different TDC phases. A bias voltage of 250 V (600 V) was applied
to the non-irradiated (irradiated) mini-module. A VCTH value of 106 (110) DAC units was used for the
non-irradiated (irradiated) mini-module. 6.2
Performance of the full-size module For the full-size 2S module, the primary goal was to check the uniformity of the response across all
strips. Figure 22 (top) shows the stub efficiency per strip for the full-size 2S module. The module – 20 – 1.2
1.4
1.6
1.8
2
2.2
2.4
2.6
2.8
3
3.2
(GeV)
T
Emulated p
0
0.2
0.4
0.6
0.8
1
Stub efficiency
d = 2.75mm
= 106 DAC units,
CTH
= 250V, V
bias
V
Non-irradiated
d = 3.05mm
= 110 DAC units,
CTH
= 600V, V
bias
V
2
cm
⁄
eq
n
14
10
×
Irradiated to 6
16.6
14.1
12.3
11.0
9.8
8.9
8.2
7.6
7.0
6.5
6.1
Angle (deg)
Figure 20. Stub efficiency for the irradiated (blue) and non-irradiated (red) modules as a function of the
beam incident angle. As expected, for larger angles of incidence, which corresponds to smaller effective
pT, the stub efficiency drops. A radius of 60 cm was used for the calculation of pT from the beam incident
angle which is approximately the radius at which the first layer of 2S modules will be installed. The stub
correlation window is set to 5 strips. 1.2
1.4
1.6
1.8
2
2.2
2.4
2.6
2.8
3
3.2
(GeV)
T
Emulated p
0
0.2
0.4
0.6
0.8
1
Stub efficiency
d = 2.75mm
= 106 DAC units,
CTH
= 250V, V
bias
V
Non-irradiated
d = 3.05mm
= 110 DAC units,
CTH
= 600V, V
bias
V
2
cm
⁄
eq
n
14
10
×
Irradiated to 6
16.6
14.1
12.3
11.0
9.8
8.9
8.2
7.6
7.0
6.5
6.1
Angle (deg) 2020 JINST 15 P030 2020 JINST 15 P03014 JINST 15 P03014 Figure 20. Stub efficiency for the irradiated (blue) and non-irradiated (red) modules as a function of the
beam incident angle. As expected, for larger angles of incidence, which corresponds to smaller effective
pT, the stub efficiency drops. A radius of 60 cm was used for the calculation of pT from the beam incident
angle which is approximately the radius at which the first layer of 2S modules will be installed. The stub
correlation window is set to 5 strips. 6.2
Performance of the full-size module 1.2
1.4
1.6
1.8
2
2.2
2.4
2.6
2.8
3
3.2
3.4
3.6
3.8
4
4.2
(GeV)
T
Emulated p
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
Stub efficiency
16.6
14.1
12.3
11.0
9.8
8.9
8.2
7.6
7.0
6.5
6.1
5.8
5.5
5.2
4.9
4.7
Angle (deg)
Correlation window
6 strips
5 strips
4 strips
Figure 21. Stub efficiency comparison of different angular scans with different correlation windows for the
irradiated module. The choice of window size leads to a shift in the turn-on pT, but the efficiency at the
plateau remains the same. A bias voltage of 600 V was applied to the irradiated mini-module. 1.2
1.4
1.6
1.8
2
2.2
2.4
2.6
2.8
3
3.2
3.4
3.6
3.8
4
4.2
(GeV)
T
Emulated p
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
Stub efficiency
16.6
14.1
12.3
11.0
9.8
8.9
8.2
7.6
7.0
6.5
6.1
5.8
5.5
5.2
4.9
4.7
Angle (deg)
Correlation window
6 strips
5 strips
4 strips Figure 21. Stub efficiency comparison of different angular scans with different correlation windows for the
irradiated module. The choice of window size leads to a shift in the turn-on pT, but the efficiency at the
plateau remains the same. A bias voltage of 600 V was applied to the irradiated mini-module. – 21 – was operated at a bias voltage of 250 V and VCTH was set to 115 DAC units. The analysis techniques
used are the same as reported for the mini-modules. The region between strips 185 and 239 has no
data because it was not scanned by the beam. The large statistical uncertainty in efficiency at the
edges is due to the limited data collected for the scans performed at the module edges. The mean stub
efficiency extracted from a linear fit, where the asymmetric errors on each measurement are taken
into account, is 97.4%, and the strip-to-strip variation of the stub efficiency was found to be 1.3%. The efficiency is approximately 2% lower than that measured in the 2S mini-module. The difference
is due to a different operational configuration of the modules and a possible remaining contamination
of events for which the module was not synchronized with the telescope. The stub efficiency per chip
is shown in figure 22 (bottom). The results demonstrate that the response of the full-size 2S module
is uniform across strips. 6.2
Performance of the full-size module The stub efficiency as a function of effective pT for the full-size 2S module
measured with data collected at the Fermilab test beam facility is shown in figure 23. The correlation
window used for stub formation was set to 5 strips. The figure shows that the behaviour of the full-
size module is similar to that of the mini-modules. From the fit, a turn-on threshold of 1.2 GeV is
obtained with a pT resolution of 7.9%. The turn-on threshold is lower compared to the non-irradiated
mini-module since the sensor separation is smaller. The efficiency at the plateau is 99%. 2020 JINST 15 P030 0 JINST 15 P03014 7
Summary A new silicon strip tracker will be installed in CMS for the HL-LHC period. The new Outer Tracker
will comprise novel detector modules with two closely spaced sensors and a new front-end ASIC that
is capable of correlating hits between the sensor layers. The performance of 2S prototype modules
has been characterized at three test beam facilities. The presence of tracking detectors at these facili-
ties has allowed for spatial matching of the tracks of the incident beam and the hits on the 2S modules. This has provided the first measurements of the absolute efficiency of these prototype detectors. Cluster efficiencies of approximately 99.5% and 98% have been measured for non-irradiated
and irradiated modules, respectively. These results are robust with respect to variations in particle
arrival times relative to the trigger. For the non-irradiated module, an increase in the mean cluster
width is observed as the beam incident angle increases. For the irradiated module, the average
cluster size is higher in general and thus the variation of cluster width with angle is less evident. The stub efficiency across all the strips of the sensors shows a uniform response. The stub
efficiencies of both the non-irradiated mini-module and the full-size module are found to be around
99%. The stub efficiencies obtained from the analysis of data from the three test beam facilities are
in agreement with each other. For the irradiated module, the stub efficiency was found to be 97%. All of the modules demonstrate the ability to reject tracks with pT < 2 GeV. The high efficiency
of the irradiated module provides evidence that the modules will be able to operate throughout the
lifetime of the HL-LHC without much loss of efficiency. – 22 – Strip number
0
127
254
381
508
635
762
889
1016
Stub efficiency
0.86
0.88
0.9
0.92
0.94
0.96
0.98
1
0.0003
±
Average efficiency = 0.9748
Chip ID
0
1
2
3
4
5
6
7
Stub efficiency
0.9
0.91
0.92
0.93
0.94
0.95
0.96
0.97
0.98
0.99
1
Figure 22. Stub efficiency of a full-size 2S module measured at the CERN beam test facility. The module
was operated at a bias voltage of 250V and the VCTH value was set to 115 DAC units. Top: stub efficiency
per strip, bottom: stub efficiency per chip computed using data from strips scanned by the beam. 7
Summary Strip number
0
127
254
381
508
635
762
889
1016
Stub efficiency
0.86
0.88
0.9
0.92
0.94
0.96
0.98
1
0.0003
±
Average efficiency = 0.9748 2020 JINST 15 P030 0 JINST 15 P03014 Figure 22. Stub efficiency of a full-size 2S module measured at the CERN beam test facility. The module
was operated at a bias voltage of 250V and the VCTH value was set to 115 DAC units. Top: stub efficiency
per strip, bottom: stub efficiency per chip computed using data from strips scanned by the beam. Acknowledgments
The tracker groups gratefully acknowledge financial support from the following funding agencies:
BMWFW and FWF (Austria); FNRS and FWO (Belgium); CERN; MSE and CSF (Croatia);
Academy of Finland, MEC, and HIP (Finland); CEA and CNRS/IN2P3 (France); BMBF, DFG,
and HGF (Germany); GSRT (Greece); NKFIA K124850, and Bolyai Fellowship of the Hungarian
Academy of Sciences (Hungary); DAE and DST (India); IPM (Iran); INFN (Italy); PAEC (Pakistan);
SEIDI, CPAN, PCTI and FEDER (Spain); Swiss Funding Agencies (Switzerland); MST (Taipei);
STFC (United Kingdom); DOE and NSF (U.S.A.). Individuals have received support from HFRI
(Greece). Acknowledgments The tracker groups gratefully acknowledge financial support from the following funding agencies:
BMWFW and FWF (Austria); FNRS and FWO (Belgium); CERN; MSE and CSF (Croatia);
Academy of Finland, MEC, and HIP (Finland); CEA and CNRS/IN2P3 (France); BMBF, DFG,
and HGF (Germany); GSRT (Greece); NKFIA K124850, and Bolyai Fellowship of the Hungarian
Academy of Sciences (Hungary); DAE and DST (India); IPM (Iran); INFN (Italy); PAEC (Pakistan);
SEIDI, CPAN, PCTI and FEDER (Spain); Swiss Funding Agencies (Switzerland); MST (Taipei);
STFC (United Kingdom); DOE and NSF (U.S.A.). Individuals have received support from HFRI
(Greece). – 23 – 0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
2.4
(GeV)
T
Emulated p
0
0.2
0.4
0.6
0.8
1
Stub Efficiency
25.5
25.3
20.0
16.6
14.1
12.3
11.0
9.8
8.9
8.2
Angle (deg)
Figure 23. Stub efficiency of the full-size 2S module as a function of particle pT measured at the Fermilab
beam test facility during the angular scan. The module was operated at a bias voltage of 250 V and the VCTH
value was set to 92. A radius of 60 cm is used to convert the beam incident angle to effective pT. 0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
2.4
(GeV)
Emulated p
0
0.2
0.4
0.6
0.8
1
Stub Efficiency
25.5
25.3
20.0
16.6
14.1
12.3
11.0
9.8
8.9
8.2
Angle (deg) Stub Efficiency 2020 JINST 15 P030 JINST 15 P03014 Figure 23. Stub efficiency of the full-size 2S module as a function of particle pT measured at the Fermilab
beam test facility during the angular scan. The module was operated at a bias voltage of 250 V and the VCTH
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(2012) 107 [arXiv:1201.4320]. – 25 – Universiteit Antwerpen, Antwerpen, Belgium W. Beaumont, D. Di Croce, X. Janssen, J. Lauwers, P. Van Mechelen, N. Van Remortel Vrije Universiteit Brussel, Brussel, Belgium
F. Blekman, S.S. Chhibra, J. De Clercq, J. D’Hondt, S. Lowette, I. Marchesini, S. Moortgat,
Q. Python, K. Skovpen, E. Sørensen Bols, P. Van Mulders 2020 JINST 15 P030 Institut für Hochenergiephysik, Wien, Austria W. Adam, T. Bergauer, D. Blöch, E. Brondolin1, M. Dragicevic, R. Frühwirth2, V. Hinger,
H. Steininger Universiteit Antwerpen, Antwerpen, Belgium Université Libre de Bruxelles, Bruxelles, Belgium g
Y. Allard, D. Beghin, B. Bilin, H. Brun, B. Clerbaux, G. De Lentdecker, H. Delannoy, W. Deng,
L. Favart, R. Goldouzian, A. Grebenyuk, A. Kalsi, J. Luetic, I. Makarenko, L. Moureaux, A. Popov,
N. Postiau, F. Robert, Z. Song, L. Thomas, P. Vanlaer, D. Vannerom, Q. Wang, H. Wang, Y. Yang
Université Catholique de Louvain, Louvain-la-Neuve, Belgium Y. Allard, D. Beghin, B. Bilin, H. Brun, B. Clerbaux, G. De Lentdecker, H. Delannoy, W. Deng,
L. Favart, R. Goldouzian, A. Grebenyuk, A. Kalsi, J. Luetic, I. Makarenko, L. Moureaux, A. Popov,
N. Postiau, F. Robert, Z. Song, L. Thomas, P. Vanlaer, D. Vannerom, Q. Wang, H. Wang, Y. Yang 0 JINST 15 P03014 Université Catholique de Louvain, Louvain-la-Neuve, Belgium Université Catholique de Louvain, Louvain-la-Neuve, Belgium O. Bondu, G. Bruno, C. Caputo, P. David, C. Delaere, M. Delcourt, A. Giammanco, G. Krintiras,
V. Lemaitre, A. Magitteri, K. Piotrzkowski, A. Saggio, N. Szilasi, M. Vidal Marono, P. Vischia,
J. Zobec ST 15 P03014 Institut Ruđer Bošković, Zagreb, Croatia Institut Ruđer Bošković, Zagreb, Croatia
V. Brigljević, S. Ceci, D. Ferenček, M. Roguljić, A. Starodumov3, T. Šuša V. Brigljević, S. Ceci, D. Ferenček, M. Roguljić, A. Starodumov3, T. Šuša Department of Physics, University of Helsinki, Helsinki, Finland
P. Eerola, J. Heikkilä Helsinki Institute of Physics, Helsinki, Finland E. Brücken, T. Lampén, P. Luukka, L. Martikainen, E. Tuominen Lappeenranta University of Technology, Lappeenranta, Finland
T. Tuuva Université de Strasbourg, CNRS, IPHC UMR 7178, Strasbourg, France
J.-L. Agram4, J. Andrea, D. Bloch, C. Bonnin, G. Bourgatte, J.-M. Brom, E. Chabert, L. Charles,
V. Cherepanov, E. Dangelser, D. Gelé, U. Goerlach, L. Gross, M. Krauth, N. Tonon Université de Lyon, Université Claude Bernard Lyon 1, CNRS-IN2P3, Institut de Physique Nu-
cléaire de Lyon, Villeurbanne, France G. Baulieu, G. Boudoul, L. Caponetto, N. Chanon, D. Contardo, P. Dené, T. Dupasquier, G. Galbit,
N. Lumb, L. Mirabito, B. Nodari, S. Perries, M. Vander Donckt, S. Viret RWTH Aachen University, I. Physikalisches Institut, Aachen, Germany C. Autermann, L. Feld, W. Karpinski, M.K. Kiesel, K. Klein, M. Lipinski, D. Meuser, A. Ostapchuk,
A. Pauls, G. Pierschel, M. Preuten, M. Rauch, N. Röwert, S. Schael, J. Schulz, G. Schwering,
M. Teroerde, M. Wlochal, V. Zhukov – 26 – RWTH Aachen University, III. Physikalisches Institut B, Aachen, Germany
C. Dziwok, G. Fluegge, T. Müller, O. Pooth, A. Stahl, T. Ziemons RWTH Aachen University, III. Physikalisches Institut B, Aachen, Germany C. Dziwok, G. Fluegge, T. Müller, O. Pooth, A. Stahl, T. Ziemons Deutsches Elektronen-Synchrotron, Hamburg, Germany
M. Aldaya, C. Asawatangtrakuldee, G. Eckerlin, D. Eckstein, T. Eichhorn, E. Gallo, M. Guthoff,
M. Haranko, A. Harb, J. Keaveney, C. Kleinwort, R. Mankel, H. Maser, M. Meyer, M. Missiroli,
C. Muhl, A. Mussgiller, D. Pitzl, O. Reichelt, M. Savitskyi, P. Schuetze, R. Stever, R. Walsh,
A. Zuber Wigner Research Centre for Physics, Budapest, Hungary T. Balázs, F. Siklér, T. Vámi, V. Veszprémi University of Delhi, Delhi, India
A. Bhardwaj, C. Jain, G. Jain, K. Ranjan University of Delhi, Delhi, India A. Bhardwaj, C. Jain, G. Jain, K. Ranjan Saha Institute of Nuclear Physics, Kolkata, India R. Bhattacharya, S. Dutta, S. Roy Chowdhury, G. Saha, S. Sarkar INFN Sezione di Baria, Università di Barib, Politecnico di Baric, Bari, Italy INFN Sezione di Baria, Università di Barib, Politecnico di Baric, Bari, Italy
P. Cariolaa, D. Creanzaa,c, M. de Palmaa,b, G. De Robertisa, L. Fiorea, M. Incea,b, F. Loddoa,
G. Maggia,c, S. Martiradonnaa, M. Mongellia, S. Mya,b, G. Selvaggia,b, L. Silvestrisa P. Cariolaa, D. Creanzaa,c, M. de Palmaa,b, G. De Robertisa, L. Fiorea, M. Incea,b, F. Loddo
G. Maggia,c, S. Martiradonnaa, M. Mongellia, S. Mya,b, G. Selvaggia,b, L. Silvestrisa INFN Sezione di Cataniaa, Università di Cataniab, Catania, Italy
S. Albergoa,b, S. Costaa,b, A. Di Mattiaa, R. Potenzaa,b, M.A. Saizua,5, A. Tricomia,b, C. Tuvea,b INFN Sezione di Cataniaa, Università di Cataniab, Catania, Italy S. Albergoa,b, S. Costaa,b, A. Di Mattiaa, R. Potenzaa,b, M.A. Saizua,5, A. Tricomia,b, C. Tuvea INFN Sezione di Firenzea, Università di Firenzeb, Firenze, Italy University of Hamburg, Hamburg, Germany 2020 JINST 15 P030 A. Benecke, H. Biskop, P. Buhmann, A. Ebrahimi, M. Eich, F. Feindt, A. Froehlich, E. Garutti,
P. Gunnellini, J. Haller, A. Hinzmann, G. Kasieczka, R. Klanner, V. Kutzner, T. Lange, M. Matysek,
M. Mrowietz, C. Niemeyer, Y. Nissan, K. Pena, A. Perieanu, O. Rieger, P. Schleper, J. Schwandt,
D. Schwarz, J. Sonneveld, G. Steinbrück, A. Tews, B. Vormwald, J. Wellhausen, I. Zoi Institut für Experimentelle Teilchenphysik, Karlsruhe, Germany Institut für Experimentelle Teilchenphysik, Karlsruhe, Germany 0 JINST 15 P03014 M. Abbas, L. Ardila, M. Balzer, C. Barth, T. Barvich, M. Baselga, T. Blank, F. Bögelspacher, E. Butz,
M. Caselle, W. De Boer, A. Dierlamm, K. El Morabit, J.-O. Gosewisch, F. Hartmann, U. Husemann,
R. Koppenhöfer, S. Kudella, S. Maier, S. Mallows, M. Metzler, Th. Muller, M. Neufeld, A. Nürnberg,
O. Sander, D. Schell, M. Schröder, T. Schuh, I. Shvetsov, H.-J. Simonis, P. Steck, M. Wassmer,
M. Weber, A. Weddigen Institute of Nuclear and Particle Physics (INPP), NCSR Demokritos, Aghia Paraskevi, Greece
G. Anagnostou, P. Asenov, P. Assiouras, G. Daskalakis, A. Kyriakis, D. Loukas, L. Paspalaki Wigner Research Centre for Physics, Budapest, Hungary
T. Balázs, F. Siklér, T. Vámi, V. Veszprémi Wigner Research Centre for Physics, Budapest, Hungary CERN, European Organization for Nuclear Research, Geneva, Switzerland Abbaneo, I. Ahmed, B. Akgun, E. Albert, J. Bendotti, G. Berruti, G. Blanchot, F. Boyer, D. Abbaneo, I. Ahmed, B. Akgun, E. Albert, J. Bendotti, G. Berruti, G. Blanchot, F. Boyer,
A. Caratelli, D. Ceresa, J. Christiansen, K. Cichy, J. Daguin, N. Deelen6, S. Detraz, D. Deyrail, D. Abbaneo, I. Ahmed, B. Akgun, E. Albert, J. Bendotti, G. Berruti, G. Blanchot, F. Boyer,
A. Caratelli, D. Ceresa, J. Christiansen, K. Cichy, J. Daguin, N. Deelen6, S. Detraz, D. Deyrail,
N. Emriskova7, F. Faccio, A. Filenius, N. Frank, T. French, T. Gadek, R. Gajanec, A. Honma,
G. Hugo, W. Hulek, L.M. Jara Casas, J. Kaplon, K. Kloukinas, A. Kornmayer, N. Koss, L. Kottelat,
D. Koukola, M. Kovacs, A. La Rosa, P. Lenoir, R. Loos, A. Marchioro, S. Marconi, S. Mersi,
S. Michelis, C. Nieto Martin, A. Onnela, S. Orfanelli, T. Pakulski, A. Perez, F. Perez Gomez,
J.-F. Pernot, P. Petagna, Q. Piazza, H. Postema, T. Prousalidi, R. Puente Rico8, S. Scarfí9,
S. Spathopoulos, S. Sroka, P. Tropea, J. Troska, A. Tsirou, F. Vasey, P. Vichoudis Caratelli, D. Ceresa, J. Christiansen, K. Cichy, J. Daguin, N. Deelen6, S. Detraz, D. Deyrail, Emriskova7, F. Faccio, A. Filenius, N. Frank, T. French, T. Gadek, R. Gajanec, A. Honma, J.-F. Pernot, P. Petagna, Q. Piazza, H. Postema, T. Prousalidi, R. Puente Rico8, S. Scarfí9,
S. Spathopoulos, S. Sroka, P. Tropea, J. Troska, A. Tsirou, F. Vasey, P. Vichoudis INFN Sezione di Torinoa,Università di Torinob, Politecnico di Torinoc, Torino, Italy R. Bellana,b, M. Costaa,b, R. Covarellia,b, G. Dellacasaa, N. Demariaa, A. Di Salvoa,c, G. Mazzaa,
E. Migliorea,b, E. Monteila,b, L. Pachera, A. Paternoa,c, A. Rivettia, A. Solanoa,b R. Bellana,b, M. Costaa,b, R. Covarellia,b, G. Dellacasaa, N. Demariaa, A. Di Salvoa,c, G. Mazzaa,
l
a b
la b
h
a A
a c A
a A S l
a b E. Migliorea,b, E. Monteila,b, L. Pachera, A. Paternoa,c, A. Rivettia, A. Solanoa,b Instituto de Física de Cantabria (IFCA), CSIC-Universidad de Cantabria, Santander, Spain
E. Curras Rivera, J. Duarte Campderros, M. Fernandez, G. Gomez, F.J. Gonzalez Sanchez,
R. Jaramillo Echeverria, D. Moya, E. Silva Jimenez, I. Vila, A.L. Virto INFN Sezione di Milano-Bicoccaa, Università di Milano-Bicoccab, Milano, Italy INFN Sezione di Milano-Bicoccaa, Università di Milano-Bicoccab, Milano, Italy F. Brivioa,b, M.E. Dinardoa,b, P. Dinia, S. Gennaia, L. Guzzi, S. Malvezzia, D. Menascea,
L. Moronia, D. Pedrinia, D. Zuoloa,b INFN Sezione di Padovaa, Università di Padovab, Padova, Italy INFN Sezione di Paviaa, Università di Bergamob, Bergamo, Italy F. De Canioa,b, L. Gaionia,b, M. Manghisonia,b, L. Rattia, V. Rea,b, E. Riceputia,b, G. Traversia,b 2020 JINST 15 P030 INFN Sezione di Perugiaa, Università di Perugiab, CNR-IOM Perugiac, Perugia, Italy INFN Sezione di Perugiaa, Università di Perugiab, CNR-IOM Perugiac, Perugia, Italy G. Baldinellia,b, F. Bianchia,b, M. Biasinia,b, G.M. Bileia, S. Bizzagliaa, M. Capraia, C. Cecchia,b,
B. Checcuccia, D. Ciangottinia, L. Fanòa,b, L. Farnesinia, M. Ionicaa, R. Leonardia,b, E. Manonia,
G. Mantovania,b, V. Mariania,b, M. Menichellia, A. Morozzia, F. Moscatellia,c, D. Passeria,b,
P. Placidia,b, A. Rossia,b, A. Santocchiaa,b, D. Spigaa, L. Storchia, C. Turrionia,b JINST 15 P03014 INFN Sezione di Pisaa, Università di Pisab, Scuola Normale Superiore di Pisac, Pisa, Italy INFN Sezione di Padovaa, Università di Padovab, Padova, Italy P. Azzia, N. Bacchettaa, D. Biselloa, T.Dorigoa, N. Pozzobona,b, M. Tosia,b INFN Sezione di Paviaa, Università di Bergamob, Bergamo, Italy W. Bertl†, L. Caminada10, K. Deiters, W. Erdmann, R. Horisberger, H.-C. Kaestli, D. Kotlinski,
U. Langenegger, B. Meier, T. Rohe, S. Streuli INFN Sezione di Firenzea, Università di Firenzeb, Firenze, Italy S
, U
,
,
y
G. Barbaglia, M. Brianzia, A. Cassesea, R. Ceccarellia,b, R. Ciaranfia, V. Ciullia,b, C. Civininia,
R. D’Alessandroa,b, E. Focardia,b, G. Latinoa,b, P. Lenzia,b, M. Meschinia, S. Paolettia,
L. Russoa,b, E. Scarlinia,b, G. Sguazzonia, L. Viliania,b INFN Sezione di Genovaa, Università di Genovab, Genova, Italy
S. Cerchia, F. Ferroa, R. Mulargiaa,b, E. Robuttia – 27 – INFN Sezione di Pisaa, Università di Pisab, Scuola Normale Superiore di Pisac, Pisa, Italy K. Androsova, P. Azzurria, G. Bagliesia, A. Bastia, R. Beccherlea, V. Bertacchia,c, L. Bianchinia,
T. Boccalia, L. Borrelloa, F. Bosia, R. Castaldia, M.A. Cioccia,b, R. Dell’Orsoa, G. Fedia,
F. Fioria,c, L. Gianninia,c, A. Giassia, M.T. Grippoa,b, F. Ligabuea,c, G. Magazzua, E. Mancaa,c,
G. Mandorlia,c, E. Mazzonia, A. Messineoa,b, A. Moggia, F. Morsania, F. Pallaa, F. Palmonaria,
F. Raffaellia, A. Rizzia,b, P. Spagnoloa, R. Tenchinia, G. Tonellia,b, A. Venturia, P.G. Verdinia T 15 P03014 Paul Scherrer Institut, Villigen, Switzerland Roeser, D. Ruini, V. Tavolaro, R. Wallny, D. Zhu
Universität Zürich, Zurich, Switzerland
T. Aarrestad, C. Amsler11, K. Bösiger, F. Canelli, V. Chiochia, A. De Cosa, R. Del Burgo, C. Galloni,
B. Kilminster, S. Leontsinis, R. Maier, G. Rauco, P. Robmann, Y. Takahashi, A. Zucchetta
National Taiwan University (NTU), Taipei, Taiwan
P.-H. Chen, W.-S. Hou, R.-S. Lu, M. Moya, J.F. Tsai
University of Bristol, Bristol, United Kingdom
D. Burns, E. Clement, D. Cussans, J. Goldstein, S. Seif El Nasr-Storey
Rutherford Appleton Laboratory, Didcot, United Kingdom
D. Braga12, J.A. Coughlan, K. Harder, K. Manolopoulos, I.R. Tomalin
Imperial College, London, United Kingdom
G. Auzinger, R. Bainbridge, J. Borg, G. Hall, T. James, M. Pesaresi, S. Summers, K. Uchida
Brunel University, Uxbridge, United Kingdom
J. Cole, C. Hoad, P. Hobson, I.D. Reid
The Catholic University of America, Washington DC, U.S.A. R. Bartek, A. Dominguez, R. Uniyal
Brown University, Providence, U.S.A. G. Altopp, B. Burkle, C. Chen, X. Coubez, Y.-T. Duh, M. Hadley, U. Heintz, N. Hinton, J. Hogan13,
A. Korotkov, J. Lee, M. Narain, S. Sagir14, E. Spencer, R. Syarif, V. Truong, E. Usai, J. Voelker
University of California, Davis, Davis, U.S.A. M. Chertok, J. Conway, G. Funk, F. Jensen, R. Lander, S. Macauda, D. Pellett, J. Thomson, Institute for Particle Physics, ETH Zurich, Zurich, Switzerland 2020 JINST 15 P030 University of Bristol, Bristol, United Kingdom
D. Burns, E. Clement, D. Cussans, J. Goldstein, S. Seif El Nasr-Storey Rutherford Appleton Laboratory, Didcot, United Kingdom
D. Braga12, J.A. Coughlan, K. Harder, K. Manolopoulos, I.R. Tomalin 0 JINST 15 P03014 Imperial College, London, United Kingdom
G. Auzinger, R. Bainbridge, J. Borg, G. Hall, T. James, M. Pesaresi, S. Summers, K. Uchida Brunel University, Uxbridge, United Kingdom
J. Cole, C. Hoad, P. Hobson, I.D. Reid The Catholic University of America, Washington DC, U.S.A. R. Bartek, A. Dominguez, R. Uniyal Brown University, Providence, U.S.A. Brown University, Providence, U.S.A. G. Altopp, B. Burkle, C. Chen, X. Coubez, Y.-T. Duh, M. Hadley, U. Heintz, N. Hinton, J. Hogan13,
A. Korotkov, J. Lee, M. Narain, S. Sagir14, E. Spencer, R. Syarif, V. Truong, E. Usai, J. Voelker A. Korotkov, J. Lee, M. Narain, S. Sagir14, E. Spencer, R. Syarif, V. Truong, E. Usai, J. Voelker M. Chertok, J. Conway, G. Funk, F. Jensen, R. Lander, S. Macauda, D. Pellett, J. Thomson,
R. Yohay15, F. Zhang University of California, Riverside, Riverside, U.S.A
G. Hanson, W. Paul Scherrer Institut, Villigen, Switzerland W. Bertl†, L. Caminada10, K. Deiters, W. Erdmann, R. Horisberger, H.-C. Kaestli, D. Kotlinski,
U. Langenegger, B. Meier, T. Rohe, S. Streuli – 28 – Institute for Particle Physics, ETH Zurich, Zurich, Switzerland
F. Bachmair, M. Backhaus, R. Becker, P. Berger, D. di Calafiori, L. Djambazov, M. Donega, C. Grab,
D. Hits, J. Hoss, W. Lustermann, M. Masciovecchio, M. Meinhard, V. Perovic, L. Perozzi, B. Ristic,
U. Roeser, D. Ruini, V. Tavolaro, R. Wallny, D. Zhu
Universität Zürich, Zurich, Switzerland
T. Aarrestad, C. Amsler11, K. Bösiger, F. Canelli, V. Chiochia, A. De Cosa, R. Del Burgo, C. Galloni,
B. Kilminster, S. Leontsinis, R. Maier, G. Rauco, P. Robmann, Y. Takahashi, A. Zucchetta
National Taiwan University (NTU), Taipei, Taiwan
P.-H. Chen, W.-S. Hou, R.-S. Lu, M. Moya, J.F. Tsai
University of Bristol, Bristol, United Kingdom
D. Burns, E. Clement, D. Cussans, J. Goldstein, S. Seif El Nasr-Storey
Rutherford Appleton Laboratory, Didcot, United Kingdom
D. Braga12, J.A. Coughlan, K. Harder, K. Manolopoulos, I.R. Tomalin
Imperial College, London, United Kingdom
G. Auzinger, R. Bainbridge, J. Borg, G. Hall, T. James, M. Pesaresi, S. Summers, K. Uchida
Brunel University, Uxbridge, United Kingdom
J. Cole, C. Hoad, P. Hobson, I.D. Reid
The Catholic University of America, Washington DC, U.S.A. R. Bartek, A. Dominguez, R. Uniyal
Brown University, Providence, U.S.A. G. Altopp, B. Burkle, C. Chen, X. Coubez, Y.-T. Duh, M. Hadley, U. Heintz, N. Hinton, J. Hogan13,
A. Korotkov, J. Lee, M. Narain, S. Sagir14, E. Spencer, R. Syarif, V. Truong, E. Usai, J. Voelker
University of California, Davis, Davis, U.S.A. M. Chertok, J. Conway, G. Funk, F. Jensen, R. Lander, S. Macauda, D. Pellett, J. Thomson,
R. Yohay15, F. Zhang
University of California, Riverside, Riverside, U.S.A. G. Hanson, W. Si
University of California, San Diego, La Jolla, U.S.A. R. Gerosa, S. Krutelyov, V. Sharma, A. Yagil, G. Zevi Della Porta
University of California, Santa Barbara - Department of Physics, Santa Barbara, U.S.A. O. Colegrove, V. Dutta, L. Gouskos, J. Incandela, S. Kyre, H. Qu, M. Quinnan, D. White
University of Colorado Boulder, Boulder, U.S.A. J.P. Cumalat, W.T. Ford, E. MacDonald, A. Perloff, K. Stenson, K.A. Ulmer, S.R. Wagner
Cornell University, Ithaca, U.S.A. Institute for Particle Physics, ETH Zurich, Zurich, Switzerland
F. Bachmair, M. Backhaus, R. Becker, P. Berger, D. di Calafiori, L. Djambazov, M. Donega, C. Grab,
D. Hits, J. Hoss, W. Lustermann, M. Masciovecchio, M. Meinhard, V. Perovic, L. Perozzi, B. Ristic,
U. Paul Scherrer Institut, Villigen, Switzerland Si University of California, Riverside, Riverside, U.S.A. G. Hanson, W. Si University of California, San Diego, La Jolla, U.S.A. R. Gerosa, S. Krutelyov, V. Sharma, A. Yagil, G. Zevi Della Porta R. Gerosa, S. Krutelyov, V. Sharma, A. Yagil, G. Zevi Della Porta University of California, Santa Barbara - Department of Physics, Santa Barbara, U.S.A. O. Colegrove, V. Dutta, L. Gouskos, J. Incandela, S. Kyre, H. Qu, M. Quinnan, D. White University of Colorado Boulder, Boulder, U.S.A. J.P. Cumalat, W.T. Ford, E. MacDonald, A. Perloff, K. Stenson, K.A. Ulmer, S.R. Wagner
Cornell University, Ithaca, U.S.A. J.P. Cumalat, W.T. Ford, E. MacDonald, A. Perloff, K. Stenson, K.A. Ulmer, S.R. Wagner Cornell University, Ithaca, U.S.A. J. Alexander, Y. Cheng, J. Chu, J. Conway, D. Cranshaw, A. Datta, K. McDermott, J. Monroy,
Y. Bordlemay Padilla, D. Quach, A. Rinkevicius, A. Ryd, L. Skinnari, L. Soffi, C. Strohman,
Z. Tao, J. Thom, J. Tucker, P. Wittich, M. Zientek J. Alexander, Y. Cheng, J. Chu, J. Conway, D. Cranshaw, A. Datta, K. McDermott, J. Monroy,
Y. Bordlemay Padilla, D. Quach, A. Rinkevicius, A. Ryd, L. Skinnari, L. Soffi, C. Strohman, J. Alexander, Y. Cheng, J. Chu, J. Conway, D. Cranshaw, A. Datta, K. McDermott, J. Monroy, y
,
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Z. Tao, J. Thom, J. Tucker, P. Wittich, M. Zientek – 29 – Fermi National Accelerator Laboratory, Batavia, U.S.A. A. Apresyan, A. Bakshi, G. Bolla†, K. Burkett, J.N. Butler, A. Canepa, H.W.K. Cheung,
J. Chramowicz, G. Derylo, A. Ghosh, C. Gingu, H. Gonzalez, S. Grünendahl, S. Hasegawa,
J. Hoff, S.Y. Hoh, Z. Hu, S. Jindariani, M. Johnson, C.M. Lei, R. Lipton, M. Liu, T. Liu, S. Los,
M. Matulik, P. Merkel, S. Nahn, J. Olsen, A. Prosser, F. Ravera, L. Ristori, R. Rivera, B. Schneider,
W.J. Spalding, L. Spiegel, S. Timpone, N. Tran, L. Uplegger, C. Vernieri, E. Voirin, H.A. Weber
University of Illinois at Chicago (UIC), Chicago, U.S.A. University of Illinois at Chicago (UIC), Chicago, U.S.A. D.R. Berry, X. Chen, S. Dittmer, A. Evdokimov, O. Evdokimov, C.E. Gerber, D.J. Hofman, C. Mills y
g (
),
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D.R. Berry, X. Chen, S. Dittmer, A. Evdokimov, O. Evdokimov, C.E. Gerber, D.J. Hofman, C. Mills 2020 JINST 15 P0301 The University of Iowa, Iowa City, U.S.A. M. Alhusseini, S. Durgut, J. Nachtman, Y. Onel, C. Rude, C. Snyder, K. Yi16 . Alhusseini, S. Durgut, J. Nachtman, Y. Onel, C. Rutgers, The State University of New Jersey, Piscataway, U.S.A. Rutgers, The State University of New Jersey, Piscataway, U.S.A. Rutgers, The State University of New Jersey, Piscataway, U.S.A. Rutgers, The State University of New Jersey, Piscataway, U.S.A. E. Bartz, A. Gandrakotra, Y. Gershtein, E. Halkiadakis, A. Hart, S. Kyriacou, A. Lath, K. Nash,
M. Osherson, S. Schnetzer, R. Stone
Texas A&M University, College Station, U.S.A. R. Eusebi
Vanderbilt University, Nashville, U.S.A. P. D’Angelo, W. Johns, K.O. Padeken
Wayne State University, Detroit, U.S.A. E. Bartz, A. Gandrakotra, Y. Gershtein, E. Halkiadakis, A. Hart, S. Kyriacou, A. Lath, K. Nash,
M. Osherson, S. Schnetzer, R. Stone E. Bartz, A. Gandrakotra, Y. Gershtein, E. Halkiadakis, A. Hart, S. Kyriacou, A. Lath, K. Nash,
M. Osherson, S. Schnetzer, R. Stone Paul Scherrer Institut, Villigen, Switzerland Rude, C. Snyder, K. Yi16 Johns Hopkins University, Baltimore, U.S.A. N. Eminizer, A. Gritsan, P. Maksimovic, J. Roskes, M. Swartz, M. Xiao 0 JINST 15 P03014 The University of Kansas, Lawrence, U.S.A. P. Baringer, A. Bean, S. Khalil, A. Kropivnitskaya, D. Majumder, E. Schmitz, G. Wilson Kansas State University, Manhattan, U.S.A. A. Ivanov, R. Mendis, T. Mitchell, A. Modak, R. Taylor Kansas State University, Manhattan, U.S.A. A. Ivanov, R. Mendis, T. Mitchell, A. Modak, R. Taylor University of Mississippi, Oxford, U.S.A. J.G. Acosta, L.M. Cremaldi, S. Oliveros, L. Perera, D. Summers University of Nebraska-Lincoln, Lincoln, U.S.A. University of Nebraska-Lincoln, Lincoln, U.S.A. K. Bloom, D.R. Claes, C. Fangmeier, F. Golf, I. Kravchenko, J. Siado University of Nebraska-Lincoln, Lincoln, U.S.A. K. Bloom, D.R. Claes, C. Fangmeier, F. Golf, I. Kravchenko, J. Siado K. Bloom, D.R. Claes, C. Fangmeier, F. Golf, I. Kravchenko, J. Siado State University of New York at Buffalo, Buffalo, U.S.A. C. Harrington, I. Iashvili, A. Kharchilava, D. Nguyen, A. Parker, S. Rappoccio, B. Roozbahani C. Harrington, I. Iashvili, A. Kharchilava, D. Nguyen, A. Parker, S. Rappoccio, B. Roozbahani Northwestern University, Evanston, U.S.A. K. Hahn, Y. Liu, K. Sung The Ohio State University, Columbus, U.S.A. The Ohio State University, Columbus, U.S.A. J. Alimena, B. Cardwell, B. Francis, C.S. Hill J. Alimena, B. Cardwell, B. Francis, C.S. Hill University of Puerto Rico, Mayaguez, U.S.A. S. Malik, S. Norberg, J.E. Ramirez Vargas Purdue University, West Lafayette, U.S.A. S. Das, M. Jones, A. Jung, A. Khatiwada, G. Negro, J. Thieman Purdue University Northwest, Hammond, U.S.A. T. Cheng, J. Dolen, N. Parashar Rice University, Houston, U.S.A. K.M. Ecklund, S. Freed, M. Kilpatrick, T. Nussbaum University of Rochester, Rochester, U.S.A. R. Demina, J. Dulemba, O. Hindrichs – 30 – E. Bartz, A. Gandrakotra, Y. Gershtein, E. Halkiadakis, A. Hart, S. Kyriacou, A. Lath, K. Nash,
M. Osherson, S. Schnetzer, R. Stone Texas A&M University, College Station, U.S.A.
R. Eusebi Vanderbilt University, Nashville, U.S.A. P. D’Angelo, W. Johns, K.O. Padeken P. D’Angelo, W. Johns, K.O. Padeken Wayne State University, Detroit, U.S.A. R. Harr, N. Poudyal Wayne State University, Detroit, U.S.A. 2020 JINST 15 P030 R. Harr, N. Poudyal †: Deceased
1: Now at CERN, European Organization for Nuclear Research, Geneva, Switzerland
2: Also at Vienna University of Technology, Vienna, Austria
3: Also at Institute for Theoretical and Experimental Physics, Moscow, Russia
4: Also at Université de Haute-Alsace, Mulhouse, France
5: Also at Horia Hulubei National Institute of Physics and Nuclear Engineering (IFIN-HH), Bucharest,
Romania
6: Also at Institut für Experimentelle Kernphysik, Karlsruhe, Germany
7: Also at Université de Strasbourg, CNRS, IPHC UMR 7178, Strasbourg, France
8: Also at Instituto de Física de Cantabria (IFCA), CSIC-Universidad de Cantabria, Santander, Spain
9: Also at École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
10: Also at Universität Zürich, Zurich, Switzerland
11: Also at Albert Einstein Center for Fundamental Physics, Bern, Switzerland
12: Also at Imperial College, London, United Kingdom
13: Now at Bethel University, St. Paul, Minnesota, U.S.A. 14: Now at Karamanoglu Mehmetbey University, Karaman, Turkey
15: Now at Florida State University, Tallahassee, U.S.A. 16: Also at Nanjing Normal University, Nanjing, China †: Deceased †: Deceased 1: Now at CERN, European Organization for Nuclear Research, Geneva, Switzerland 0 JINST 15 P03014 2: Also at Vienna University of Technology, Vienna, Austria 2: Also at Vienna University of Technology, Vienna, Austria 3: Also at Institute for Theoretical and Experimental Physics, Moscow, Russia 3: Also at Institute for Theoretical and Experimental Physics, Moscow, Russia 4: Also at Université de Haute-Alsace, Mulhouse, France Also at Université de Haute-Alsace, Mulhouse, Franc 5: Also at Horia Hulubei National Institute of Physics and Nuclear Engineering (IFIN-HH), Bucharest,
Romania 6: Also at Institut für Experimentelle Kernphysik, Karlsruhe, Germany 6: Also at Institut für Experimentelle Kernphysik, Karlsruhe, Germany 7: Also at Université de Strasbourg, CNRS, IPHC UMR 7178, Strasbourg, France 8: Also at Instituto de Física de Cantabria (IFCA), CSIC-Universidad de Cantabria, Santander, Spain 9: Also at École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland 11: Also at Albert Einstein Center for Fundamental Physics, Bern, Switzerland 12: Also at Imperial College, London, United Kingdom 13: Now at Bethel University, St. Paul, Minnesota, U.S.A. 14: Now at Karamanoglu Mehmetbey University, Karaman, Turkey 15: Now at Florida State University, Tallahassee, U.S.A. 15: Now at Florida State University, Tallahassee, U.S.A. 16: Also at Nanjing Normal University, Nanjing, China 16: Also at Nanjing Normal University, Nanjing, China – 31 –
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https://www.life-science-alliance.org/content/lsa/2/2/e201900392.full.pdf
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English
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Inhibition of the deubiquitinase USP8 corrects a Drosophila PINK1 model of mitochondria dysfunction
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Inhibition of the deubiquitinase USP8 corrects a
Drosophila PINK1 model of mitochondria dysfunction Sophia von Stockum1, Alvaro Sanchez-Martinez2
, Samantha Corr`a3,4, Joy Chakraborty3, Elena Marchesan1,
Lisa Locatello3
, Caterina Da Rè3,4, Paola Cusumano3,4, Federico Caicci3, Vanni Ferrari3, Rodolfo Costa3,4
,
Luigi Bubacco3, Maria Berica Rasotto3, Ildiko Szabo3, Alexander J Whitworth2
, Luca Scorrano3,5, Elena Ziviani1,3 cytoplasmic protein Parkin, mutated in familial PD and encoding
an E3 ubiquitin ligase (Shimura et al, 2000), translocates in a
PINK1-dependent
manner
to
dysfunctional
mitochondria
(Narendra et al, 2008; Vives-Bauza et al, 2010b; Ziviani et al, 2010). In this process, kinase PINK1, also mutated in familial PD (Silvestri
et al, 2005), phosphorylates Parkin (Sha et al, 2010), its targets
(Wang et al, 2011; Chen & Dorn, 2013), and ubiquitin itself (Koyano
et al, 2014) promoting Parkin translocation (Narendra et al, 2010;
Ziviani et al, 2010) and Parkin activity (Lazarou et al, 2013; Koyano
et al, 2014; Zhang et al, 2014). On depolarized mitochondria, Parkin
ubiquitinates the mitochondrial pro-fusion protein Mitofusin
(MFN) (Gegg et al, 2010; Poole et al, 2010; Tanaka et al, 2010; Ziviani
et al, 2010; Sarraf et al, 2013) leading to p97/VCP–mediated ret-
rotranslocation and proteosomal degradation (Tanaka et al, 2010). In addition, Parkin ubiquitinates the mitochondrial protein
translocase TOM20, mitochondrial VDAC/Porin and Fis1 (Sarraf et
al, 2013), and it also promotes the degradation of Miro (Wang et al,
2011), a protein that couples mitochondria to microtubules. Se-
lected mitochondria are, therefore, deprived of their pro-fusion
protein MFN, isolating them from the mitochondrial network,
before degradation via autophagy. This mechanism is consistent
with observations showing that mitochondria cluster around the
perinuclear area (Vives-Bauza et al, 2010b) and fragment before
mitophagy (Twig et al, 2008a; Poole et al, 2008). Genetic studies in
Drosophila showed that down-regulation of MFN or promotion of
mitochondrial fission by expressing pro-fission protein DRP1
rescues Parkin KO phenotypes, and those of kinase PINK1 (Deng et
al, 2008; Poole et al, 2008), which acts upstream of Parkin (Clark et
al, 2006; Park et al, 2006; Yang et al, 2006). This genetic interaction
can be in part explained biochemically by the fact that Parkin
ubiquitinates MFN to control its steady-state levels (Gegg et al,
2010; Tanaka et al, 2010; Ziviani et al, 2010; Rakovic et al, 2011) that
are elevated in Parkin and PINK1 KO models (Ziviani et al, 2010). on 23 October, 2024
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Published Online: 15 April, 2019 | Supp Info: 1Fondazione Ospedale San Camillo, IRCCS, Venezia, Italy
2MRC Mitochondrial Biology Unit, Cambridge Biomedical Campus, Cambridge, UK
3Department of Biology,
University of Padova, Padova, Italy
4Neurogenetics and Behavior of Drosophila Lab, Department of Biology, University of Padova, Padova, Italy
5Dulbecco-Telethon
Institute, Venetian Institute of Molecular Medicine, Padova, Italy Inhibition of the deubiquitinase USP8 corrects a
Drosophila PINK1 model of mitochondria dysfunction Thus, interventions that restore MFN levels can ameliorate Parkin
and PINK1 phenotypes, presumably by impinging on the numerous Aberrant mitochondrial dynamics disrupts mitochondrial function
and contributes to disease conditions. A targeted RNA interference
screen for deubiquitinating enzymes (DUBs) affecting protein
levels of multifunctional mitochondrial fusion protein Mitofusin
(MFN) identified USP8 prominently influencing MFN levels. Ge-
netic and pharmacological inhibition of USP8 normalized the
elevated MFN protein levels observed in PINK1 and Parkin-
deficient models. This correlated with improved mitochondrial
function, locomotor performance and life span, and prevented
dopaminergic neurons loss in Drosophila PINK1 KO flies. We
identified a novel target antagonizing pathologically elevated
MFN levels, mitochondrial dysfunction, and dopaminergic neu-
ron loss of a Drosophila model of mitochondrial dysfunction. DOI 10.26508/lsa.201900392 | Received 1 April 2019 | Revised 4 April
2019 | Accepted 5 April 2019 | Published online 15 April 2019 DOI 10.26508/lsa.201900392 | Received 1 April 2019 | Revised 4 April
2019 | Accepted 5 April 2019 | Published online 15 April 2019 Correspondence: elena.ziviani@unipd.it A targeted siRNA screening identifies DUBs affecting MFN protein
levels We next assessed the impact of USP8 down-regulation on MFN levels
in vivo. To this aim, we drove efficient whole body USP8 knockdown (KD)
by using the Actin5C driver (Act-GAL4>USP8-RNAi), achieving significant
USP8 down-regulation at 29°C (Fig S1F). Attempts to increase USP8
down-regulation efficiency by using the stronger GAL4 driver daugh-
terless (da) caused larvae lethality, suggesting that USP8 expression
levels in vivo are tightly regulated. Act-GAL4>USP8-RNAi on the other
hand was viable with no apparent locomotor defects. As previously
observed in vitro, levels of MFN were reduced in vivo in USP8
down-regulating flies (Fig 1F). We also found decreased MFN levels
in protein extracts coming from flies carrying heterozygous USP8
gene deletion (USP8−/+) (Mukai et al, 2010), further supporting that
the effect is specific for USP8 (Fig 1G). Steady-state levels of MFN protein in Drosophila PINK1 or Parkin KO
background are increased (Ziviani et al, 2010), and interventions
that decrease MFN levels can ameliorate Drosophila PINK1 and
Parkin phenotypes (Celardo et al, 2016; Deng et al, 2008; Poole et al,
2008). Given the importance of MFN in inter-organellar commu-
nication (Cosson et al, 2012; de Brito & Scorrano, 2008; Filadi et al,
2015) and mitophagy (Chen & Dorn, 2013), we set out to identify
regulators of its steady-state levels. We designed an unbiased loss-
of-function screen using dsRNA to inhibit the expression of 35
known or predicted fly DUBs. Fly DUBs were identified by domain
similarity and based on the list of 79 human DUBs (Dupont et al,
2009) (Table 1). We transiently expressed flag-tagged MFN in S2R+
cells to mimic pathologically elevated MFN and down-regulated
each of the 35 DUBs. To assess the effect of DUB silencing on steady-
state MFN levels, we performed Western blotting analysis on cell
lysates and quantified the levels of unmodified MFN normalized for
the loading control and expressed it as fold change (Fig 1A). Flag-
tagged MFN exhibited mitochondrial subcellular localization, and
its expression in S2R+ cells resulted in an elongated mitochondrial
network (Fig S1A). We identified two DUBs whose down-regulation
resulted in decreased MFN levels (CG5798/USP8 and CG5384/USP14)
and two DUBs, whose down-regulation resulted in increased MFN
levels (CG5505/USP36, CG2904/Echinus) (Fig 1B). Down-regulation of
Parkin or PINK1 increased MFN levels, as previously described
(Tanaka et al, 2010; Ziviani et al, 2010). MFN functions that in the fruit fly include both promotion of fusion
and ER–mitochondria crosstalk (Debattisti et al, 2014). MFN functions that in the fruit fly include both promotion of fusion
and ER–mitochondria crosstalk (Debattisti et al, 2014). controlling Parkin recruitment to depolarized mitochondria after
CCCP treatment (Durcan et al, 2014). More recently, it has been found
that it can regulate basal autophagy in the absence of CCCP, although
its role has not been thoroughly characterized in this process and it is
controversial (Jacomin et al, 2015). USP8 is also highly expressed in
the brain and up-regulated in neurodegenerative conditions (Paiardi
et al, 2014), which makes it of neurological interest. To identify other mechanisms regulating MFN levels, we per-
formed an RNA interference screen for deubiquitinating enzymes
(DUBs) that affect steady-state levels of MFN. DUBs participate in
important reversible signaling pathways (Salmena & Pandolfi, 2007)
and are attractive druggable candidates (Hussain et al, 2009;
Colland, 2010). We identified USP8, an evolutionary conserved DUB
whose down-regulation correlates with decreased MFN levels. USP8
has previously been linked to PINK1/Parkin–dependent mitophagy
in cell culture and under intoxicating conditions (Durcan et al, 2014),
but no in vivo studies have been reported. Here, we demonstrate
that in vivo under basal conditions, genetic and pharmacological
inhibition of USP8 ameliorates Drosophila phenotypes deriving
from loss of function of PINK1 and Parkin. A targeted siRNA screening identifies DUBs affecting MFN protein
levels Of the two DUBs causing
decreased MFN levels, USP8 was the highest scoring hit that de-
creased MFN levels (Fig 1B). USP8 interacts with many substrates such
as the epidermal growth factor receptor, an essential regulator of
proliferation and differentiation, and regulates endosomal trafficking
by ubiquitin-mediated sorting of the endocytosed cargoes (Mizuno
et al, 2005; Row et al, 2006; Williams & Urbe, 2007). Moreover, USP8
knockdown protects from α-synuclein–induced locomotor deficits
and cell loss in an α-synuclein fly model of PD (Alexopoulou et al,
2016). It was also shown that USP8 regulates induced mitophagy by USP8 down-regulation correlates with decreased MFN protein
levels We next validated if USP8 down-regulation correlated with changes
in MFN protein levels. Upon efficient USP8 down-regulation in fly
cells, as assessed by qPCR (Fig S1B), steady-state levels of en-
dogenous (Figs 1C and S1C) or exogenously expressed tagged MFN
were decreased (Fig 1D) and mitochondria appeared accordingly
fragmented (Fig S1D). The effect was specific for USP8 because re-
expression of USP8 in USP8 RNAi cells restored MFN levels (Fig 1D). In contrast, in cells overexpressing USP8, levels of exogenously
expressed (Fig 1D) and endogenous MFN were increased (Fig 1E) and
mitochondria were elongated and clumped, accumulating in the
perinuclear area (Fig S1E). Protective effect of USP8 inhibition
von Stockum et al. Introduction Mitochondria dysfunction plays critical role in neurodegenerative
conditions affecting the elderly, such as Parkinson’s disease (PD)
(Moore et al, 2005; Bueler, 2009; Vives-Bauza et al, 2010a; Ryan
et al, 2015). Mitochondria function directly correlates with
mitochondria dynamics and balanced remodeling of the
mitochondrial network through fission and fusion events to
control mitochondria shape and ultrastucture. Intuitively,
fusion maintains the mitochondrial network and allows
intermixing of matrix contents, such as mtDNA and metabolites;
fission is needed to populate new cells with new mitochondria
(Twig et al, 2008b; Gomes & Scorrano, 2008; Malena et al, 2009) and
plays a substantial role in the mitochondria quality control. A key
aspect of mitochondrial quality control is a well-characterized
process called mitophagy that segregates and selectively elimi-
nates damaged mitochondria via autophagy (Twig et al, 2008a;
Twig & Shirihai, 2011). During stress-induced mitophagy, the https://doi.org/10.26508/lsa.201900392
vol 2 | no 2 | e201900392
1 of 16 1 of 16 © 2019 von Stockum et al. © 2019 von Stockum et al. https://doi.org/10.26508/lsa.201900392
vol 2 | no 2 | e201900392 USP8 down-regulation ameliorates the phenotype of PINK1 KO
flies We addressed whether USP8 knockdown in PINK1 KO flies pre-
vented the multiple phenotypes recapitulating key features of
locomotor and cellular defects manifested in the flies as de-
generation of dopaminergic (DA) neurons and reduced climbing
ability. We also assessed the flight muscle, mitochondria ultra-
structure, male fertility, and life span, all degenerated or affected in
PINK1 KO flies (Clark et al, 2006; Park et al, 2006; Yang et al, 2006). Immunostaining for the specific DA neuronal marker tyrosine hy-
droxylase (TH) allowed the inspection of the DA neuronal network
composed of well-characterized DA neuron clusters (PPM1, PPM2,
PPM3, PPL1, PPL2, and VUM) in brains (Fig 2A). PINK1 KO showed the
expected reduction in TH staining and exhibited a small but sig-
nificant decrease in the number of DA neurons in the PPL1 DA
neuronal cluster (Fig 2B and C) (Park et al, 2006; Wang et al, 2006;
Yang et al, 2006). Accordingly, dopamine levels measured from
PINK1 KO heads were significantly lower compared with control flies
(Fig 2D). USP8 down-regulation completely prevented the loss of https://doi.org/10.26508/lsa.201900392
vol 2 | no 2 | e201900392 2 of 16 Table 1. Complete list of the 75 human known or predicted DUBs and their
fly homologue, when known or predicted, based on sequence similarity. Where available, Entrez/PubMed gene ID and fly gene name is provided. Table 1. Complete list of the 75 human known or predicted DUBs and their
fly homologue, when known or predicted, based on sequence similarity. Where available, Entrez/PubMed gene ID and fly gene name is provided. Table 1. Continued Gene name
Gene ID
Fly homologue
Fly gene name
USP39
10713
CG7288
USP40
55230
CG5486
USP64E/USP47
USP41
373856
CG5486
USP64E/USP47
USP42
84132
CG5505
USP36/SCRAWNY
USP43
124739
CG30421
USP15-31
USP44
84101
CG5798
USP8/USPY
USP45
85015
CG4165
USP16-45
USP46
64854
CG7023
USP12-46
USP47
55031
CG5486
USP64E/USP47
USP48
84196
CG1490
USP7
USP49
25862
CG5798
USP8/USPY
USP50
373509
CG5798
USP8/USPY
USP51
158880
CG4166
NOT
USP52
9924
CG8232
PAN2
USP53
54532
CG2904
ECHINUS
USP54
159195
CG2904
ECHINUS
OTUB1
55611
CG4968
CYLD
1540
CG5603
TNFAIP3/A20
7128
CG9448
TRABID
OTUD1
220213
CG6091
YOD1
55432
CG4603
OTUD3
23252
CG6091
OTUD4
54726
CG12743
OTU
OTUD6A
139562
CG7857
OTUD6B
51633
CG7857
OTUD7A
161725
CG9448
TRABID
OTUD7B
56957
CG9448
TRABID
TRABID
54764
CG9448
TRABID
ATXN3
4287
CG13379
ATX3L
N.A. Protective effect of USP8 inhibition
von Stockum et al. USP8 down-regulation ameliorates the phenotype of PINK1 KO
flies (F) Equal amounts of protein (70 μg), isolated from wild-type (Ctrl) flies or those down-regulating USP8 (USP8) separated
by SDS–PAGE and immunoblotted using the indicated antibodies. Representative of n = 8. Graph bar shows mean ± SEM of ratio between densitometric levels of MFN and
those of Actin relatively to control from at least three independent experiments. Means are significantly different according to the t test; P = 0.0044 (**), n = 8. The flies were
raised at 29°C to allow efficient down-regulation of USP8. (G) Equal amounts of protein (70 μg), isolated from wild-type (Ctrl) flies and those carrying heterozygous deletion
of USP8 (USP8−/+) separated by SDS–PAGE and immunoblotted using the indicated antibodies. Representative of n = 5. Graph bar shows mean ± SEM of ratio between
densitometric levels of MFN and those of Actin relatively to control from at least four independent experiments. Means are significantly different according to the t test; P =
0.0069 (**), n = 5. Source data are available for this figure. Source data are available for this figure. the previous results and confirm that loss of USP8 ameliorates
PINK1 KO phenotypes. To independently validate the previous results, we analyzed a
bona fide genetic mutant for USP8. Heterozygous USP8 gene de-
letion (USP8−/+) in PINK1 KO background also completely prevented
the loss of DA neurons (Fig 3A and B), restored dopamine levels to
wild-type (Fig 3C), corrected thoracic muscle fiber disorganization
(Fig 3D) and mitochondrial structure (Fig 3E), ameliorated the
shorter longevity (Fig 3F), and completely corrected the locomotor
defects (Fig 3G). Thus, these observations support the specificity of USP8 down-regulation ameliorates the phenotype of PINK1 KO
flies CG13379
JOSD1
9929
CG3781
JOSD2
126119
CG3781
AMSH/STAMBP
10617
CG2224
AMSH-LP
57559
CG2224 Gene name
Gene ID
Fly homologue
Fly gene name
UCHL1
7345
CG4265
UCHL3
7347
CG4265
BAP1
8314
CG8445
CALYPSO
UCHL5/UCH37
51377
CG3431
DUB3
377630
CG5505
USP36/SCRAWNY
USP1
7398
CG15817
USP1
USP2
9099
CG14619
USP3
9960
CG5798
UBPY/USP8
USP4
7375
CG8334
USP5
8078
CG12082
USP6
9098
CG8334
USP7/HAUSP
7874
CG1490
USP7
USP8/USPY
9101
CG5798
UBPY/USP8
USP9X/FAM
8239
CG1945
FAT FACETS
USP10
9100
CG32479
USP11
8237
CG8334
USP12
219333
CG7023
USP12-46
USP13
8975
CG12082
USP5
USP14
9097
CG5384
USP15
9958
CG12082
USP16
10600
CG4165
USP16-45
USP18
11274
CG5486
USP64E/USP47
USP19
10869
CG8334
USP20
10868
CG8494
USP21
27005
CG14619
USP22
23326
N/A
USP24
23358
CG1945
FAT FACETS
USP25
29761
CG5794
PUF/USP34
USP26
83844
CG5798
USP8/USPY
USP27X
389856
CG4166
NOT
USP28
57646
CG5794
PUF/USP34
USP29
57663
CG5798
USP8/USPY
USP30
84749
CG3016
USP31
57478
CG30421
USP15-31
USP32
84669
CG8334
USP33
23032
CG8494
USP20-33
USP34
9736
CG5794
PUF/USP34
USP35
57558
CG8830
DUBAI
USP36
57602
CG5505
USP37
57695
CG5798
USP8/USPY
USP38
84640
CG8830
DUBAI
(Continued on following page) PINK1 KO DA neurons (Fig 2B and C), restoring dopamine to wild-
type levels (Fig 2D). Moreover, USP8 down-regulation ameliorated
the shorter longevity (Fig 2E), corrected thoracic muscle fiber
disorganization with enlarged electron transparent mitochondria
and irregular myofibril arrays (Park et al, 2006) (Fig 2F) typical of the
PINK1 KO flies (Park et al, 2006). More importantly, ultrastructural
transmission electron microscopy (TEM) analysis showed that the
mitochondrial cristae, fragmented and sparely packed in PINK1
mutants, were recovered with highly increased electron-dense
staining intensity (Fig 2G). USP8 knockdown also ameliorated the
PINK1 climbing defect (Fig 2H). Protective effect of USP8 inhibition
von Stockum et al. https://doi.org/10.26508/lsa.201900392
vol 2 | no 2 | e201900392
3 o 3 of 16 Figure 1. A targeted siRNA screening identified DUB
USP8 whose down-regulation correlates with
decreased MFN levels. (A) siRNA screen to identify DUBs affecting
pathologically elevated MFN protein levels. Protein
extracts from Drosophila S2R+ cells expressing equal
amounts of Flag-MFN and treated with 2 μg dsRNA probe
were separated by SDS–PAGE and immunoblotted using
an anti-Flag antibody. Densitometric analysis of MFN
signal normalized to loading control and expressed as
fold change (FC) versus control dsRNA was used as read
out to identify DUBs whose down-regulation affects MFN
protein levels. USP8 down-regulation ameliorates the phenotype of PINK1 KO
flies (B) Volcano plot constructed by plotting
the negative log of the FDR corrected P value (qval) on
the y-axis against the log of the FC calculated in (A). Those points that are found toward the top of the plot
far to either the left- or right-hand side represent values
with large FC and high statistical significance. A
threshold of P < 0.05 and 0.75 < FC > 1.3 led to the
identification of four DUBs whose down-regulation
resulted in either decreased MFN levels (USP8 FC = 0.345 ±
0.04, qval = 0.024; USP14 FC = 0.537 ± 0.06, qval = 0.044) or
increased MFN levels (Echinus FC = 1.784 ± 0.13, qval =
0.024; USP36 FC = 1.524 ± 0.12, qval = 0.040). Down-
regulation of PINK1 or Parkin led to increased MFN levels
(FC = 2.724 ± 0.44, qval = 0.045; and FC = 1.994 ± 0.28, qval =
0.045, respectively). (C) S2R+ cells were transfected with
the indicated siRNA (Ctrl and USP8) and lysed after 3 d. Equal amounts of protein (30 μg) were separated by
SDS–PAGE and immunoblotted using the indicated
antibodies. Representative of n = 6. Graph bar shows
mean ± SEM of ratio between densitometric levels of
MFN and those of Actin from at least eight independent
experiments. Means are significantly different according
to t test; P = 0.0025 (**), n = 6. (D) S2R+ cells were
transfected with the indicated plasmid (MFN-Flag, USP8)
and siRNA (Ctrl and USP8) and lysed after 3 d. Equal
amounts of protein (30 μg) were separated by SDS–PAGE
and immunoblotted using the indicated antibodies. Representative of n = 5. Graph bar shows mean ± SEM of
ratio between densitometric levels of Flag (MFN) and
those of Actin relatively to control from at least four
independent experiments. One-way ANOVA; P < 0.0001
(****), followed by Tukey’s multiple comparison test. n =
5. (E) S2R+ cells were transfected with the indicated
plasmids (empty vector, ev or USP8) and lysed after 3 d. Equal amounts of protein (30 μg) were separated by
SDS–PAGE and immunoblotted using the indicated
antibodies. Representative of n = 4. Graph bar shows
mean ± SEM of ratio between densitometric levels of
MFN and those of Actin relatively to control from at least
four independent experiments. Means are significantly
different according to the t test; P = 0.0313 (*), n = 4. USP8 down-regulation ameliorates the phenotype of PINK1 KO
flies (F) Equal amounts of protein (70 μg), isolated from wild-type (Ctrl) flies or those down-regulating USP8 (USP8) separated
by SDS–PAGE and immunoblotted using the indicated antibodies. Representative of n = 8. Graph bar shows mean ± SEM of ratio between densitometric levels of MFN and
those of Actin relatively to control from at least three independent experiments. Means are significantly different according to the t test; P = 0.0044 (**), n = 8. The flies were
raised at 29°C to allow efficient down-regulation of USP8. (G) Equal amounts of protein (70 μg), isolated from wild-type (Ctrl) flies and those carrying heterozygous deletion
of USP8 (USP8−/+) separated by SDS–PAGE and immunoblotted using the indicated antibodies. Representative of n = 5. Graph bar shows mean ± SEM of ratio between
densitometric levels of MFN and those of Actin relatively to control from at least four independent experiments. Means are significantly different according to the t test; P =
0.0069 (**), n = 5. d
il bl
f
hi
fi different according to the t test; P = 0.0313 (*), n = 4. (F) Equal amounts of protein (70 μg), isolated from wild-type (
by SDS–PAGE and immunoblotted using the indicated antibodies. Representative of n = 8. Graph bar shows mea
those of Actin relatively to control from at least three independent experiments. Means are significantly differen
raised at 29°C to allow efficient down-regulation of USP8. (G) Equal amounts of protein (70 μg), isolated from wild
of USP8 (USP8−/+) separated by SDS–PAGE and immunoblotted using the indicated antibodies. Representativ
densitometric levels of MFN and those of Actin relatively to control from at least four independent experiments. 0.0069 (**), n = 5. Source data are available for this figure. Figure 1. A targeted siRNA screening identified DUB
USP8 whose down-regulation correlates with
decreased MFN levels. Figure 1. A targeted siRNA screening identified DUB
USP8 whose down-regulation correlates with
decreased MFN levels. Figure 1. A targeted siRNA screening identified DUB
USP8 whose down-regulation correlates with
decreased MFN levels. (A) siRNA screen to identify DUBs affecting
pathologically elevated MFN protein levels. Protein
extracts from Drosophila S2R+ cells expressing equal
amounts of Flag-MFN and treated with 2 μg dsRNA probe
were separated by SDS–PAGE and immunoblotted using
an anti-Flag antibody. USP8 down-regulation ameliorates the phenotype of PINK1 KO
flies Densitometric analysis of MFN
signal normalized to loading control and expressed as
fold change (FC) versus control dsRNA was used as read
out to identify DUBs whose down-regulation affects MFN
protein levels. (B) Volcano plot constructed by plotting
the negative log of the FDR corrected P value (qval) on
the y-axis against the log of the FC calculated in (A). Those points that are found toward the top of the plot
far to either the left- or right-hand side represent values
with large FC and high statistical significance. A
threshold of P < 0.05 and 0.75 < FC > 1.3 led to the
identification of four DUBs whose down-regulation
resulted in either decreased MFN levels (USP8 FC = 0.345 ±
0.04, qval = 0.024; USP14 FC = 0.537 ± 0.06, qval = 0.044) or
increased MFN levels (Echinus FC = 1.784 ± 0.13, qval =
0.024; USP36 FC = 1.524 ± 0.12, qval = 0.040). Down-
regulation of PINK1 or Parkin led to increased MFN levels
(FC = 2.724 ± 0.44, qval = 0.045; and FC = 1.994 ± 0.28, qval =
0.045, respectively). (C) S2R+ cells were transfected with
the indicated siRNA (Ctrl and USP8) and lysed after 3 d. Equal amounts of protein (30 μg) were separated by
SDS–PAGE and immunoblotted using the indicated
antibodies. Representative of n = 6. Graph bar shows
mean ± SEM of ratio between densitometric levels of
MFN and those of Actin from at least eight independent
experiments. Means are significantly different according
to t test; P = 0.0025 (**), n = 6. (D) S2R+ cells were
transfected with the indicated plasmid (MFN-Flag, USP8)
and siRNA (Ctrl and USP8) and lysed after 3 d. Equal
amounts of protein (30 μg) were separated by SDS–PAGE
and immunoblotted using the indicated antibodies. Representative of n = 5. Graph bar shows mean ± SEM of
ratio between densitometric levels of Flag (MFN) and
those of Actin relatively to control from at least four
independent experiments. One-way ANOVA; P < 0.0001
(****), followed by Tukey’s multiple comparison test. n =
5. (E) S2R+ cells were transfected with the indicated
plasmids (empty vector, ev or USP8) and lysed after 3 d. Equal amounts of protein (30 μg) were separated by
SDS–PAGE and immunoblotted using the indicated
antibodies. Representative of n = 4. USP8 down-regulation ameliorates the phenotype of PINK1 KO
flies Graph bar shows
mean ± SEM of ratio between densitometric levels of
MFN and those of Actin relatively to control from at least
four independent experiments. Means are significantly
trl) flies or those down-regulating USP8 (USP8) separated
n ± SEM of ratio between densitometric levels of MFN and
according to the t test; P = 0.0044 (**), n = 8. The flies were
-type (Ctrl) flies and those carrying heterozygous deletion
e of n = 5. Graph bar shows mean ± SEM of ratio between
Means are significantly different according to the t test; P = (A) siRNA screen to identify DUBs affecting
pathologically elevated MFN protein levels. Protein
extracts from Drosophila S2R+ cells expressing equal
amounts of Flag-MFN and treated with 2 μg dsRNA probe
were separated by SDS–PAGE and immunoblotted using an anti-Flag antibody. Densitometric analysis of MFN
signal normalized to loading control and expressed as
fold change (FC) versus control dsRNA was used as read
out to identify DUBs whose down-regulation affects MFN
protein levels. (B) Volcano plot constructed by plotting
the negative log of the FDR corrected P value (qval) on p
p
g
y
different according to the t test; P = 0.0313 (*), n = 4. (F) Equal amounts of protein (70 μg), isolated from wild-type (Ctrl) flies or those down-regulating USP8 (USP8) separated
by SDS–PAGE and immunoblotted using the indicated antibodies. Representative of n = 8. Graph bar shows mean ± SEM of ratio between densitometric levels of MFN and
those of Actin relatively to control from at least three independent experiments. Means are significantly different according to the t test; P = 0.0044 (**), n = 8. The flies were
raised at 29°C to allow efficient down-regulation of USP8. (G) Equal amounts of protein (70 μg), isolated from wild-type (Ctrl) flies and those carrying heterozygous deletion
of USP8 (USP8−/+) separated by SDS–PAGE and immunoblotted using the indicated antibodies. Representative of n = 5. Graph bar shows mean ± SEM of ratio between
densitometric levels of MFN and those of Actin relatively to control from at least four independent experiments. Means are significantly different according to the t test; P =
0.0069 (**), n = 5. Source data are available for this figure. different according to the t test; P = 0.0313 (*), n = 4. Protective effect of USP8 inhibition
von Stockum et al. USP8 whose down-regulation correlates with
decreased MFN levels.
(A) siRNA screen to identify DUBs affecting
pathologically elevated MFN protein levels. Protein
extracts from Drosophila S2R+ cells expressing equal
amounts of Flag-MFN and treated with 2 μg dsRNA probe
were separated by SDS–PAGE and immunoblotted using
an anti-Flag antibody. Densitometric analysis of MFN
signal normalized to loading control and expressed as
fold change (FC) versus control dsRNA was used as read
out to identify DUBs whose down-regulation affects MFN
protein levels. (B) Volcano plot constructed by plotting
the negative log of the FDR corrected P value (qval) on
the y-axis against the log of the FC calculated in (A).
Those points that are found toward the top of the plot
far to either the left- or right-hand side represent values
with large FC and high statistical significance. A
threshold of P < 0.05 and 0.75 < FC > 1.3 led to the
identification of four DUBs whose down-regulation
resulted in either decreased MFN levels (USP8 FC = 0.345 ±
0.04, qval = 0.024; USP14 FC = 0.537 ± 0.06, qval = 0.044) or
increased MFN levels (Echinus FC = 1.784 ± 0.13, qval =
0.024; USP36 FC = 1.524 ± 0.12, qval = 0.040). Down-
regulation of PINK1 or Parkin led to increased MFN levels
(FC = 2.724 ± 0.44, qval = 0.045; and FC = 1.994 ± 0.28, qval =
0.045, respectively). (C) S2R+ cells were transfected with
the indicated siRNA (Ctrl and USP8) and lysed after 3 d.
Equal amounts of protein (30 μg) were separated by
SDS–PAGE and immunoblotted using the indicated
antibodies. Representative of n = 6. Graph bar shows
mean ± SEM of ratio between densitometric levels of
MFN and those of Actin from at least eight independent
experiments. Means are significantly different according
to t test; P = 0.0025 (**), n = 6. (D) S2R+ cells were
transfected with the indicated plasmid (MFN-Flag, USP8)
and siRNA (Ctrl and USP8) and lysed after 3 d. Equal
amounts of protein (30 μg) were separated by SDS–PAGE
and immunoblotted using the indicated antibodies.
Representative of n = 5. Graph bar shows mean ± SEM of
ratio between densitometric levels of Flag (MFN) and
those of Actin relatively to control from at least four
independent experiments. One-way ANOVA; P < 0.0001
(****), followed by Tukey’s multiple comparison test. n =
5. (E) S2R+ cells were transfected with the indicated
plasmids (empty vector, ev or USP8) and lysed after 3 d.
Equal amounts of protein (30 μg) were separated by
SDS–PAGE and immunoblotted using the indicated
antibodies. Representative of n = 4. Graph bar shows
mean ± SEM of ratio between densitometric levels of
MFN and those of Actin relatively to control from at least
four independent experiments. Means are significantly
Ctrl) flies or those down-regulating USP8 (USP8) separated
n ± SEM of ratio between densitometric levels of MFN and
t according to the t test; P = 0.0044 (**), n = 8. The flies were
-type (Ctrl) flies and those carrying heterozygous deletion
e of n = 5. Graph bar shows mean ± SEM of ratio between
Means are significantly different according to the t test; P = USP8 down-regulation rescues mitochondria defects of PINK1 KO
flies To verify if USP8 down-regulation also correlates to the amelio-
ration of mitochondrial function, impaired in PINK1 KO/KD models https://doi.org/10.26508/lsa.201900392
vol 2 | no 2 | e201900392
4 of 16 https://doi.org/10.26508/lsa.201900392
vol 2 | no 2 | e201900392
4 of 16 Protective effect of USP8 inhibition
von Stockum et al. Figure 2. USP8 down-regulation corrects DA neuron loss, life span, muscle degeneration, and locomotor impairment of PINK1-deficient flies. (A) Confocal images (projection, Z stack) of whole-mount adult brain (left panel) showing DA neuron clusters marked with an anti-TH antibody. Immunostaining
for the specific DA neuronal marker TH allows the inspection of the DA neuronal network composed by well-characterized DA neuron clusters (PPM1, PPM2, PPM3,
PPL1, PPL2, and VUM) in brains (right panel). (B) Whole brains of 15-d-old male flies of the indicated genotypes were immunostained with anti-TH antibody. Panel shows
confocal images of PPL1 cluster DA neurons of the indicated genotypes. Representative of n = 15. (C) Bar graph shows the number of DA neurons in the PPL1
cluster of the brains of the indicated genotypes. One-way ANOVA, P < 0.0001 (****) followed by Tukey’s multiple comparison test; n = 15. (D) Relative dopamine amount from
15-d-old adult heads of the indicated genotype normalized to control flies. One-way ANOVA, P = 0.0073 (**) followed by Tukey’s multiple comparison test. n = 4. (E)
Life span analysis of adult males of the indicated genotypes. Male flies of the indicated genotypes were collected during 12 h after hatching and transferred to
fresh food every 2 d and dead flies were counted in the same interval. At least 100 flies per genotype were used for the analysis. Log-rank, Mantel–Cox test (Ctrl versus PINK1
KO P < 0.0001; Ctrl versus PINK1 KO USP8 RNAi P < 0.0001; Ctrl versus USP8 RNAi P > 0.05; PINK1 KO versus PINK1 KO USP8 RNAi P < 0.0001; PINK1 KO versus USP8 RNAi P <
0.0001; and PINK1 KO USP8 RNAi versus USP8 RNAi P < 0.0001 P < 0.0001). (F) Ultrastructural analysis of the indirect flight muscles from fly thoraces of the indicated
genotypes. Images show TEM images of thorax muscles from flies of the indicated genotypes. Representative of n = 3. (G) Enlarged TEM images of flight muscle
mitochondria of the indicated genotypes. Representative of n = 3. USP8 down-regulation rescues mitochondria defects of PINK1 KO
flies (G) Enlarged TEM images of flight muscle
mitochondria of the indicated genotypes. Representative of n = 3. (H) Graph bar shows mean ± SEM of the climbing performance of flies of the indicated genotype from at
least three independent experiments. One-way ANOVA, P < 0.0001 (****); Tukey’s multiple comparison test; n = 3. Figure 2. USP8 down-regulation corrects DA neuron loss, life span, muscle degeneration, and locomotor impairment of PIN
( )
(
)
(
) Figure 2. USP8 down-regulation corrects DA neuron loss, life span, muscle degeneration, and locomotor impairment of PINK1-deficient flies. (A) Confocal images (projection, Z stack) of whole-mount adult brain (left panel) showing DA neuron clusters marked with an anti-TH antibody. Immunostaining
for the specific DA neuronal marker TH allows the inspection of the DA neuronal network composed by well-characterized DA neuron clusters (PPM1, PPM2, PPM3,
PPL1, PPL2, and VUM) in brains (right panel). (B) Whole brains of 15-d-old male flies of the indicated genotypes were immunostained with anti-TH antibody. Panel shows
confocal images of PPL1 cluster DA neurons of the indicated genotypes. Representative of n = 15. (C) Bar graph shows the number of DA neurons in the PPL1
cluster of the brains of the indicated genotypes. One-way ANOVA, P < 0.0001 (****) followed by Tukey’s multiple comparison test; n = 15. (D) Relative dopamine amount from
15-d-old adult heads of the indicated genotype normalized to control flies. One-way ANOVA, P = 0.0073 (**) followed by Tukey’s multiple comparison test. n = 4. (E)
Life span analysis of adult males of the indicated genotypes. Male flies of the indicated genotypes were collected during 12 h after hatching and transferred to
fresh food every 2 d and dead flies were counted in the same interval. At least 100 flies per genotype were used for the analysis. Log-rank, Mantel–Cox test (Ctrl versus PINK1
KO P < 0.0001; Ctrl versus PINK1 KO USP8 RNAi P < 0.0001; Ctrl versus USP8 RNAi P > 0.05; PINK1 KO versus PINK1 KO USP8 RNAi P < 0.0001; PINK1 KO versus USP8 RNAi P <
0.0001; and PINK1 KO USP8 RNAi versus USP8 RNAi P < 0.0001 P < 0.0001). (F) Ultrastructural analysis of the indirect flight muscles from fly thoraces of the indicated
genotypes. Images show TEM images of thorax muscles from flies of the indicated genotypes. Representative of n = 3. USP8 down-regulation rescues mitochondria defects of PINK1 KO
flies (H) Graph bar shows mean ± SEM of the climbing performance of flies of the indicated genotype from at
least three independent experiments. One-way ANOVA, P < 0.0001 (****); Tukey’s multiple comparison test; n = 3. n-regulation corrects DA neuron loss, life span, muscle degeneration, and locomotor impairment of PINK1-deficient flies. j
i
k)
f
h l
d l b
i
(l f
l)
h
i
l
k d
i h
i
ib d Figure 2. USP8 down-regulation corrects DA neuron loss, life span, muscle degeneration, and locomotor impairment of PINK1-deficient flies. (A) Confocal images (projection, Z stack) of whole-mount adult brain (left panel) showing DA neuron clusters marked with an anti-TH antibody. Immunostaining
for the specific DA neuronal marker TH allows the inspection of the DA neuronal network composed by well-characterized DA neuron clusters (PPM1, PPM2, PPM3,
PPL1, PPL2, and VUM) in brains (right panel). (B) Whole brains of 15-d-old male flies of the indicated genotypes were immunostained with anti-TH antibody. Panel shows
confocal images of PPL1 cluster DA neurons of the indicated genotypes. Representative of n = 15. (C) Bar graph shows the number of DA neurons in the PPL1
cluster of the brains of the indicated genotypes. One-way ANOVA, P < 0.0001 (****) followed by Tukey’s multiple comparison test; n = 15. (D) Relative dopamine amount from
15-d-old adult heads of the indicated genotype normalized to control flies. One-way ANOVA, P = 0.0073 (**) followed by Tukey’s multiple comparison test. n = 4. (E)
Life span analysis of adult males of the indicated genotypes. Male flies of the indicated genotypes were collected during 12 h after hatching and transferred to
fresh food every 2 d and dead flies were counted in the same interval. At least 100 flies per genotype were used for the analysis. Log-rank, Mantel–Cox test (Ctrl versus PINK1
KO P < 0.0001; Ctrl versus PINK1 KO USP8 RNAi P < 0.0001; Ctrl versus USP8 RNAi P > 0.05; PINK1 KO versus PINK1 KO USP8 RNAi P < 0.0001; PINK1 KO versus USP8 RNAi P <
0.0001; and PINK1 KO USP8 RNAi versus USP8 RNAi P < 0.0001 P < 0.0001). (F) Ultrastructural analysis of the indirect flight muscles from fly thoraces of the indicated
genotypes. Images show TEM images of thorax muscles from flies of the indicated genotypes. Representative of n = 3. Protective effect of USP8 inhibition
von Stockum et al. USP8 down-regulation rescues mitochondria defects of PINK1 KO
flies One-way ANOVA,
P = 0.0002 (***); Tukey’s multiple comparison test; n = 5. (D) TEM images of thorax muscles from flies of the
indicated genotypes. Representative of n = 3. (E)
Enlarged TEM image of flight muscle mitochondria of
the indicated genotypes. Representative of n = 3. (F) Life
span analysis of adult males of the indicated
genotypes. Male flies of the indicated genotypes were
collected during 12 h after hatching and transferred to
fresh food every 2 d and dead flies were counted in the
same interval. At least 100 flies per genotype were used
for the analysis. Log-rank, Mantel–Cox test (Ctrl versus
PINK1 KO P < 0.0001; Ctrl versus PINK1 KO USP8−/+ P <
0.0001; Ctrl versus USP8−/+ P > 0.05; PINK1 KO versus
PINK1 KO USP8−/+ P < 0.0001; PINK1 KO versus USP8−/+
P < 0.0001; and PINK1 KO USP8−/+ versus USP8−/+ P <
0.0001 P < 0.0001). (G) Graph bar shows mean ± SEM of
the climbing performance of flies of the indicated
genotype from at least three independent experiments. One-way ANOVA, P < 0.0001 (****); Tukey’s multiple
comparison test; n = 3. (A) Whole brains of 15-d-old male flies of the indicated
genotypes were immunostained with anti-TH antibody. Panel shows confocal images of DA neuron of the PPL1
cluster of the indicated genotypes. Representative of
n = 11. (B) Bar graph shows the number of DA neurons in
the PPL1 cluster of the brains of the indicated
genotypes. One-way ANOVA, P < 0.0001 (****); Tukey’s
multiple comparison test; n = 11. (C) Relative dopamine
amount from 15-d-old adult heads of the indicated
genotype normalized to control flies. One-way ANOVA,
P = 0.0002 (***); Tukey’s multiple comparison test; n = 5. (D) TEM images of thorax muscles from flies of the mitochondrial function (Fig S2C–E). Because USP8 participates in a
multiplicity of pathways (Alexopoulou et al, 2016; Durcan & Fon,
2015; Mizuno et al, 2005; Row et al, 2006), the beneficial effects on
mitochondrial function measured in situ might be indirect. We,
therefore, compared the function of mitochondria purified from
PINK1-mutant (KO) flies with that recorded in mitochondria isolated
from PINK1 KO flies where we down-regulated USP8 (Fig 4A and B) or
from double heterozygous USP8-deficient (USP8−/+), PINK1 KO flies
(Fig 4C and D). USP8 down-regulation rescues mitochondria defects of PINK1 KO
flies Because USP8 participates in a
the other hand, USP8 RNAi (Fig 4A and B) or heterozygou
Figure 3. Heterozygous USP8 gene deletion
DA neuron loss, life span, muscle degenerati
locomotor impairment of PINK1-deficient flie
(A) Whole brains of 15-d-old male flies of the in
genotypes were immunostained with anti-TH a
Panel shows confocal images of DA neuron of
cluster of the indicated genotypes. Represent
n = 11. (B) Bar graph shows the number of DA ne
the PPL1 cluster of the brains of the indicated
genotypes. One-way ANOVA, P < 0.0001 (****);
multiple comparison test; n = 11. (C) Relative do
amount from 15-d-old adult heads of the indi
genotype normalized to control flies. One-way
P = 0.0002 (***); Tukey’s multiple comparison te
(D) TEM images of thorax muscles from flies o
indicated genotypes. Representative of n = 3. Enlarged TEM image of flight muscle mitochon
the indicated genotypes. Representative of n = 3
span analysis of adult males of the indicated
genotypes. Male flies of the indicated genotyp
collected during 12 h after hatching and transf
fresh food every 2 d and dead flies were counte
same interval. At least 100 flies per genotype w
for the analysis. Log-rank, Mantel–Cox test (Ct
PINK1 KO P < 0.0001; Ctrl versus PINK1 KO USP
0.0001; Ctrl versus USP8−/+ P > 0.05; PINK1 KO
PINK1 KO USP8−/+ P < 0.0001; PINK1 KO versus
P < 0.0001; and PINK1 KO USP8−/+ versus USP
0.0001 P < 0.0001). (G) Graph bar shows mean
the climbing performance of flies of the indic
genotype from at least three independent expe
One-way ANOVA, P < 0.0001 (****); Tukey’s mu
comparison test; n = 3. Figure 3. Heterozygous USP8 gene deletion corrects
DA neuron loss, life span, muscle degeneration, and
locomotor impairment of PINK1-deficient flies. ,
p
,
g
,
locomotor impairment of PINK1-deficient flies. (A) Whole brains of 15-d-old male flies of the indicated
genotypes were immunostained with anti-TH antibody. Panel shows confocal images of DA neuron of the PPL1
cluster of the indicated genotypes. Representative of
n = 11. (B) Bar graph shows the number of DA neurons in
the PPL1 cluster of the brains of the indicated
genotypes. One-way ANOVA, P < 0.0001 (****); Tukey’s
multiple comparison test; n = 11. (C) Relative dopamine
amount from 15-d-old adult heads of the indicated
genotype normalized to control flies. USP8 down-regulation rescues mitochondria defects of PINK1 KO
flies (G) Enlarged TEM images of flight muscle
mitochondria of the indicated genotypes. Representative of n = 3. (H) Graph bar shows mean ± SEM of the climbing performance of flies of the indicated genotype from at
least three independent experiments. One-way ANOVA, P < 0.0001 (****); Tukey’s multiple comparison test; n = 3. lacking PINK1, mitochondrial dysfunction is mirrored also by
changes in mitochondrial membrane potential (Δψm) (Gandhi et al,
2009; Morais et al, 2009; Mortiboys et al, 2008). When we measured
latent mitochondrial dysfunction using a well-established assay
based on the response of Δψm to the ATPase inhibitor oligomycin,
as expected (Gandhi et al, 2009; Morais et al, 2009), we noticed that
PINK1-deficient mitochondria sustain their Δψm by hydrolyzing
cytosolic ATP and therefore depolarize after oligomycin treatment
(Fig S2C–E). Although down-regulation of USP8 had no effect on
Δψm, in PINK1-deficient cells, it fully prevented the oligomycin-
induced depolarization, further confirming its beneficial effects on (Clark et al, 2006; Gandhi et al, 2009; Morais et al, 2014; Park et al,
2006),
we
measured
mitochondrial
respiration
in
digitonin-
permeabilized cells, where mitochondria are directly accessible
to substrates. In line with what has been previously reported
(Gandhi et al, 2009; Morais et al, 2009), we found that ADP-
stimulated glutamate-supported respiration (state 3) was signifi-
cantly reduced in cells lacking PINK1 (Fig S2A). State 3/basal (state
4) respiration ratio, also known as respiratory control ratio (RCR),
was reduced (Fig S2B). USP8 down-regulation did not perturb
mitochondrial respiration per se; however, it corrected the respi-
ration defects of the PINK1-deficient cells (Fig S2A and B). In cells (Clark et al, 2006; Gandhi et al, 2009; Morais et al, 2014; Park et al,
2006),
we
measured
mitochondrial
respiration
in
digitonin-
permeabilized cells, where mitochondria are directly accessible
to substrates. In line with what has been previously reported
(Gandhi et al, 2009; Morais et al, 2009), we found that ADP-
stimulated glutamate-supported respiration (state 3) was signifi-
cantly reduced in cells lacking PINK1 (Fig S2A). State 3/basal (state
4) respiration ratio, also known as respiratory control ratio (RCR),
was reduced (Fig S2B). USP8 down-regulation did not perturb
mitochondrial respiration per se; however, it corrected the respi-
ration defects of the PINK1-deficient cells (Fig S2A and B). In cells https://doi.org/10.26508/lsa.201900392
vol 2 | no 2 | e201900392 Protective effect of USP8 inhibition
von Stockum et al. 5 of 16 mitochondrial function (Fig S2C–E). Protective effect of USP8 inhibition
von Stockum et al. https://doi.org/10.26508/lsa.201900392
vol 2 | no 2 | e201900392
6 USP8 down-regulation rescues mitochondria defects of PINK1 KO
flies (A) Representative traces of oxygen consumption of intact
isolated mitochondria extracted from flies of the indicated
genotype and subjected to 10 mM/5 mM pyruvate/malate
200 μM ADP, 2 μg/ml oligomycin, and 200 nM FCCP, 2 μM
antimycinA, respectively. Representative of n = 5. (B)
Quantitative analysis of respiratory fitness of isolated
mitochondria extracted from flies of the indicated genotype
treated as in (A). Graph shows mean ± SEM (n = 5 independent
experiments) of RCR relative to ctrl. One-way ANOVA, P =
0.0074 (**); Tukey’s multiple comparison test; n = 5. (C)
Representative traces of oxygen consumption of intact
isolated mitochondria extracted from flies of the indicated
genotype and subjected to 10 mM/5 mM pyruvate/malate
200 μM ADP, 2 μg/ml oligomycin, and 200 nM FCCP, 2 μM
antimycinA, respectively. Representative of n = 5. (D)
Quantitative analysis of respiratory fitness of isolated
mitochondria extracted from flies of the indicated genotype
treated as in (G). Graph shows mean ± SEM (n = 5
independent experiments) of RCR relative to ctrl. One-way
ANOVA, P = 0.0064 (**); Tukey’s multiple comparison test; n =
5. (E) Blue Native PAGE of mitochondrial extracts from flies of
the indicated genotypes. Respiratory complexes were
separated in a non-denaturing polyacrylamide gel. Representative of n = 3. (F) Densitometric analysis of (E). Graph bar shows mean ± SEM of ratio between densitometric
levels of complex I (CI) and those of complex V (CV). One-way
ANOVA, P = 0.0282 (**); Tukey’s multiple comparison test; n =
3. (G) Graph shows mean ± SEM (n = 4 independent
experiments) of complex I activity relatively to citrate
synthase (CS) activity in isolated 2.5 μM alamethicin-treated
mitochondria extracted from flies of the indicated genotype. One-way ANOVA, P = 0.0012 (**); Tukey’s multiple comparison
test; n = 4. (H) Graph shows mean ± SEM (n = 7 independent
experiments) of complex I activity relatively to CS activity in
isolated 2.5 μM alamethicin-treated mitochondria extracted
from flies of the indicated genotype. One-way ANOVA, P <
0.0001 (****); Tukey’s multiple comparison test; n = 7. PINK1-null mutant males are sterile, as a consequence of
spermatogenesis defects deriving from mitochondrial dysfunction
(Clark et al, 2006; Deng et al, 2008; Greene et al, 2003; Park et al,
2006). Of note, heterozygous USP8 gene deletion favours the re-
storing of sperm production of the PINK1 KO, rescuing male sterility
(Fig S3). USP8 down-regulation rescues mitochondria defects of PINK1 KO
flies As expected, glutamate-supported ADP-stimulated
respiration was reduced, resulting in lower RCR in isolated PINK1 KO
mitochondria (Gandhi et al, 2009; Morais et al, 2009) (Fig 4A–D). On the other hand, USP8 RNAi (Fig 4A and B) or heterozygous USP8
gene deletion (Fig 4C and D) in PINK1 KO flies normalized ADP-
stimulated respiration and RCR. Blue Native PAGE (BN-PAGE) of mitochondrial extracts lent
further biochemical support to the measured functional amelio-
ration. Extracts from PINK1-deficient flies displayed reduced levels
of respiratory complex I, which was corrected by heterozygous
deletion of USP8 (Fig 4E and F). PINK1 mutants show reduced
enzymatic activity of complex I (Morais et al, 2014; Pogson et al,
2014). Both USP8 fly lines (USP8+/−and USP8 RNAi) restored
complex I activity of PINK1 mutants (Fig 4G and H). https://doi.org/10.26508/lsa.201900392
vol 2 | no 2 | e201900392
6 of 16 Protective effect of USP8 inhibition
von Stockum et al. 6 of 16 PINK1-null mutant males are sterile, as a consequence of
a large amount PINK1-null mutant males are sterile, as a consequence of
spermatogenesis defects deriving from mitochondrial dysfunction
(Clark et al, 2006; Deng et al, 2008; Greene et al, 2003; Park et al,
2006). Of note, heterozygous USP8 gene deletion favours the re-
storing of sperm production of the PINK1 KO, rescuing male sterility
(Fig S3). The seminal vesicles of Ctrl and USP8−/+ males were well
developed, swollen, and brownish in color (Fig S3A and B), whereas
those of PINK1 KO were reduced in volume and more transparent
(Fig S3C). Puncturing the vesicles of Ctrl and USP8−/+ males released
a large amount of sperm (Fig S3E and F), whereas sperm was almost
absent in PINK1 KO vesicles (Fig S3G). Rescued males (PINK1 KO,
USP8−/+) showed an intermediate pattern, with swollen, opaque
vesicles (Fig S3D) releasing some sperm groups (Fig S3H). The
fluorescence staining revealed a difference among the four male
groups also in the accessory glands’ wall, whose cells appeared
alive (green) in ctrl and USP8−/+ males (Fig S3I and J) and dead (red)
in PINK1 KO (Fig S3K). In rescued males (PINK1 KO, USP8−/+), part of
the accessory glands’ cells was alive (Fig S3L). The result of
Figure 4. USP8 down-regulation rescues PINK1-deficient
mitochondria respiratory defects ex vivo. g
g
mitochondria respiratory defects ex vivo.
(A) Representative traces of oxygen consumption of intact
isolated mitochondria extracted from flies of the indicated
genotype and subjected to 10 mM/5 mM pyruvate/malate
200 μM ADP, 2 μg/ml oligomycin, and 200 nM FCCP, 2 μM
antimycinA, respectively. Representative of n = 5. (B)
Quantitative analysis of respiratory fitness of isolated
mitochondria extracted from flies of the indicated genotype
treated as in (A). Graph shows mean ± SEM (n = 5 independent
experiments) of RCR relative to ctrl. One-way ANOVA, P =
0.0074 (**); Tukey’s multiple comparison test; n = 5. (C)
Representative traces of oxygen consumption of intact
isolated mitochondria extracted from flies of the indicated
genotype and subjected to 10 mM/5 mM pyruvate/malate
200 μM ADP, 2 μg/ml oligomycin, and 200 nM FCCP, 2 μM
antimycinA, respectively. Representative of n = 5. (D)
Quantitative analysis of respiratory fitness of isolated
mitochondria extracted from flies of the indicated genotype
treated as in (G). Graph shows mean ± SEM (n = 5
independent experiments) of RCR relative to ctrl. One-way
ANOVA, P = 0.0064 (**); Tukey’s multiple comparison test; n =
5. (E) Blue Native PAGE of mitochondrial extracts from flies of
the indicated genotypes. Respiratory complexes were
separated in a non-denaturing polyacrylamide gel.
Representative of n = 3. (F) Densitometric analysis of (E).
Graph bar shows mean ± SEM of ratio between densitometric
levels of complex I (CI) and those of complex V (CV). One-way
ANOVA, P = 0.0282 (**); Tukey’s multiple comparison test; n =
3. (G) Graph shows mean ± SEM (n = 4 independent
experiments) of complex I activity relatively to citrate
synthase (CS) activity in isolated 2.5 μM alamethicin-treated
mitochondria extracted from flies of the indicated genotype.
One-way ANOVA, P = 0.0012 (**); Tukey’s multiple comparison
test; n = 4. (H) Graph shows mean ± SEM (n = 7 independent
experiments) of complex I activity relatively to CS activity in
isolated 2.5 μM alamethicin-treated mitochondria extracted
from flies of the indicated genotype. One-way ANOVA, P <
0.0001 (****); Tukey’s multiple comparison test; n = 7. USP8 down-regulation rescues mitochondria defects of PINK1 KO
flies (A) Representative traces of oxygen consumption of intact
isolated mitochondria extracted from flies of the indicated
genotype and subjected to 10 mM/5 mM pyruvate/malate
200 μM ADP, 2 μg/ml oligomycin, and 200 nM FCCP, 2 μM
antimycinA, respectively. Representative of n = 5. (B)
Quantitative analysis of respiratory fitness of isolated
mitochondria extracted from flies of the indicated genotype
treated as in (A). Graph shows mean ± SEM (n = 5 independent
experiments) of RCR relative to ctrl. One-way ANOVA, P =
0.0074 (**); Tukey’s multiple comparison test; n = 5. (C)
Representative traces of oxygen consumption of intact
isolated mitochondria extracted from flies of the indicated
genotype and subjected to 10 mM/5 mM pyruvate/malate
200 μM ADP, 2 μg/ml oligomycin, and 200 nM FCCP, 2 μM
antimycinA, respectively. Representative of n = 5. (D)
Quantitative analysis of respiratory fitness of isolated
mitochondria extracted from flies of the indicated genotype
treated as in (G). Graph shows mean ± SEM (n = 5
independent experiments) of RCR relative to ctrl. One-way
ANOVA, P = 0.0064 (**); Tukey’s multiple comparison test; n =
5. (E) Blue Native PAGE of mitochondrial extracts from flies of
the indicated genotypes. Respiratory complexes were
separated in a non-denaturing polyacrylamide gel. Representative of n = 3. (F) Densitometric analysis of (E). Graph bar shows mean ± SEM of ratio between densitometric
levels of complex I (CI) and those of complex V (CV). One-way
ANOVA, P = 0.0282 (**); Tukey’s multiple comparison test; n =
3. (G) Graph shows mean ± SEM (n = 4 independent
experiments) of complex I activity relatively to citrate
synthase (CS) activity in isolated 2.5 μM alamethicin-treated
mitochondria extracted from flies of the indicated genotype. One-way ANOVA, P = 0.0012 (**); Tukey’s multiple comparison
test; n = 4. (H) Graph shows mean ± SEM (n = 7 independent
experiments) of complex I activity relatively to CS activity in
isolated 2.5 μM alamethicin-treated mitochondria extracted
from flies of the indicated genotype. One-way ANOVA, P <
0.0001 (****); Tukey’s multiple comparison test; n = 7. P
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f 16 Figure 4. USP8 down-regulation rescues PINK1-deficient
mitochondria respiratory defects ex vivo. mitochondria respiratory defects ex vivo. Protective effect of USP8 inhibition
von Stockum et al. Pharmacological inhibition of USP8 corrects PINK1-deficient flies The genetic experiments showed that USP8 inhibition amelio-
rates all the phenotypes that we tested that are associated to
Drosophila PINK1 KO. We, therefore, decided to test in vivo the
effect of DUBs-IN-2 (ChemScene LLC), a potent and membrane-
permeant USP8 drug inhibitor. DUBs-IN-2 is highly selective for
USP8 with a half maximal inhibitory concentration (IC50) of 0.28
μM (Colombo et al, 2010) and small or no effect on USP7 (IC50 >
100 μM for USP7). The compound has been described as an
inhibitor of human USP8, which shares about ~45% sequence
homology to the fly ortholog. DUBs-IN-2 was mixed in the fly food with the food-coloring patent blue V (E131) to monitor drug
ingestion (Fig S4A). Increasing inhibitor concentrations did not
affect the food uptake of flies as measured by E131 absorbance in
fly lysates (Fig S4B) and did not affect locomotor behavior in a
control background (Fig S4C). Remarkably, DUBs-IN-2 adminis-
tered to adult PINK1-deficient flies significantly suppressed the
locomotor deficits (Fig 5A). Dose–response curve indicated the
best rescue of PINK1 KO climbing performance upon 10 μM DUBs-
IN-2 administration (Fig S4C). DUBs-IN-2 administration to PINK1
KO flies also prevented loss of DA neurons (Fig 5B and C), re-
stored dopamine levels (Fig 5D), and it modestly ameliorated
longevity (Fig 5E). fluorescence staining proves that the effect is not limited to sperm
production, but it is also extended to the functionality of the ac-
cessory glands, that play a crucial role on both male fertilization
success and female fertility (Simmons & Fitzpatrick, 2012). fluorescence staining proves that the effect is not limited to sperm
production, but it is also extended to the functionality of the ac-
cessory glands, that play a crucial role on both male fertilization
success and female fertility (Simmons & Fitzpatrick, 2012). Taken together, these analyses show that the mitochondrial-
defective phenotype of PINK1 KO flies can be recovered by de-
creasing USP8 expression, including complex I levels and activity. USP8 down-regulation rescues mitochondria defects of PINK1 KO
flies The seminal vesicles of Ctrl and USP8−/+ males were well
developed, swollen, and brownish in color (Fig S3A and B), whereas
those of PINK1 KO were reduced in volume and more transparent
(Fig S3C). Puncturing the vesicles of Ctrl and USP8−/+ males released a large amount of sperm (Fig S3E and F), whereas sperm was almost
absent in PINK1 KO vesicles (Fig S3G). Rescued males (PINK1 KO,
USP8−/+) showed an intermediate pattern, with swollen, opaque
vesicles (Fig S3D) releasing some sperm groups (Fig S3H). The
fluorescence staining revealed a difference among the four male
groups also in the accessory glands’ wall, whose cells appeared
alive (green) in ctrl and USP8−/+ males (Fig S3I and J) and dead (red)
in PINK1 KO (Fig S3K). In rescued males (PINK1 KO, USP8−/+), part of
the accessory glands’ cells was alive (Fig S3L). The result of a large amount of sperm (Fig S3E and F), whereas sperm was almost
absent in PINK1 KO vesicles (Fig S3G). Rescued males (PINK1 KO,
USP8−/+) showed an intermediate pattern, with swollen, opaque
vesicles (Fig S3D) releasing some sperm groups (Fig S3H). The
fluorescence staining revealed a difference among the four male
groups also in the accessory glands’ wall, whose cells appeared
alive (green) in ctrl and USP8−/+ males (Fig S3I and J) and dead (red)
in PINK1 KO (Fig S3K). In rescued males (PINK1 KO, USP8−/+), part of
the accessory glands’ cells was alive (Fig S3L). The result of https://doi.org/10.26508/lsa.201900392
vol 2 | no 2 | e201900392
7 of 16 Protective effect of USP8 inhibition
von Stockum et al. 7 of 16 USP8 down-regulation corrects pathologically elevated MFN
levels of PINK1 and Parkin KO flies PINK1 loss-of-function results in increased MFN protein levels
(Tanaka et al, 2010; Ziviani et al, 2010), altered mitochondrial
morphology (Mortiboys et al, 2008; Narendra et al, 2008; Tanaka et
al, 2010; Ziviani et al, 2010), impaired mitophagy (Gegg et al, 2010; Protective effect of USP8 inhibition
von Stockum et al. https://doi.org/10.265 Figure 5. Pharmacological USP8 inhibition corrects DA
neuron loss, life span, muscle degeneration, and
locomotor impairment of PINK1-deficient flies. Figure 5.
Pharmacological USP8 inhibition corrects DA
neuron loss, life span, muscle degeneration, and
locomotor impairment of PINK1-deficient flies. (A) Graph bar shows mean ± SEM of the climbing
performance of 3-d-old flies of the indicated genotype or
treated with DUBs-IN-2 or DMSO for 48 h from at least
four independent experiments. Two-way ANOVA P <
0.0001 (****); Tukey’s multiple comparison test; n = 8. (B)
Whole brains of 15-d-old male flies of the indicated
genotypes or treated with DUBs-IN-2 for 15 d were
immunostained with anti-TH antibody. Panel shows
(projection, Z stack) confocal images of PPL1 cluster DA
neurons of the indicated genotypes. Representative of n
= 9. (C) Bar graph shows the number of PPL1 cluster DA
neurons in brains of the indicated genotypes treated
with DUBs-IN-2 or DMSO for 15 d. Two-way ANOVA, P =
0.0004 (***); Tukey’s multiple comparison test; n = 15. (D)
Relative dopamine amount from 15 d old adult heads of
the indicated genotype treated with DUBs-IN-2 or DMSO
for 15 d normalized to control flies. Two-way ANOVA P =
0.0217 (*); Tukey’s multiple comparison test; n = 3. (E) Life
span analysis of male flies of the indicated genotypes
treated with DUBs-IN-2 or DMSO. At least 100 flies were
used for the analysis. Log-rank, Mantel–Cox test (Ctrl
versus PINK1 KO P < 0.0001; Ctrl versus PINK1 KO+DUBs-
IN-2, P < 0.0001; Ctrl versus Ctrl+DUBs-IN-2 P > 0.05; PINK1
KO versus PINK1 KO+DUBs-IN-2 P < 0.001; PINK1 KO versus
Ctrl+DUBs-IN-2 P < 0.0001; and PINK1 KO+DUBs-IN-2
versus Ctrl+DUBs-IN-2 P < 0.0001). Protective effect of USP8 inhibition
von Stockum et al. Protective effect of USP8 inhibition
von Stockum et al. https://doi.org/10.26508/lsa.201900392
vol 2 | no 2 | e201900392
8 of 16 8 of 16 Narendra et al, 2008; Ziviani et al, 2010), and oxidative phosphor-
ylation (Morais et al, 2009, 2014), with mitochondrial Ca2+ overload
and increased reactive oxygen species production (Gandhi et al,
2009). Similar phenotypes are caused by altered MFN, which
prompted us to investigate whether USP8 down-regulation cor-
rected pathologically elevated MFN levels of PINK1 KO flies. Indeed,
USP8 down-regulation in vivo completely normalized increased
MFN levels of PINK1 KO (Fig 6A). Pharmacological inhibition of USP8
also led to reduced PINK1 KO MFN protein levels in flies, indicating
that the inhibitor phenocopied genetic inhibition of USP8 (Fig 6B). Like PINK1, Parkin KO/KD also results in increased MFN protein
levels (Gegg et al, 2010; Tanaka et al, 2010; Ziviani et al, 2010). Discussion Interventions that decrease MFN levels in PINK1 or Parkin KO flies
can ameliorate the multiple phenotypes that are associated with
the KO backgrounds (Deng et al, 2008; Poole et al, 2008; Liu et al,
2011; Vilain et al, 2012; Celardo et al, 2016). We, therefore, conducted
an RNAi-based screening to identify DUBs that regulate MFN protein
levels. We found USP8, a DUB previously identified in the regulation
of endosomal trafficking (Mizuno et al, 2005; Row et al, 2006), CCCP-
induced mitophagy (Durcan et al, 2014) and basal autophagy
(Jacomin et al, 2015), and which down-regulation is protective from Figure 6. USP8 down-regulation corrects pathologically elevated MFN levels of PINK1 and Parkin KO flies. (A) Equal amounts of protein (70 μg), isolated from flies of the indicated phenotype were separated by SDS–PAGE and immunoblotted using the indicated antibodies. Representative of n = 6. Graph bar shows mean ± SEM of ratio between densitometric levels of MFN and those of Actin from at least six independent experiments. One-way
ANOVA, P = 0.0003 (***); Tukey’s multiple comparison test; n = 6. (B) Equal amounts of protein (70 μg), isolated from flies treated with DUBs-IN-2 or DMSO for 48 h were
separated by SDS–PAGE and immunoblotted using the indicated antibodies. Representative of n = 3. Graph bar shows mean ± SEM of ratio between densitometric levels of
MFN and those of Actin from at least three independent experiments. Two-way ANOVA, P < 0.00001 (****); Tukey’s multiple comparison test; n = 3. (C) Equal amounts of
protein (70 μg), isolated from flies of the indicated phenotype were separated by SDS–PAGE and immunoblotted using the indicated antibodies. Representative of n = 9. Graph bar shows mean ± SEM of ratio between densitometric levels of MFN and those of Actin from at least nine independent experiments. One-way ANOVA, P < 0.0001
(****); Tukey’s multiple comparison test; n = 9. (D) TEM images of thorax muscles from flies of the indicated genotypes. Thoraces were dissected from 3-d-old adult flies and
fixed in 2% paraformaldehyde and 2.5% gluteraldehyde. The samples were rinsed, dehydrated, and embedded using Epon. Ultrathin sections were examined using TEM. Representative of n = 3. (E) Bar graph shows the number of DA neurons in the PPL1 cluster of the brains of the indicated genotypes. One-way ANOVA, P < 0.0001 (****);
Tukey’s multiple comparison test; n = 10. Discussion One-way ANOVA, P < 0.0001 (****); n = 3. (G) Graph bar shows mean ± SEM of the climbing performance of flies of the indicated genotype from at least three
independent experiments. Two-way ANOVA, P < 0.0001 (****); Tukey’s multiple comparison test; n = 7. Figure 6. USP8 down-regulation corrects pathologically elevated MFN levels of PINK1 and Parkin KO flies. (A) Equal amounts of protein (70 μg), isolated from flies of the indicated phenotype were separated by SDS–PAGE and immunoblotted using the indicated antibodies. Representative of n = 6. Graph bar shows mean ± SEM of ratio between densitometric levels of MFN and those of Actin from at least six independent experiments. One-way
ANOVA, P = 0.0003 (***); Tukey’s multiple comparison test; n = 6. (B) Equal amounts of protein (70 μg), isolated from flies treated with DUBs-IN-2 or DMSO for 48 h were
separated by SDS–PAGE and immunoblotted using the indicated antibodies. Representative of n = 3. Graph bar shows mean ± SEM of ratio between densitometric levels of
MFN and those of Actin from at least three independent experiments. Two-way ANOVA, P < 0.00001 (****); Tukey’s multiple comparison test; n = 3. (C) Equal amounts of
protein (70 μg), isolated from flies of the indicated phenotype were separated by SDS–PAGE and immunoblotted using the indicated antibodies. Representative of n = 9. Graph bar shows mean ± SEM of ratio between densitometric levels of MFN and those of Actin from at least nine independent experiments. One-way ANOVA, P < 0.0001
(****); Tukey’s multiple comparison test; n = 9. (D) TEM images of thorax muscles from flies of the indicated genotypes. Thoraces were dissected from 3-d-old adult flies and
fixed in 2% paraformaldehyde and 2.5% gluteraldehyde. The samples were rinsed, dehydrated, and embedded using Epon. Ultrathin sections were examined using TEM. Representative of n = 3. (E) Bar graph shows the number of DA neurons in the PPL1 cluster of the brains of the indicated genotypes. One-way ANOVA, P < 0.0001 (****);
Tukey’s multiple comparison test; n = 10. (F) Graph bar shows mean ± SEM of the climbing performance of flies of the indicated genotype from at least three independent
experiments. One-way ANOVA, P < 0.0001 (****); n = 3. (G) Graph bar shows mean ± SEM of the climbing performance of flies of the indicated genotype from at least three
independent experiments. Figure 5.
Pharmacological USP8 inhibition corrects DA
neuron loss, life span, muscle degeneration, and
locomotor impairment of PINK1-deficient flies. We,
therefore, assessed the effect of USP8 KD in a Parkin loss-of-
function model of pathologically elevated MFN levels. USP8 KD
corrected elevated MFN levels of Parkin KO flies (Fig 6C). It also
recovered the disorganized muscle fibers with irregular arrange-
ment of myofibrils and the swollen mitochondria of Parkin flies (Fig 6D), and normalized the number of DA neurons that are de-
creased in Parkin KO background (Fig 6E). Interestingly, USP8 KD or
inhibition did not correct climbing defects in Parkin KO flies (Fig 6F),
nor in PINK1:Parkin double KO (Fig 6G). Discussion (F) Graph bar shows mean ± SEM of the climbing performance of flies of the indicated genotype from at least three independent
experiments. One-way ANOVA, P < 0.0001 (****); n = 3. (G) Graph bar shows mean ± SEM of the climbing performance of flies of the indicated genotype from at least three
independent experiments. Two-way ANOVA, P < 0.0001 (****); Tukey’s multiple comparison test; n = 7. Figure 6. USP8 down-regulation corrects pathologically elevated MFN levels of PINK1 and Parkin KO flies. Figure 6. USP8 down-regulation corrects pathologically elevated MFN levels of PINK1 and Parkin KO flies. (A) Equal amounts of protein (70 μg), isolated from flies of the indicated phenotype were separated by SDS–PAGE and immunoblotted using the indicated antibodies. Representative of n = 6. Graph bar shows mean ± SEM of ratio between densitometric levels of MFN and those of Actin from at least six independent experiments. One-way
ANOVA, P = 0.0003 (***); Tukey’s multiple comparison test; n = 6. (B) Equal amounts of protein (70 μg), isolated from flies treated with DUBs-IN-2 or DMSO for 48 h were
separated by SDS–PAGE and immunoblotted using the indicated antibodies. Representative of n = 3. Graph bar shows mean ± SEM of ratio between densitometric levels of
MFN and those of Actin from at least three independent experiments. Two-way ANOVA, P < 0.00001 (****); Tukey’s multiple comparison test; n = 3. (C) Equal amounts of
protein (70 μg), isolated from flies of the indicated phenotype were separated by SDS–PAGE and immunoblotted using the indicated antibodies. Representative of n = 9. Graph bar shows mean ± SEM of ratio between densitometric levels of MFN and those of Actin from at least nine independent experiments. One-way ANOVA, P < 0.0001
(****); Tukey’s multiple comparison test; n = 9. (D) TEM images of thorax muscles from flies of the indicated genotypes. Thoraces were dissected from 3-d-old adult flies and
fixed in 2% paraformaldehyde and 2.5% gluteraldehyde. The samples were rinsed, dehydrated, and embedded using Epon. Ultrathin sections were examined using TEM. Representative of n = 3. (E) Bar graph shows the number of DA neurons in the PPL1 cluster of the brains of the indicated genotypes. One-way ANOVA, P < 0.0001 (****);
Tukey’s multiple comparison test; n = 10. (F) Graph bar shows mean ± SEM of the climbing performance of flies of the indicated genotype from at least three independent
experiments. Discussion USP8 KD also
prevented Parkin KO DA neurons loss and normalized mitochon-
drial morphological defects, although it did not ameliorate Parkin
climbing performance (Fig 6). It has been shown that the knockdown of MFN is able to rescue
the mitochondrial defects and the overall phenotypes of Dro-
sophila PINK1 KO flies (Deng et al, 2008; Poole et al, 2008). More
recently, it was shown that MFN knockdown can suppress loss of DA
neurons of the PPL1 cluster and thorax deformation resulting from
crushed thoracic muscle of the PINK1 KO flies, but not the mito-
chondrial defects (Celardo et al, 2016). We found that normalizing
MFN levels of PINK1 KO flies by driving efficient whole body MFN KD
(Debattisti et al, 2014) ameliorated the disorganized muscle fibers
and mitochondria ultrastructure of PINK1 KO flies, but dopamine
content and climbing performance were only modestly recovered,
even if MFN levels of PINK1 KO flies were completely corrected (Fig
S5). This result indicates that MFN normalization deriving from USP8
KD likely contributes to the amelioration of the PINK1 phenotype
but does not explain the full recovery of the multiple phenotypes
that are associated with PINK1 loss. Indeed, our in vivo analysis
indicates that USP8 KD has a broader protective effect than MFN KD
and unlike MFN KD (Celardo et al, 2016; Vilain et al, 2012), it cor-
relates with full correction of mitochondrial respiratory defects,
complex I content and activity, and mitochondrial membrane
potential of PINK1 KO flies (Figs 4 and S2). Previous examination of
the PINK1-mutant phenotype demonstrated that although de-
creasing mitochondrial fusion rescues morphological mitochon-
drial defects of PINK1 flies, manipulation of mitochondrial fusion (or
fission) does not rescue other PINK1-related phenotypes such as
the reduced activity of complex I, loss of mitochondrial membrane
potential, ATP content, and defective neurotransmitter release
(Vilain et al, 2012; Vos et al, 2012). In light of this, we hypothesize that
the protective effect of USP8 inhibition comes from a combination
of signaling pathways, which directly or indirectly impinges on MFN
levels and mitochondrial function. In mammals, it is established
that USP8 is involved in endosomal trafficking (Clague et al, 2013),
although its activity can have opposing effects. Discussion Two-way ANOVA, P < 0.0001 (****); Tukey’s multiple comparison test; n = 7. Protective effect of USP8 inhibition
von Stockum et al. https://doi.org/10.26508/lsa.201900392
vol 2 | no 2 | e201900392
9 of 16 9 of 16 mitochondria and to promote stress-induced mitophagy in vitro
(Durcan et al, 2014). Thus, USP8 down-regulation in this context inhibits
Parkin recruitment to mitochondria, causing a delay in mitochondria
clearance by mitophagy. In light of these seemingly opposing phe-
notypic outcomes, it is clear that USP8 has pleiotropic effects that
depends on the specific genetic repertoire of the cell/tissue, varies in
response to physiological versus pathological conditions, or might
simply operate differently in cell lines versus the whole organism. Our model is consistent with a role of USP8 in controlling mi-
tochondrial
function
via
Parkin-independent
regulation
of
pathologically elevated MFN protein levels. Yet, it does not ex-
clude MFN-unrelated pathways that nevertheless impinge on
mitochondrial function via Parkin, like the mitochondrial-derived
vesicle pathway regulating mitochondria quality control
(McLelland et al, 2014), or the endosomal–lysosomal pathway that
can also play a role in selective degradation of dysfunctional
mitochondria (Hammerling et al, 2017a, 2017b). Interestingly, it was
shown in the latter that the autophagic activity is increased when
the endosomal activity is impaired, sustaining the hypothesis that
there is crosstalk between the various degradation pathways to
ensure effective clearance. It is tempting to hypothesis an en-
hancement of autophagy deriving from USP8 KD to complement
for impaired endosomal-mediated quality control. For these
reasons, future studies need to be conducted in vivo to validate
this hypothesis and clearly dissect coordination and timing of
activation of these pathways in different tissues under physio-
logical and pathological conditions. α-synuclein–induced locomotor deficits in flies (Alexopoulou et al,
2016). Our data show that inhibition of USP8 in vitro and in vivo
correlated with decreased mitochondrial fusion protein MFN, one
of the bona fide Parkin targets (Gegg et al, 2010; Poole et al, 2010;
Tanaka et al, 2010; Ziviani et al, 2010; Sarraf et al, 2013) (Fig 1),
ameliorated PINK1 KO phenotypes in vivo (Figs 2, 3, and 5) and PINK1
KO mitochondrial dysfunction (Fig 4), and corrected MFN protein
levels, increased in PINK1 KO models (Fig 6). Interestingly, USP8 KD
also corrected MFN protein levels of Parkin KO flies, indicating that
the effect on the levels of MFN is Parkin independent. Discussion For instance,
deubiquitination by USP8 was reported to slow the degradation of
substrates (Mizuno et al, 2005; Mukai et al, 2010), but also to fa-
cilitate endosomal trafficking and lysosomal degradation (Row et
al, 2007; Ali et al, 2013). shRNA against USP8 in SH-SY5Y neuro-
blastoma cells promotes α-synuclein degradation by the lysosome,
which exerts a protective effect in vivo in an α-synuclein fly model
of PD (Alexopoulou et al, 2016). It was also reported that USP8 is
required for lysosomal biogenesis and productive autophagy in
Drosophila larval fat body but inhibits basal autophagy in vitro in
HeLa cells (Jacomin et al, 2015). Finally, deubiquitination of Parkin
by USP8 is required for Parkin recruitment to CCCP-intoxicated Because of their involvement in the regulation of important
signaling pathways, DUBs are emerging as extremely attractive
druggable candidates (Sugiura et al, 2013). In recent years, many
DUBs emerged as therapeutic targets to compensate for impaired
mitophagy in PD (Bingol et al, 2014; Cornelissen et al, 2014; Wang et
al, 2015; Chakraborty et al, 2018). Mitophagy is triggered by ubiquitin
modification of mitochondrial proteins, which is in principle
subject to suppression by deubiquitination. It is, therefore, rea-
sonable that inhibition of specific DUBs should induce mitophagy
and that it does so by deubiquitination mitochondrial proteins. Clinical trials for specific inhibitors of the ubiquitin–proteosome
system have already been approved in cancer therapy for the
treatment of multiple myeloma (Colland, 2010). Moreover, high-
throughput screening of small chemical libraries identified non-
selective DUB inhibitors as potent inducers of apoptosis in various
cancer cells (Liu et al, 2003; Brancolini, 2008; Engels et al, 2009;
Hussain et al, 2009; Py et al, 2013). Similarly, specific DUB inhibitors
(or activators) can affect cellular response to stimuli that induce
cell death. In this respect, the identification of a specific DUB that
normalizes mitochondrial function might be instrumental to de-
velop specific isopeptidase inhibitors that can modulate the
fundamental biological process of mitochondria physiology and
fitness, supporting the potential of USP8 inhibitors as therapeutics. Protective effect of USP8 inhibition
von Stockum et al. Cell culture and transfection Drosophila S2R+ cells were cultured in Schneider’s medium
(Invitrogen) supplemented with 10% heat-inactivated fetal calf
serum (Sigma-Aldrich). The cells were maintained at 25°C and https://doi.org/10.26508/lsa.201900392
vol 2 | no 2 | e201900392
10 of 16 10 of 16 passaged routinely before they reached confluence, to maintain a
logarithmic growth. The cells were transfected using TransFectin
lipid reagent (Bio-Rad) or Effectene (QIAGEN) following the man-
ufacturer’s instructions. In brief, 0.6 million cells were plated in six-
well plate and transfected with 2 μg DNA/5 μl TransFectin or 1 μg
DNA/10 μl Effectene + 8 μl Enhancer, 1 d after plating. The cells were
collected 24–48 h after transfection. 500 μM copper sulfate solution
was added to the cells to induce plasmid expression when
required. under ambient conditions on an Andromeda iMIC spinning disk live
cell microscope with confocal resolution (TILL Photonics, 60X ob-
jective). For confocal z-axis stacks, 40 images separated by 0.2 μm
along the z-axis were acquired. For measurements of mitochondrial membrane potential, the
cells were loaded with 25 nM tetramethylrhodamine methyl ester
(TMRM) for 30 min at room temperature, and the dye was present
during the experiment together with the multidrug resistance in-
hibitor cyclosporine H (1 μM). The cells were then observed using an
Olympus IX81 inverted microscope equipped with a cell imaging
system. Sequential images of TMRM fluorescence were acquired
every 60 s with a 40× objective (Olympus). Where indicated,
oligomycin (2.5 μg/ml; Sigma-Aldrich) or the uncoupler carbonyl
cyanide p-trifluoromethoxyphenylhydrazone (CCCP, 10 μM; Sigma-
Aldrich) was added. TMRM fluorescence analysis over the mito-
chondrial regions of interest was performed using ImageJ. A
reduction in TMRM fluorescence represents mitochondrial mem-
brane depolarization. In the graph bars, we indicated TMRM fluo-
rescence after 30-min oligomycin administration in the cells of the
indicated genotypes. The cells were always loaded in the presence
of the multidrug resistance inhibitor cyclosporine H. Plasmids MitoDsRed was subcloned from pDsRed2-Mito vector (Clontech)
into pAct-PPA expression plasmid. C-terminal Flag tag MFN was
obtained by amplification from cDNA clone (RE04414) and subcl-
oned into pAct-PPA expression plasmid. CG5798/USP8 was am-
plified from cDNA clone and subcloned into pMt copper-inducible
vector (Invitrogen). Mitochondria morphology analysis Quantification of mitochondria length was performed by using
ImageJ software. To measure mitochondrial length, we created
maximum-intensity projections of z-series with 0.2-μm increments. Quantification was then performed by using “Squassh” (Segmen-
tation and QUAntification of Subcellular SHapes), a plugin com-
patible with the image processing software ImageJ or Fiji, freely
available from http://mosaic.mpi-cbg.de/?q=downloads/imageJ. Squassh is a segmentation method that enables both colocaliza-
tion and shape analyses of subcellular structures in fluorescence
microscopy images (Rizk et al, 2014). For our analysis, segmentation
was performed with the minimum intensity threshold set to 0.15
and the regularization weight to 0.015. Gene silencing Drosophila dsRNA probes were prepared using MEGA script kit
(Ambion) following the manufacturer’s instructions. The fol-
lowing primers have been used to prepare the RNAi probes:
PINK1 CAATGTGACTTCTCCAGCGA and TCGTAGCGTTTCATCAGCAG;
Parkin CTGTTGCAATTTGGAGGGA and CTTTGGCACGGACTCTTTCT;
and MFN GGAACCTCTTTATTCTCTAT and GGTTTGCTTTGCCCCAA-
CAT. CG5798/USP8 dsRNA probe was acquired from the Sheffield
RNAi Screening Facility. 1.2 millions cells were plated on a six-well
plate and treated with 7 μg RNAi probe in serum-free medium. 2 h
after the probe treatment, complete medium was added to the
wells, and the cells were cultured for 2 d before being transfected
with indicated fly expression plasmids as previously described. Total RNA extraction and qRT-PCR Total RNA extraction and qRT PCR
Total RNA was extracted from Drosophila S2R+ cells using TRI
Reagent (Sigma-Aldrich) according to the manufacturer’s in-
structions. The RNA pellet was dissolved in 5–10 μl RNAase-free
water. Total RNA was extracted from approximately 10 flies using
Trizol (Life Technologies) and further purified by precipitation with
LiCl 8M. RNA samples were checked for integrity by capillary
electrophoresis (RNA 6000Nano LabChip; Agilent Technologies). For
each sample, 1 μg of RNA was used for first-strand cDNA synthesis,
using 10 μM deoxynucleotides, 10 μM oligo-dT, and SuperScript II
(Life Technologies). qRT-PCRs were performed in triplicate in a 7500
Real-Time PCR System (Life Technologies) using SYBR Green Immunoblotting Western blotting was performed using standard techniques. In
brief, the cells were collected in lysis buffer (50 mM Tris–HCl, pH 8,
150 mM HCl, 1 mM MgCl2, 2 mM EGTA, 1% Triton X, 10% glycerol, 10
mM NEM, 10 μM MG132 and protease inhibitor cocktail by Roche)
and incubated on ice for 30 min before being centrifuged at
maximum speed at 4°C. Ten to twelve flies were homogenized
using a mortar and pestle in protein extraction buffer (200–300 μl,
150 mM NaCl, 5 mM EDTA, pH 8.0, 50 mM Tris, pH 8.0, 1% NP-40, 0.1%
SDS 0.1, supplemented with 10 μM MG132, 10 mM NEM, and pro-
tease inhibitor cocktail). The following commercial antibodies
were used: anti-Flag (1:1,000; Cell Signaling Technology), anti-
Actin (1:10,000; Chemicon) has been described before. Anti-
Drosophila Mitofusin (1:2000) was raised in rabbit against an
N-terminal peptide, DTVDKSGPGSPLSRF. For detection, secondary
antibodies conjugated with HRP (Chemicon) were used (1:3,000),
and immunoreactivity was visualized with ECL chemiluminescence
(Amersham). The mitochondria morphology score was assigned as in Pogson
et al (2014). Briefly, a morpho score is assigned to each imaged cell
according to the morphology of its mitochondrial network. Num-
bers represent the designated “morphology score”: 0 = cell with a
full complement of mitochondria; 1 = cell with a full complement of
mitochondria and some clumped mitochondria; 2 = cell with a
reduced mitochondrial network and some clumped mitochondria;
3 = cell with a clumped mitochondrial network; and 4 = cell with a
complete clumped mitochondrial network. Protective effect of USP8 inhibition
von Stockum et al. Immunostaining of whole-mounted brains Brains of 15-d-old male control or mutant flies were dissected in
ice-cold PBS and fixed in 4% PFA at room temperature for 20 min. Samples were washed six times for 10 min with PBS + 0.3% Triton
X-100, permeabilized with PBS + 1% Triton X-100 for 10 min, and
blocked with PBS + 0.3% Triton X-100 containing 1% BSA overnight at
4°C. For immunostaining of DA neurons, rabbit anti-TH antibody
(Millipore) diluted 1:100 in PBS + 0.3% Triton X-100 containing 0.3%
BSA was added and incubated over three nights at 4°C. Brains were
washed and blocked again as described above, despite the blocking
this time being carried out at RT for 1 h. The immunoreaction was
revealed with Cy3-conjugated anti-rabbit IgG (Jackson Immuno-
Research) at a working dilution of 1:500 in PBS + 0.3% Triton X-100
containing 0.3% BSA overnight at 4°C. After another six washing
steps, whole brains were mounted with Vectashield (Vector Lab-
oratories). Z-stack images were obtained by a Zeiss LSM700 con-
focal microscope. Drug treatment
h
ifi The specific USP8 inhibitor DUBs-IN-2 (ChemScene LLC) was ad-
ministered to flies in the food. DUBs-IN-2 (or DMSO) was diluted in
water to the desired concentration and used to reconstitute dry
Formula 4-24 Instant Drosophila Medium (Carolina Biological
Supply). 1-d-old male mutant or control flies in groups of 10 were
fed on the supplemented food for 48 h and subsequently climbing
assay was performed. In the case of DA neuron staining and
measurement of dopamine levels, mutant and control flies were
aged for 15 d on the supplemented food that was exchanged every 2
d adding fresh drug or vehicle. The use of non-harmful food col-
oring demonstrated food uptake and excluded the possibility that
smell or taste of the drug prevented the latter. Toxic concentrations
were excluded beforehand by performing dose-dependent viability
curves on control flies. Isolation of mitochondria Mitochondria were extracted from whole flies by differential cen-
trifugation. Each sample was homogenized using a Dounce
glass–glass potter and a loose-fitting pestle in a mannitol–sucrose
buffer (225 mM mannitol, 75 mM sucrose, 5 mM Hepes, and 0.1 mM
EGTA, pH 7.4) supplemented with 2% BSA. The samples were then
centrifuged at 1,500 g at 4°C for 6 min. The pellet was discarded by
filtering the sample through a fine mesh, and the supernatant was
centrifuged at 7,000 g at 4°C for 6 min. The resulting pellet was
resuspended in mannitol–sucrose buffer without BSA before being
centrifuged at 7,000 g under the same conditions as above and
resuspended in a small volume of mannitol–sucrose buffer. Protein
concentration was measured using the biuret test. Drosophila head dopamine amount measurement (HPLC)
Drosophila heads of 15-d-old male flies were dissected out and
collected separately in 10 μl of ice-cold 0.2 N perchloric acid. The
tissue was homogenized by sonication for 15 s and kept on ice for 20
min, then centrifuged at 12,000 g for 10 min, and the supernatant
was collected. The samples were further diluted and 5 μl was in-
jected into a HPLC system equipped with a rheodyne injector and a
guard cell, set to +350 mV (E1 = +150 mV, E2 = −350 mV, s: 2 nA). A C18
ion-pair, reverse phase analytical column (4.6 × 250 mm; 5 μm
particle size; Agilent Technologies) was used for the separation of
biogenic amines with a flow rate of 0.8 ml/min. Composition of the
mobile phase was 75 mM sodium phosphate monobasic mono-
hydrate, 6% acetonitrile, 1.7 mM 1-octane sulfonic acid, and 25 μM
EDTA (pH 3 ± 0.01). Dopamine values were determined by comparing
with the standard peak value. Drosophila stocks and procedures Drosophila were raised under standard conditions at 25°C unless
differently stated on agar, cornmeal, yeast food. park25 mutants
and UAS-Parkin have been described before (Greene et al, 2003). PINK1B9 mutants (Park et al, 2006) were provided by Dr. J Chung
(KAIST). w1118 and Act-GAL4 strains were obtained from the
Bloomington Drosophila Stock Center. UAS-USP8 RNAi and UAS-
Marf RNAi lines were obtained from the VDRC Stock Center. Usp8−/+ and UAS-Usp8 (uspy) lines were kindly provided by S
Goto (Mukai et al, 2010). Climbing assays
Cli
bi Climbing assays were performed as previously described (Greene et
al, 2003). For the climbing assay upon drug treatment, groups of 10
flies were collected and placed into an empty vial (12 × 5 cm) with a
line drawn at 6 cm from the bottom of the tube. The flies were gently
tapped to the bottom of the tube, and the number of flies that
successfully climbed above the 6-cm mark after 10 s was noted. Fifteen separate and consecutive trials were performed for each
experiment, and the results were averaged. At least 40 flies were
tested for each genotype or condition. Data collection and analysis
were performed blind to the conditions of the experiments unless
otherwise indicated. Mitochondrial respiration Rates of mitochondrial respiration were measured using the
Oxytherm System (Hansatech) with magnetic stirring and ther-
mostatic control maintained at 25°C. Isolated Drosophila mito-
chondria (1 mg/ml) were incubated in 120 mM KCl, 5 mM Pi-Tris,
3 mM Hepes, 1 mM EGTA, and 1 mM MgCl2, pH 7.2, and additions were
made as indicated in the figure legends. O2 consumption was
calculated according to the slope of the registered graph and
plotted as ng atoms: O2 × min−1 × mg−1. RCR (ADP-stimulated res-
piration over basal respiration) was calculated. Live imaging Cells were grown on imaging dishes (Chamber Slide Lab-Tek II 8;
Thermo Fisher Scientific) or coverslips. After appropriate treatment,
when indicated, the cells were treated with the selective mito-
chondrial dye Mitotracker (50 nM; Molecular Probe) for 10 min,
washed three times with PBS, and imaged live in growing medium https://doi.org/10.26508/lsa.201900392
vol 2 | no 2 | e201900392
11 of 16 Protective effect of USP8 inhibition
von Stockum et al. 11 of 16 Protective effect of USP8 inhibition
von Stockum et al. chemistry (Promega). The 2−ΔΔCt (RQ, relative quantification) method
implemented in the 7500 Real-Time PCR System software was used to
calculate the relative expression ratio (ref.). The USP8 oligonucleo-
tides primer used were USP8_F (CACCCATTCAAATTGTCGAG) and
USP8_R (TCGATGGTCTCAATGTCGTT). Rp49 was used as endogenous
control and the oligonucleotides used were Rp49 F (ATCGGTTACG-
GATCGAACAA) and R (GACAATCTCCTTGCGCTTCT). chemistry (Promega). The 2−ΔΔCt (RQ, relative quantification) method
implemented in the 7500 Real-Time PCR System software was used to
calculate the relative expression ratio (ref.). The USP8 oligonucleo-
tides primer used were USP8_F (CACCCATTCAAATTGTCGAG) and
USP8_R (TCGATGGTCTCAATGTCGTT). Rp49 was used as endogenous
control and the oligonucleotides used were Rp49 F (ATCGGTTACG-
GATCGAACAA) and R (GACAATCTCCTTGCGCTTCT). Immunostaining of whole-mounted brains Measurement of food uptake Dry Formula 4-24 Instant Drosophila Medium (Carolina Biological
Supply) was reconstituted with a mix of water and food-coloring
patent blue V (E131) (1:1) previously supplemented with DMSO or the
desired DUBs-IN-2 concentration. Three groups of 10 male 1–3-d-
old w1118 flies were kept in the food vials for 48 h. Afterward, the
flies were weighed and homogenized in 20 volumes of PBS with an
electric potter. The homogenate was centrifuged for 10 min at
15.000 g and absorbance of the supernatant was measured at
640 nm. Sperm content and reproductive apparatus viability assay L Locatello: conceptualization, formal analysis, and methodology. L Locatello: conceptualization, formal analysis, and methodology. C Da Rè: conceptualization, data curation, formal analysis, and
methodology. The anatomy of male reproductive apparatus was analyzed on 10
males per group. To this aim, the reproductive apparatus was re-
moved, placed on a slide with few drops of Drosophila Ringer’s
solution (182 mM KCl, 46 mM NaCl, 3 mM CaCl2 2H2O, and 10 mM
Tris–HCl, pH 7.2) and freshly examined under a light microscope. To
verify the presence of sperm inside the seminal vesicles, these were
then removed from the whole apparatus and gently punctured with
a needle to let the sperm pouring out. Five more intact apparatuses
per group were stained with a dead/alive cell viability kit (Molecular
Probes) that allows differentiation between live green cells, per-
meable to green SYBR 14 nucleic acid stain, and red dead cells,
permeable to propidium iodide nucleic acid stain, which penetrates
through compromised membranes. C Da Rè: conceptualization, data curation, formal analysis, and
methodology. P Cusumano: conceptualization and methodology. F Caicci: data curation and methodology. V Ferrari: methodology. R Costa: supervision and writing—review and editing. L Bubacco: conceptualization, data curation, formal analysis, and
writing—review and editing. writing—review and editing. MB Rasotto: conceptualization, data curation, formal analysis, su-
pervision, and methodology. I Szabo: conceptualization, supervision, and writing—review and
editing. I Szabo: conceptualization, supervision, and writing—review and
editing. AJ Whitworth: conceptualization, data curation, formal analysis,
supervision, funding acquisition, methodology, and writing—review
and editing. AJ Whitworth: conceptualization, data curation, formal analysis,
supervision, funding acquisition, methodology, and writing—review
and editing. Life span analysis Male flies of the indicated genotypes were collected during 12 h
after hatching and grouped into 20 flies per food vial. At least 100
flies were used for the analysis (exact numbers are indicated in the
figure legends). The flies were transferred to fresh food (and fresh
drug for the inhibitor treatment) every 2 d, and dead flies were
counted in the same interval. BN PAGE Pellets of mitochondria isolated from adult male flies of the in-
dicated genotypes were suspended at 10 mg × ml−1 in 1× native PAGE
sample buffer (Invitrogen) supplemented with protease inhibitor
mixture (Sigma-Aldrich), solubilized with 2% (wt/vol) digitonin and
immediately centrifuged at 100,000 g for 25 min at 4°C. The
supernatants were supplemented with native PAGE 5% G-250
sample additive (Invitrogen) and quickly loaded onto a blue native
polyacrylamide 3–12% gradient gel (Invitrogen). After electropho-
resis, the gels were fixed in 50% methanol + 10% acetic acid for 20
min at RT, stained in 0,025% Coomassie + 10% acetic acid overnight
at RT and destained with 10% acetic acid. Electron microscopy Thoraces were prepared from 3-d-old adult flies and fixed over-
night in 2% paraformaldehyde and 2.5% gluteraldehyde. After
rinsing in 0.1 M cacodylate buffer with 1% tannic acid, the samples
were postfixed in 1:1 2% OsO4 and 0.2 M cacodylate buffer for 1 h. The
samples were rinsed, dehydrated in an ethanol series, and https://doi.org/10.26508/lsa.201900392
vol 2 | no 2 | e201900392
12 of 16 Protective effect of USP8 inhibition
von Stockum et al. 12 of 16 embedded using Epon. Ultrathin sections were examined using a
transmission electron microscope. embedded using Epon. Ultrathin sections were examined using a
transmission electron microscope. collected and processed simultaneously and, therefore, no ran-
domization was appropriate (GraphPad Prism. ****P < 0.0001, ***P <
0.001, **P < 0.01, and *P < 0.05). Please refer to the enclosed
document for detailed statistical tests. Supplementary Information is available at https://doi.org/10.26508/lsa.
201900392. Supplementary Information is available at https://doi.org/10.26508/lsa. 201900392. Acknowledgements This work was supported by grants from the Italian Ministry of Health
“Ricerca Finalizzata” (GR-2011-02351151), Rita Levi Montalcini “Brain Gain”
program, and Michael J Fox RRIA 2014 (Grant ID 9795) to E Ziviani and by ERC
FP7-282280, FP7 CIG PCIG13-GA-2013-618697, and Italian Ministry of Research
FIRB RBAP11Z3YA_005 to L Scorrano. AJ Whitworth is funded by MRC Core
funding (MC_UU_00015/6). We would like to acknowledge Francesco Boldrin
from the EM facility for the help and technical support. We thank the
Sheffield RNAi Screening Facility, Biomedical Sciences, University of Shef-
field, for providing the RNAi library and reagents used in this study sup-
ported by the Wellcome Trust (grant reference number 084757)” Author Contributions S von Stockum: data curation, formal analysis, validation, investi-
gation, methodology, and writing—review and editing. A Sanchez-martinez: data curation, formal analysis, validation,
investigation, and methodology. A Sanchez-martinez: data curation, formal analysis, validation,
investigation, and methodology. S Corr`a: data curation, formal analysis, methodology, and writing—
review and editing. J Chakraborty: formal analysis and methodology. E Marchesan: data curation and methodology. Protective effect of USP8 inhibition
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described at https://creativecommons.org/
licenses/by/4.0/). Wang X, Winter D, AshrafiG, Schlehe J, Wong YL, Selkoe D, Rice S, Steen J,
LaVoie MJ, Schwarz TL (2011) PINK1 and Parkin target Miro for https://doi.org/10.26508/lsa.201900392
vol 2 | no 2 | e201900392 Protective effect of USP8 inhibition
von Stockum et al. Protective effect of USP8 inhibition
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Corneal Viscoelastic Properties from Finite-Element Analysis of In Vivo Air-Puff Deformation
|
PloS one
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cc-by
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Introduction keratocyte density or the presence of hydrophilic proteoglycans
may result in the viscoelastic failure or abnormal repair [19]. The
demand for measuring biomechanical properties
of
biological tissue in-vivo and non-invasively is high, because
abnormal tissue biomechanics play a key role in a wide range of
diseases. The stress distribution [1] around and stiffness [2] of
tumor tissue largely determine its progression. Biomechanical
properties are also indicative of muscle function [3] and the effects
of disease, wound healing [4], aging or cosmetics [5]. Today, most information regarding available corneal biome-
chanical properties was assessed ex vivo [7,8,9,10], where changes
in the hydration state [9] and other non-physiological conditions
affect the measurement. In vivo approaches to measure corneal biomechanical proper-
ties include stepwise indentation with a cantilever [11]; ultrasonic
[12] and magnetic resonance [13] techniques; corneal optical
coherence elastography [14]; phase-sensitive [15] Optical Coher-
ence
Tomography
(OCT);
and
Brillouin
microscopy
[16]. Drawbacks of several of these techniques include that they only
can be operated at low speed, have a low spatial resolution or
require contact with the patient’s corneal surface. In ophthalmology, ocular biomechanics are essential for basic
research, clinical evaluation, prognosis and treatment. Patholog-
ical weakening of the cornea appears to be responsible for the
corneal bulging, and dramatic visual degradation in keratoconus. Corneal collagen cross-linking is an emerging treatment to
increase corneal stiffness in this disease [6]. Theoretical models
that integrate individual mechanical, geometrical and structural
patient data have the potential to improve clinical outcomes of eye
surgery, but depend largely on the identification of pre-operative
biomechanical parameters. Most frequently only the elastic tissue
properties are evaluated, more specifically the elasticity modulus. However, also time-dependent mechanical properties are expected
to matter, along with active remodeling processes. For example,
the progressive deformation of the cornea (ectasia) occurring in
keratoconus [17] and after some laser refractive procedures [18],
may result from an altered stress distribution of the cornea
inducing viscoelastic deformation until the new steady state is
reached. Also certain treatments such as UV corneal collagen
cross-linking (CXL) likely modify both elastic and viscoelastic
properties. Changes in the degree of collagen interweaving, Studying the dynamic deformation following an air-puff has
recently been proposed in different biomedical areas (skin [5],
bacteria [20], cornea [21], soft tissue tumors [22]) to non-
invasively assess biomechanical properties, but also in other fields
to study chicken embryogenesis [23], fruit firmness [24] or meat
tenderness [25]. Abstract Biomechanical properties are an excellent health marker of biological tissues, however they are challenging to be measured
in-vivo. Non-invasive approaches to assess tissue biomechanics have been suggested, but there is a clear need for more
accurate techniques for diagnosis, surgical guidance and treatment evaluation. Recently air-puff systems have been
developed to study the dynamic tissue response, nevertheless the experimental geometrical observations lack from an
analysis that addresses specifically the inherent dynamic properties. In this study a viscoelastic finite element model was
built that predicts the experimental corneal deformation response to an air-puff for different conditions. A sensitivity
analysis reveals significant contributions to corneal deformation of intraocular pressure and corneal thickness, besides
corneal biomechanical properties. The results show the capability of dynamic imaging to reveal inherent biomechanical
properties in vivo. Estimates of corneal biomechanical parameters will contribute to the basic understanding of corneal
structure, shape and integrity and increase the predictability of corneal surgery. Citation: Kling S, Bekesi N, Dorronsoro C, Pascual D, Marcos S (2014) Corneal Viscoelastic Properties from Finite-Element Analysis of In Vivo Air-Puff
Deformation. PLoS ONE 9(8): e104904. doi:10.1371/journal.pone.0104904
Editor: Craig Boote, Cardiff University, United Kingdom
Received May 1, 2014; Accepted July 15, 2014; Published August 14, 2014 si N, Dorronsoro C, Pascual D, Marcos S (2014) Corneal Viscoelastic Properties from Finite-Element Analysis of In Vivo Air-Puff
9(8): e104904. doi:10.1371/journal.pone.0104904 Received May 1, 2014; Accepted July 15, 2014; Published August 14, 2014 Received May 1, 2014; Accepted July 15, 2014; Published August 14, 2014 Received May 1, 2014; Accepted July 15, 2014; Published August 14, 2014 Copyright: 2014 Kling et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its
Supporting Information files. Funding: Spanish Government FIS2011-25637, European Research Council ERC-2011 AdG-294099 to SM. FPI-BES-2009-024560 Pre-doctoral Fellowship to SK. 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. * Email: susana@io.cfmac.csic.es Sabine Kling, Nandor Bekesi, Carlos Dorronsoro, Daniel Pascual, Susana Marcos*
Instituto de O´ ptica ‘‘Daza de Valde´s’’, Madrid, Spain Sabine Kling, Nandor Bekesi, Carlos Dorronsoro, Daniel Pascual, Susana Marcos
Instituto de O´ ptica ‘‘Daza de Valde´s’’, Madrid, Spain August 2014 | Volume 9 | Issue 8 | e104904 Introduction In most cases the degree of deformation of the
sample is empirically related to mechanical parameters, and the
inherent mechanical parameters of the tissue were rarely retrieved. To our knowledge, only Boyer et al [5] proposed an analytical
estimation of the ‘‘restricted Young’s modulus’’ from experimental
deformation curves in skin. Air puff applanation of the cornea is a frequent technique in
ophthalmology to measure intraocular pressure, yet requiring
correction formulae to account for corneal thickness and stiffness. August 2014 | Volume 9 | Issue 8 | e104904 PLOS ONE | www.plosone.org 1 Corneal Biomechanical Evaluation by Air-Puff Finite Element Modelling Recently, high speed Optical Coherence Tomography [26] and
Scheimpflug imaging systems [27] have been proposed allowing
dynamic imaging of corneal cross-sections during the air-puff
deformation event. Experimental studies confirm that the spatio-
temporal corneal deformation pattern depends on the inherent
mechanical properties, as well as on corneal geometry and
intraocular pressure [21]. were normal patients (35.4 years of age on average) and signed an
informed consent after receiving an explanation regarding the
nature of the study. All protocols are in accordance with the tenets
of the Declaration of Helsinki and had been approved by the
Institutional Review Boards (CSIC Ethics Committee, Bioethics
Subcommittee, Madrid, Spain). Ex vivo porcine corneas were
measured under different hydration (after photosensitizer 0.125%-
riboflavin-20%-dextran instillation), stiffness (after UV collagen
cross-linking), boundary conditions (corneal button, eye globe
ex vivo, eye globe in vivo) and intraocular pressures (IOP) in order
to evaluate the effect of the different parameters on the corneal
deformation pattern. For this study we used a subset of these
experimental data: (1) ex vivo cross-linked pig eye globes (n = 5),
(2) ex vivo human eye globes (n = 5); and (3) human eyes in vivo
(n = 9). Corneal experimental input parameters include the un-
deformed corneal geometry, the temporal profile of corneal apex
indentation, and the spatial corneal deformation pattern. Average
deformation values of each condition were used as input to the
simulation. Some studies have used forward biomechanical modeling of the
corneal tissue to predict the corneal response to incisional surgery
[28] or laser ablation [29]. On the other hand, inverse modeling is
used to retrieve inherent material properties matching the
response of the model to the experimental response. Previous
studies have obtained elastic properties from inverse modeling of
topographic differences before and after refractive surgery [30]. Spatial air-puff characterization In order to determine the geometry-dependent spatial shape of
the air-puff at different stages of the corneal deformation, the
maximum air speed (115 m/s) was estimated by Bernoulli’s
equation at the stagnation point of the airflow: vmax~
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2
r (pstagnation{patmosphere)
s
ð1Þ ð1Þ where r is density, pstagnation is the measured absolute stagnation
pressure of the air puff, and patmosphere is the static atmospheric
pressure. This value is comparable with the air speed measured
previously [32] using a hot wire anemometer next to the tube end
(.100 m/s). Methods Corneal deformation was studied using a finite element model
with a two-dimensional axis-symmetric geometry. Initial corneal
curvature, thickness dimensions and the dynamic deformation
response were available from previous experimental Scheimpflug
cross-sectional images of the anterior eye segment (see Figure 1). To simulate the experimental conditions accordingly, the pressure
characteristics provided by the air-puff system were determined:
First, the temporal pressure profile was measured experimentally
and
then
a
computational
fluid
dynamics
simulation
was
performed to determine the spatial pressure profile. Inverse
modeling was performed in order to find the biomechanical
parameter set that best represented the different experimental
conditions. Finally a sensitivity analysis was performed in order to
determine the parameters that were correlated the most to the
corneal deformation response following an air-puff. Temporal air-puff characterization A pressure sensor (MPX2301DT1, Freescale Seminconductor
Inc, Tempe, AZ, USA) was used to measure the central temporal
pressure distribution of the air-puff at 11-mm distance (typical
position of the cornea) from the air-tube. The temporal pressure
profile was fit by a linear function for pressure increase
(slope = 5.38 mmHg/ms; r = 0.9883; p,0.001) as well as for
pressure
decrease
(slope
= 28.38 mmHg/ms;
r = 0.9897;
p = 0.03). Although the overall air-puff duration was 27.50 ms,
only 20.63 ms (i.e. 97% of the air-pressure) contributed effectively
to the corneal deformation. Air Puff Imaging The Corvis system (Oculus, Wetzlar, Germany) uses high-speed
Scheimpflug imaging to capture the spatio-temporal corneal
deformation following an air-puff. Typically 140 images are taken
during the ,30 ms deformation event (i.e. at a speed of about
4330 images/sec) with a resolution of 6406480 pixels. The spatial
and temporal deformation profiles are highly reproducible (RMS
difference 0.015 mm and 0.012 mm, respectively). To retrieve the spatial air pressure distribution, an axis-
symmetric computational fluid dynamics (CFD) simulation was
performed using the finite volume method (FVM) implemented in
the Fluent module of the ANSYS Release 14.0 software package. CFD Geometry. Prior experimental data show a correlation
between the maximum deformation amplitude of the cornea and
the peak-to-peak distance. Figure 2 shows a polynomial fit
between these two deformation parameters and indicates that
the deformed corneal geometries can be parameterized using
maximum corneal indentation as a free parameter. For the current
model, six representative positions were sampled. Figure 3 shows
the mesh of the modeled air volume for the cornea in its maximal
deformed state, along with the resulting airflow velocity distribu-
tion. The geometrical parameters of the meshes for the six
different corneal geometries are shown in Table 1. Introduction Also the anisotropic properties of corneal tissue have been
determined from inverse modeling of corneal inflation experi-
ments [31]. In this study we present a finite element model that predicts the
corneal deformation pattern upon air-puff ejection and which has
allowed, for the first time to our knowledge, to retrieve both, the
elastic and viscoelastic properties of the cornea. The model was
validated with experimental data of porcine and human eyes and
the
sensitivity
of
corneal
deformation
to
geometrical
and
biomechanical parameters was studied. Being able to retrieve
dynamic material properties will open a new way for tissue
characterization in vivo. Experimental data Experimental corneal deformation data following an air-puff
were taken from a previous study in porcine eyes ex vivo and
human eyes in vivo and ex vivo [21]. Porcine eyes were obtained
from a local slaughterhouse (Patel S.A.U., Barcelona, Spain). Human donor eyes for explicit therapeutic or scientific use were
obtained through a collaborative agreement with Universidad
Auto´noma de Madrid and Transplant Services Foundation (Banco
de Sangre y Tejidos, Barcelona, Spain, http://www.bancsang.net)
and used within less than 12 hours post mortem. Human subjects CFD Boundary conditions. The air-puff emitting tube and
the cornea were considered as wall type boundary conditions (i.e. August 2014 | Volume 9 | Issue 8 | e104904 PLOS ONE | www.plosone.org 2 Corneal Biomechanical Evaluation by Air-Puff Finite Element Modelling Figure 1. Schematic illustration of the simulation procedure. (a) Corneal geometrical data from Scheimpflug images are used to define the
model geometry. Inverse modeling is performed to account for the effect of applying the IOP. (b) The temporal pressure profile measured
experimentally and the spatial pressure profile obtained from CFD (Computation Fluid Dynamics) simulation are applied to the cornea as a function
of time, location and current deformed shape. (c) The finite element model is solved for the current parameter set and simulation results are
compared to the experimentally measured deformation. A step-wise optimization approach is used to find the parameter set that leads to the most
similar deformation. doi:10.1371/journal.pone.0104904.g001 Figure 1. Schematic illustration of the simulation procedure. (a) Corneal geometrical data from Scheimpflug images are used to define the
model geometry. Inverse modeling is performed to account for the effect of applying the IOP. (b) The temporal pressure profile measured
experimentally and the spatial pressure profile obtained from CFD (Computation Fluid Dynamics) simulation are applied to the cornea as a function
of time, location and current deformed shape. (c) The finite element model is solved for the current parameter set and simulation results are
compared to the experimentally measured deformation. A step-wise optimization approach is used to find the parameter set that leads to the most
similar deformation. d i 10 1371/j
l
0104904 001 doi:10.1371/journal.pone.0104904.g001 air-puff characterization. As inertial effects are expected to
dominate over viscous effects, turbulent flow was assumed and
described using the Reynolds stress model. The Reynolds
averaged momentum equations for the mean velocity are: the cornea was simulated as a rigid body not considering fluid-
structure interaction). Experimental data A velocity inlet was modeled at the end of
the air tube and a pressure outlet around the cornea. The initial
flow velocity of the air-puff (115 m/s) was calculated from the
stagnation pressure, i.e. the peak central pressure measured in the August 2014 | Volume 9 | Issue 8 | e104904 August 2014 | Volume 9 | Issue 8 | e104904 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 3 Corneal Biomechanical Evaluation by Air-Puff Finite Element Modelling Figure 2. Correlation between the peak distance and apex
indentation obtained from an ample experimental data set,
including corneal response under different stiffness, thickness
and IOPs. doi:10.1371/journal.pone.0104904.g002 p"~pz 2
3 m Luk
Lxk
ð3Þ ð3Þ In the differential stress model, ruiuj is made to satisfy a
transport equation. A separate transport equation must be solved
for each of the six Reynolds stress components of ruiuj. The
differential equation Reynolds stress transport is: Lruiuj
Lt
z L
Lxk
ukruiuj
{ L
Lxk
dkimzrCS
k
e ukui
Luiuj
Lxi
~Pij{ 2
3 LijrezWijzPij,b
ð4Þ ð4Þ ~Pij{ 2
3 LijrezWijzPij,b Figure 2. Correlation between the peak distance and apex
indentation obtained from an ample experimental data set,
including corneal response under different stiffness, thickness
and IOPs. where Pij and Pij,b are shear and buoyancy turbulence production
terms of the Reynolds stresses respectively, Wij is the pressure-
strain tensor, and C is a constant. doi:10.1371/journal.pone.0104904.g002 doi:10.1371/journal.pone.0104904.g002 doi:10.1371/journal.pone.0104904.g002 The pressure distribution along the corneal surface was
obtained for each of the six deformed corneal geometries. Figure 4
presents the resulting spatial pressure profiles for different corneal
apex
indentations,
which
were
then
interpolated
(with
an
indentation step-width of 20 mm) into a 2D spatial pressure
surface and used as an input in further analysis. The shape of the
pressure distribution did not change significantly with varying flow
speed, but it was altered as the cornea deforms (see Figure 5b). Lrui
Lt z L
Lxj
(ruiuj){ L
Lxj
m Lui
Lxj
z Luj
Lxi
~
Lp
Lxi
{ L
Lxj
ruiuj
zSMi
ð2Þ
" ð2Þ In order to account for measurement inaccuracies of the
maximal pressure and hence the estimation of the air speed the
spatial pressure function was normalized and a scaling factor Pmax
introduced. Experimental data This was necessary as the sensor, due to the geometry
of its housing, tends to overestimate the pressure in high-speed
dynamic measurements. In the structural finite element simula-
tions, the applied surface pressure was a function of apex
indentation, distance from the apex and time. where p’’ is a modified pressure, SMi is the sum of body forces and
ruiuj corresponds to the fluctuating Reynolds stress contribution. Unlike eddy viscosity models, the modified pressure has no
turbulence contribution and it is related to the static (thermody-
namic) pressure by: Structural finite element simulation Figure 3. Geometry model of the cornea at maximal deforma-
tion. Mesh of cells of the modeled air volume (left) and streamlines
(right) colored by the flow velocity distribution. doi:10.1371/journal.pone.0104904.g003 Geometry. A 2D axis-symmetric eye model was defined
corresponding to the different corneal conditions and boundaries
(see Table 2 for parameter details of the eye model). The outer
coat of a half eye globe was considered – consisting of cornea,
limbus and sclera – and modeled by 8-node elements with
quadratic displacement behavior. Thereby 400 elements were
used to represent the cornea (where corneal thickness was divided
into 8 elements), 56 elements to represent the limbus, and 640
elements to represent the sclera. Initial corneal curvatures were
adjusted to match the experimental values after IOP (15 mmHg)
application. Scleral geometry was taken from the literature [33]
and the limbus was defined by connecting cornea and sclera. The
ocular humors were modeled by a single fluid compartment, which
consisted of 137 hydrostatic fluid elements. Material models. The corneal tissue was modeled by a
linear viscoelastic material. Thereby only the shear response was
considered, as it is typically dominant over the volumetric
response. s~
ðt
0
2G(t{t) de
dt dt
ð5Þ ð5Þ Figure 3. Geometry model of the cornea at maximal deforma-
tion. Mesh of cells of the modeled air volume (left) and streamlines
(right) colored by the flow velocity distribution. doi:10.1371/journal.pone.0104904.g003 where s is the Cauchy stress, e the deviatoric strain and t past
time, which was described by a two parameter Prony series: where s is the Cauchy stress, e the deviatoric strain and t past
time, which was described by a two parameter Prony series: August 2014 | Volume 9 | Issue 8 | e104904 PLOS ONE | www.plosone.org August 2014 | Volume 9 | Issue 8 | e104904 4 Corneal Biomechanical Evaluation by Air-Puff Finite Element Modelling Table 1. Geometrical parameters of deformed corneas and the corresponding mesh size. Case n6
Apex indentation
[mm]
Peak distance
[mm]
Number of cells in mesh
Average cell size
[mm2]
0
0
0
5600
0.0464
1
0.5
3
5754
0.0452
2
0.6
4
5703
0.0457
3
0.8
5
5858
0.0446
4
1
5.3
5858
0.0446
5
1.5
5.5
6925
0.0378
doi:10.1371/journal.pone.0104904.t001 variables suitable for adequate convergence of the optimization
routines. The aqueous and the vitreous humors were defined as a
hydrostatic fluid with a pressure equal to the IOP. Structural finite element simulation The corneal density was set to [37] rnormal = 1062 kg/m3 for a
700 mm corneal thickness and scaled according to the thickness
values in the experiments. A material damping of 10 ms was used. Boundary conditions and loads. For the simulations of the
ex vivo whole globe the sclera was fixed mimicking the fixation of
the eye in the eye-holder in the experiments. For the in vivo
condition the eye globe was damped along on the vertical
symmetry
axis
representing
the
ocular
muscles
and
other
surrounding fatty tissue. It was assumed that those external
damping factors can be summarized in a single vertical damping
element, while horizontal damping effects were neglected. The
vertical damping was implemented by a mass-less longitudinal
spring-damper, which was modeled by a uniaxial tension-
compression element, defined by a spring constant (5?106 N/m)
and a damping coefficient (1.0). The intraocular pressure was
applied in the model to the interior surfaces of cornea, limbus and
sclera according to the experimental data (15, 20, 25 and
35 mmHg). Boundary conditions and loads. For the simulations of the
ex vivo whole globe the sclera was fixed mimicking the fixation of
the eye in the eye-holder in the experiments. For the in vivo
condition the eye globe was damped along on the vertical
symmetry
axis
representing
the
ocular
muscles
and
other
surrounding fatty tissue. It was assumed that those external
damping factors can be summarized in a single vertical damping
element, while horizontal damping effects were neglected. The
vertical damping was implemented by a mass-less longitudinal
spring-damper, which was modeled by a uniaxial tension-
compression element, defined by a spring constant (5?106 N/m)
and a damping coefficient (1.0). The intraocular pressure was
applied in the model to the interior surfaces of cornea, limbus and
sclera according to the experimental data (15, 20, 25 and
35 mmHg). Figure 4. Spatial pressure distribution of the air-puff along the
corneal surface for different deformed shapes. doi:10.1371/journal.pone.0104904.g004 Air-puff application. A pressure load was applied on the
element edges of the anterior corneal surface according to the
spatial pressure distribution of the air-puff at the different
indentation depths. This was necessary because the fluid dynamics
characteristics
change
significantly
as
the
cornea
deforms. Furthermore the pressure variation with time (measured experi-
mentally with the pressure sensor) was considered by multiplying
the current pressure with the normalized temporal pressure
profile. Figure 4. Structural finite element simulation Fluid-structure
interaction was considered between the ocular tissues and the
ocular humors by relating the solid stress and displacements to the
pressure imposed on the interface by the fluid pressure load
(neglecting friction): G(t)~G0:½aG
1 : exp ({ t
tG
1
)
ð6Þ ð6Þ with G0~G?zG1and aG
i ~ Gi
G0 where G(t) is the Prony shear
modulus, aG
1 is the relative modulus, G0,G?,G1 are the instanta-
neous,
the
infinite
and
the
current
shear
elastic
moduli,
respectively and tG
1 mthe relaxation times for the Prony compo-
nent. The shear modulus (G) denomination was only used to
express viscoelasticity. In order to compare to the commonly used
elasticity modulus (E), we used the following equation for isotropic
materials G~
E
2(1zn) where v is the Poisson’s ratio. Limbus and
sclera were described by a purely elastic material: s~k:e, where e
represents strain. It should be noted that these material models
represent the macroscopic response of the biological tissues. The
microscopic structure was not considered for the relatively small
strains present during the deformation. Instead, the cornea was
divided in two equal layers (anterior and posterior, extending 50%
of the entire corneal depth each) and a different elastic modulus
was assigned to each, based on the considerable differences in the
morphology of the anterior and posterior cornea (collagen
interweaving, susceptibility to swelling, mechanical structure
[34]) that have been reported in the literature [16,35] in both,
virgin and cross-linked corneas. In fact, changes in the elasticity
across the corneal depth are likely gradual [16], but the definition
of two layers with each of them representing the mean corneal
stiffness in the anterior or posterior cornea respectively, allows
capturing the physiological reality while keeping the number of Then the fluid pressure load vector
f pr
e
was added to the basic
equation of motion, MS
½
€ue
f
gz CS
½
:ue
f
gz KS
½
ue
f
g~ fS
f
gz f pr
e
ð7Þ ð7Þ where
MS
½
is the structural mass matrix,
CS
½
the structural
damping matrix,
€ue
f
g the elemental acceleration vector,
:ue
f
gthe
elemental velocity vector,
ue
f
gthe elemental displacement vector
and
fS
f
g the structural load vector. Biomechanical parameters of the sclera and limbus (see
Table 2) were defined by data obtained from the literature [36]. Multi-step optimization A multiple step optimization approach (schematic shown in
Figure 6) was applied to find the biomechanical parameter set that
best matched the experimentally observed behavior. Thereby in
each step, the corresponding parameters were first scanned within
their limits and then further optimized by a gradient-based
approach. Generally, the spatial profile is dominated by the elastic
properties, while the recovery part of the temporal deformation
profile is dominated by the viscoelastic properties (see A, B in
Figure 7). For each step, the deformed geometry obtained from
the FE-simulation was exported, analyzed in terms of the temporal
and spatial deformation profile and compared to the experimental
data. Figure 7 shows the result of this analysis. We found that (a)
increasing the elastic modulus decreases the maximal corneal
indentation depth and leads to a delayed indentation in the
temporal profile; (b) increasing the Prony constant has little impact
on the spatial profile, but increases the hysteresis in the temporal
profile; (c) decreasing the time constant t increases the amount of
corneal indentation and determines the hysteresis and duration of
the deformation event in the temporal profile; (d) increasing the
stiffness in the anterior region of the cornea with respect to the
posterior region has a little impact on the temporal profile, but
increases the vertical distance between corneal bending points and
corneal apex, especially at higher IOPs. In the beginning, the elasticity modulus was the only variable
considered to account for the deformation behavior. Then the
biomechanical description of the material model was refined in
consecutive steps, adding viscoelastic properties and a difference
between anterior and posterior corneal stiffness. Thereby the fact
that two deformation profiles (temporal, spatial) were available,
and that the optimization variables had differential effects on each,
reduced the amount of possible local minima. The multi-step
procedure allowed us to obtain better comparable parameter sets
across the different tested conditions. Pre-optimization. The different patterns were subsequently
used to optimize the geometrical and biomechanical parameter set
of the FE-model, as illustrated in Figure 6. In a step prior to the
optimization, Pmax was adjusted in the range from 65.8 mmHg
(115 m/s) to 111 mmHg (156 m/s) using the deformation profiles
of the cross-linked porcine cornea at 15, 20, 25 and 35 mmHg. This led to a value of Pmax = 72.5 mmHg, which was then kept
constant for all conditions (see ‘‘pre-optimization’’ in Figure 6). Table 2. Structural finite element simulation Spatial pressure distribution of the air-puff along the
corneal surface for different deformed shapes. doi:10.1371/journal.pone.0104904.g004 Load steps. In the first load step the IOP was applied to the
model. Then, in the next step the load modeling the air-puff was
applied. In order to achieve sufficient temporal resolution, this step PLOS ONE | www.plosone.org August 2014 | Volume 9 | Issue 8 | e104904 August 2014 | Volume 9 | Issue 8 | e104904 5 Corneal Biomechanical Evaluation by Air-Puff Finite Element Modelling Figure 5. Air-puff characterization. (a) Experimentally measured temporal air-puff profile; (b) Results from CFD simulation showing the air-puff as
a function of apex indentation and location along the cornea (horizontal distance from the apex). doi:10.1371/journal.pone.0104904.g005 Figure 5. Air-puff characterization. (a) Experimentally measured temporal air-puff profile; (b) Results from CFD simulation showing the air-puff as
a function of apex indentation and location along the cornea (horizontal distance from the apex). doi:10.1371/journal.pone.0104904.g005 was divided into 49 sub-steps, so the applied pressure was updated
every 625 ms. was divided into 49 sub-steps, so the applied pressure was updated
every 625 ms. Pattern of the cost-function for the different optimization
parameters. In order to define the individual steps of the
optimization procedure, we studied the pattern of influence for
each optimization variable on the temporal and on the spatial
corneal deformation profiles. Changing a given variable subse-
quently, while keeping the other parameters constant allowed then
identifying systematic effects. Multi-step optimization Biomechanical and geometrical model parameters used to simulate the human and pig corneal deformation. Human (virgin)
Porcine (cross-linked)
Corneal thickness (mm)
558
211
Anterior curvature (mm)
8.03
8.06
Posterior curvature (mm)
6.86
7.54
Corneal diameter (mm)
10
12
Scleral diameter (mm)
19.5
23.5
doi:10.1371/journal.pone.0104904.t002
PLOS ONE | www.plosone.org
6
August 2014 | Volume 9 | Issue 8 | e104904 l and geometrical model parameters used to simulate the human and pig corneal deformation. Table 2. Biomechanical and geometrical model parameters used to simulate the human and pig corne Corneal Biomechanical Evaluation by Air-Puff Finite Element Modelling Figure 6. Schematic of the optimization process. Pre-optimization step (0), where the maximal air-puff pressure is adjusted; Multi-step
optimization, comprising adjustment of initial elastic modulus and corneal geometry (I); adjustment of viscoelastic parameters (II) and further
refinement of the elastic modulus and geometry (I); adjustment of the anterior and posterior elastic moduli (III), followed by a further refinement of
the elastic moduli and geometry (I). In human eyes and additional step was incorporated to account for damping by ocular muscles and external
tissue (IV). In the illustration the size of the loops is positively correlated with the dominance of the parameter adjusted therein. The resulting
parameter values, shown in blue, represent an example of the output parameters at each step for human eyes ex vivo and in vivo. doi:10.1371/journal.pone.0104904.g006 Figure 6. Schematic of the optimization process. Pre-optimization step (0), where the maximal air-puff pressure is adjusted; Multi-step
optimization, comprising adjustment of initial elastic modulus and corneal geometry (I); adjustment of viscoelastic parameters (II) and further
refinement of the elastic modulus and geometry (I); adjustment of the anterior and posterior elastic moduli (III), followed by a further refinement of
the elastic moduli and geometry (I). In human eyes and additional step was incorporated to account for damping by ocular muscles and external
tissue (IV). In the illustration the size of the loops is positively correlated with the dominance of the parameter adjusted therein. The resulting
parameter values, shown in blue, represent an example of the output parameters at each step for human eyes ex vivo and in vivo. doi:10.1371/journal.pone.0104904.g006 Optimization. In the first step of the optimization a very
simplified material model - linear elasticity - was assumed. Multi-step optimization doi:10.1371/journal.pone.0104904.g008 For the in-vivo human cornea a fourth step was necessary,
which included the adjustment of the two elements (elastic and
viscous parameters) of the external damping element (see ‘‘extra
step for in vivo eyes’’ in Figure 6). For the in-vivo human cornea a fourth step was necessary,
which included the adjustment of the two elements (elastic and
viscous parameters) of the external damping element (see ‘‘extra
step for in vivo eyes’’ in Figure 6). constants. This second step was iteratively coupled with the first
step in order to correct for the viscoelastic component, the effects
of which were initially accounted for by the previously adjusted
elastic modulus (see central part in Figure 6). The viscoelastic
parameters were adjusted in order to reproduce the hysteresis in
the temporal deformation profile, which is observed after the air-
puff stops, i.e. in the zone of indirect air-puff response. Multi-step optimization It
included the adjustment of geometrical parameters and the elastic
modulus: the initial anterior radius of curvature, central corneal
thickness and elastic modulus were adjusted so that after applying the intraocular pressure, both the modeled and experimental
corneal geometry within the 8-mm diameter central zone and the
maximal indentation depth were identical. In the second step the material model was refined adding
viscoelastic properties expressed by a Prony and a relaxation time Figure 7. Effect of the change of different biomechanical parameters on the spatial deformation profiles (upper row) and temporal
deformation profiles (lower row) at IOP = 15 mmHg. (a) Elastic properties dominate the maximal indentation depth. (b) Viscoelastic properties
dominate the amount of hysteresis when the air-pressure has decreased to zero. (c) The ratio between anterior and posterior stiffness dominates the
distance between corneal apex and bending points. doi:10.1371/journal.pone.0104904.g007 Figure 7. Effect of the change of different biomechanical parameters on the spatial deformation profiles (upper row) and temporal
deformation profiles (lower row) at IOP = 15 mmHg. (a) Elastic properties dominate the maximal indentation depth. (b) Viscoelastic properties
dominate the amount of hysteresis when the air-pressure has decreased to zero. (c) The ratio between anterior and posterior stiffness dominates the
distance between corneal apex and bending points. doi:10.1371/journal.pone.0104904.g007 August 2014 | Volume 9 | Issue 8 | e104904 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 7 7 Corneal Biomechanical Evaluation by Air-Puff Finite Element Modelling Figure 8. Temporal (a, c) and spatial (c, d) corneal deformation with air-puff. Dotted lines represent experimental corneal deformations and
continuous lines simulated corneal deformations Panels (a, b) show data for porcine corneas: simulated and experimental data at different IOPs. Panels (c, d) show data for human corneas: simulated response with and without ocular muscle damping, compared to in vivo experimental
deformations measured in patients and ex vivo deformations measured in an enucleated whole globe. doi:10.1371/journal.pone.0104904.g008 Figure 8. Temporal (a, c) and spatial (c, d) corneal deformation with air-puff. Dotted lines represent experimental corneal deformations and
continuous lines simulated corneal deformations Panels (a, b) show data for porcine corneas: simulated and experimental data at different IOPs. Panels (c, d) show data for human corneas: simulated response with and without ocular muscle damping, compared to in vivo experimental
deformations measured in patients and ex vivo deformations measured in an enucleated whole globe. August 2014 | Volume 9 | Issue 8 | e104904 Condition The latter two parameters represent accumulated contributions of muscles and fatty tissue and were not further interpreted. doi:10.1371/journal.pone.0104904.t003 25.5 MPa; Eposterior = 0.85 MPa (see Table 3). Both the decreased
corneal
apex
indentation
(Dfem20.676 mm
versus
Dexp2
0.666 mm) with increased IOP from 15 to 35 mmHg and the
decreased peak distance, i.e. horizontal distance between the
corneal bending points at maximal deformation (Dfem22.04 mm
versus Dexp22.34 mm) are well reproduced by the model. deformation is highly relevant in the clinical practice, where
diagnostics is currently performed from geometrical deformation
parameters. Corneal response simulation under different conditions
The finite element model could well reproduce average Corneal response simulation under different conditions
The finite element model could well reproduce average
experimental data from a previous study [21] of corneas under
different intraocular pressures (IOPs) and following collagen cross-
linking. Corneal
deformation
in vivo
–
human
eye
model. Defining a damping element for the fixation of the
ocular globe allowed us to describe the effect of ocular muscles and
fatty tissue. Corneal deformation in vivo (average across 9 eyes)
was compared to simulations where damping of the entire ocular
globe was considered, while corneal deformation ex vivo was
compared to simulations without damping (see Figure 8 c, d). Corneal deformation for different intraocular pressures
(IOPs) – porcine eye model. The corneal response upon air-
puff ejection was simulated for different IOPs (ranging from 15 to
35 mmHg, i.e. covering the IOP range from physiological values
to those found in severe glaucoma). We found that the maximum
corneal apex indentation was 1.13 mm for the lowest IOP and
0.46 mm for the highest IOP. Air-puff maximum pressure and, to
a lesser extent, differences in stiffness between the anterior and
posterior cornea were found to play a major role in the predicted
corneal deformation. Figure 8 a, b shows the simulated corneal
deformation compared to experimental corneal deformation21 in
cross-linked porcine corneas ex vivo for different IOPs. The
reconstructed elasticity moduli from the model were: Eanterior = We found that the retro-bulbar tissue contributed 0.112 mm to
the apex displacement and induced a hysteresis (additional to the
viscoelastic hysteresis of the cornea) in the temporal profile in the
region of indirect air-puff response. Computing techniques ANSYS APDL structural mechanics code (ANSYS, Inc.,
Canonsburg, PA) was used for the mechanical simulations and
the FLUENT module for the CFD simulations. The analysis of the
deformed corneal shape was performed in Matlab (The Math-
Works, Natick, MA). The relation between corneal indentation and eye globe compres-
sion (i.e. general deformation from a spherical to an elliptical eye
shape resulting from scleral deformation) depended on the IOP, but
also on the difference between the anterior and posterior corneal
stiffness. At 15 mmHg, corneal deformation contributed 90.4% to
the overall indentation, while at 35 mmHg only 44.9%. We also
found that increasing the difference between anterior and posterior
corneal stiffness produced larger corneal deformation (i.e. for
Eanterior/Eposterior =30: corneal deformation =90.4%; for Eanterior/
Eposterior =1: corneal deformation =83.7%). Sensitivity Analysis After selecting the model parameter set that best represented the
virgin human cornea in vivo condition, a sensitivity analysis was
performed in order to determine the geometrical and mechanical
parameters that dominate the shape and amount of corneal
deformation following the air-puff. Seven parameters were
selected (corneal thickness, stiffness, curvature, density, IOP and
two viscoelastic constants) and changed consecutively within
physiological or pathological ranges. For each parameter variation
the effect on the overall predicted corneal deformation was
determined by analyzing the coordinates of the deformed shape,
including changes in the maximal corneal indentation and peak
distance. Evaluation of the factors that dominate the corneal The third step provides a further refinement the model
accounting for physiological differences in the anterior and
posterior collagen interweaving, which result in an in-depth
variation of corneal stiffness. This difference in stiffness between
the anterior and posterior corneal regions was introduced in order
to adjust the spatial corneal deformation profile (see Figure 7 c). In
normally hydrated corneas the anterior cornea is approximately
16% more rigid. In cross-linked corneas the difference between the
anterior and posterior corneal stiffness is larger, as typically only
the 60% of the entire cornea is stiffened after treatment [38]. PLOS ONE | www.plosone.org August 2014 | Volume 9 | Issue 8 | e104904 August 2014 | Volume 9 | Issue 8 | e104904 8 Corneal Biomechanical Evaluation by Air-Puff Finite Element Modelling Corneal Biomechanical Evaluation by Air-Puff Finite Element Modelling Table 3. Corneal biomechanical parameters obtained from finite element analysis: Elasticity modulus represents the static material
properties; The difference between anterior and posterior cornea describes the differences in collagen interweaving and the
resulting higher anterior corneal rigidity; The relaxation time – which must lie within the temporal scale that was analyzed –
describes the point of time at which the corneal stiffness decreased by the factor of the relative modulus; The muscle spring and
damping constants describe the static and dynamic displacement, respectively, due to whole the eye movement in in vivo
measurements. Human
Porcine
Condition
virgin
cross-linked
Elasticity modulus (Mpa)
posterior cornea
0.40
0.85
Difference between anterior and posterior
1.12
30
Relaxation time (ms)
10
1.5
Relative modulus
0.3
0.7
Muscle spring constant (Nm)
350
-
Muscle damping constant (kg/s)
100
-
The latter two parameters represent accumulated contributions of muscles and fatty tissue and were not further interpreted. doi:10.1371/journal.pone.0104904.t003 Table 3. Air-puff modeling Figure 5 (a) depicts the experimentally measured temporal
pressure profile at the center of the air-puff. A maximal air
pressure of 120 mmHg at the corneal surface was present. The
geometry-dependent spatial pressure profile was obtained from a
separate computational fluid dynamics (CFD) simulation. Figure 5
(b) shows the resulting spatial air-pressure profile expressed as a
function of maximal corneal indentation at the apex and distance
from the apex. p
The
viscoelastic
effect
was
evidenced
by
the
remaining
deformation (between 18.7 and 24.1% of the overall deformation
at different IOPs), which gradually decreased with time after the
air-puff event. The viscoelasticity of the cross-linked porcine
corneas ex vivo was described by a relative modulus of 0.7 and a
relaxation time constant of 1.5 ms, and contributed with 46% to
the maximal apex indentation. Sensitivity Analysis Corneal biomechanical parameters obtained from finite element analysis: Elasticity modulus represents the static material
properties; The difference between anterior and posterior cornea describes the differences in collagen interweaving and the
resulting higher anterior corneal rigidity; The relaxation time – which must lie within the temporal scale that was analyzed –
describes the point of time at which the corneal stiffness decreased by the factor of the relative modulus; The muscle spring and
damping constants describe the static and dynamic displacement, respectively, due to whole the eye movement in in vivo
measurements. Retrieved corneal biomechanical parameters Table 3 summarizes the viscoelastic parameter set retrieved for
the porcine and human cornea. The retrieved corneal biome-
chanical parameters
varied across different treatments
and
conditions. In virgin human corneas, the differences in anterior
and posterior corneal stiffness are consistent with recently reported August 2014 | Volume 9 | Issue 8 | e104904 PLOS ONE | www.plosone.org 9 Corneal Biomechanical Evaluation by Air-Puff Finite Element Modelling Table 4. Parameter gradient from the sensitivity analysis. Geometrical/biomechanical parameters
Change within the physiologic range
D peak distance (mm)
D apex indentation (mm)
Thickness (mm)
D 100 mm
20.7168
0.2011
Stiffness (MPa)
D 0.2 MPa
20.7218
0.3764
Relative modulus (no unit)
D 1
0.8929
0.1000
Relaxation time (ms)
D 10 ms
0.4300
20.2324
IOP (mmHg)
D 40 mmHg
22.472
0.2320
Curvature (mm)
D 1 mm
0.1875
20.0163
Density (106g/m3)
D 500 kg/m3
0.0008
0.0000
The table lists changes in the peak distance and apex indentation for parameter (thickness, stiffness, relative modulus, relaxation time, IOP, curvature, density) for values
within the expected physiological/pathological ranges. doi:10.1371/journal.pone.0104904.t004 Geometrical/biomechanical parameters
Change within the physiologic range
D Change within the physiologic range
D peak distance (mm)
D apex indentation (mm) The table lists changes in the peak distance and apex indentation for parameter (thickness, stiffness, relative modulus, relaxation time, IOP, curvature, density) for values
within the expected physiological/pathological ranges. doi:10.1371/journal.pone.0104904.t004 The table lists changes in the peak distance and apex indentation for parameter (thickness, stiffness, relative modulus, relaxation time, IOP, curvature, density) for values
within the expected physiological/pathological ranges. doi:10.1371/journal.pone.0104904.t004 rigidity of a material, while static material properties represent the
elasticity at infinity. Dynamic properties of corneal tissue are likely
dominated by the extracellular matrix, while static properties give
information on the collagen structure. Combining static and
dynamic analysis therefore might allow a better understanding of
the interaction between the extracellular matrix with the collagen
structure. The air-puff corneal imaging technique addresses
dynamic properties of tissue in a similar time range (20 ms) as
ultrasound-based elastography (50 MHz) and magnetic resonance
imaging (300 MHz) and in a longer time range than Brillouin
microscopy (GHz). Thereby the new technique surpasses the first
two in patient comfort, the second two in acquisition rates, and
allows retrieving corneal viscoelasticity and elasticity. Mean
corneal Young’s modulus as determined in this study from air-
puff deformation was 0.71 MPa for the virgin human cornea and
13.2 MPa for the cross-linked porcine cornea. What determines the corneal response? A sensitivity analysis was performed for the human eye in vivo
(i.e. with damping) in order to evaluate which geometrical and
biomechanical parameters determine the corneal deformation
with an air-puff. Table 4 presents the predicted dependency
(gradients) of the apex indentation and peak distance (distance
between the highest lateral points of the cornea at maximal
deformation) on different geometrical and biomechanical param-
eters. Dependencies on absolute parameter variations were also
investigated. Table 4 represents data for parameter variations
within the estimated physiological range. The sclera also had a
significant effect, increasing the apex indentation by 0.20 mm
when changing its rigidity by 90%. Retrieved corneal biomechanical parameters These values fall
within the range of corneal stiffness reported from ultrasound
elastography ranging from 0.19 MPa to 20.0 MPa [41,42]. Young’s moduli obtained from magnetic resonance imaging were
lower (0.04 to 0.19 MPa) [12], suggesting that the time range in
which the measurements are acquired (ultrasound and air-puff
measurements
are
performed
more
rapidly
than
magnetic
resonance measurements) play an important role in the observed
stiffness of the corneal tissue. Further factors that might affect the
experimental assessment of corneal stiffness include the post-
mortem time and the tissue hydration [9]. corneal stiffness gradient [35]. In cross-linked porcine corneas the
difference between anterior and posterior cornea was 26.8 times
higher than in virgin corneas, consistent with the stiffening effect of
the cross-linking occurring in the anterior cornea. Discussion and Conclusions New imaging acquisition techniques allow capturing the
dynamic geometrical deformation response of the cornea following
an air-puff. To our knowledge, we have presented for the first time
numerical estimates of corneal elastic and viscoelastic parameters,
based on Scheimpflug imaging of the spatio-temporal dynamic
corneal deformation and sophisticated Finite Element Modeling. The simulation reproduces with high accuracy the corneal
deformation patterns, while the estimated biomechanical param-
eters are independent of geometry. Overall, cross-linked porcine
corneas were 9.38 times more rigid than virgin human corneas. The much higher stiffness of the anterior cornea than of the
posterior in cross-linked corneas can be primarily attributed to the
cross-linking treatment - literature reports an increase in corneal
stiffness between 72% and 329% after CXL [39] - but also to the
dehydration and hence a higher density in the anterior corneal
region produced by the photosensitizer solution. In a recent
publication [9] we showed that the corneal hydration state affects
its biomechanical response. Time scales are not only relevant when comparing different
measurement systems, but also in regard to the physiological
interpretation. Elastic properties, i.e. static properties, are deter-
mined by the collagen matrix and depend on the microstructural
arrangement as well as the number of cross-links. These material
properties determine the long-term corneal resistance against the
IOP. Viscoelastic properties, i.e. time-dependent properties,
describe the rearrangement of the extracellular matrix (mainly
due to diffusion of water) upon changes in stress. Generally the
shorter the time scale where material properties are analyzed, the
harder the material and the larger the viscoelastic contribution. Thereby the time scale of interest for corneal material properties
depends on the time scale of the treatment or pathology: For
refractive surgery short-term viscoelasticity is probably more
important, while in keratoconus long-term viscoelasticity is of
interest. Future air-puff systems may be provided with alternatives
to allow a viscoelastic analysis at different time scales. In addition, by simulating different boundary conditions, the
movement of the cornea in response to the air puff could be
isolated from the additive effects of scleral deformation and
movement of the whole eye observed in the Scheimpflug images. The dynamic response [8,10] of a material can be very different
from its static [7] behavior, as viscoelastic materials typically
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tomographic vibrography. Corneal Biomechanical Evaluation by Air-Puff Finite Element Modelling multiple meridians. An assumption of this study was that changes
in the mechanical response of the cornea could be described by the
parameters listed in Table 3. Although more parameters generally
allow a more detailed description, when comparing different
conditions it is important to have unique parameter sets. These
typically only can be obtained using a limited number of
optimization parameters. entered as input parameters in the simulation, which increases the
complexity of the finite element simulation. Comparison of the
gradients obtained from sensitivity analysis with clinical data -
based on thickness changes after LASIK surgery, and over a
physiological IOP range (personal communication by Oculus) -
show good agreement. Although the multi-step optimization approach has allowed a
systematic retrieval of the model parameters, a potential limitation
of this method might be the identification of a local rather than a
global minimum. Nevertheless, the characteristic pattern how
individual parameters changed the cost-function as well as the fact
that a single parameter set (CXL porcine corneas) allowed an
accurate reproduction of the corneal response at different IOPs
(15–35 mmHg), suggesting that the solution is robust and likely to
be unique. We believe that finite element modeling is an extremely
valuable tool for the analysis of in vivo dynamic corneal
deformation. Inverse simulation together with state of the art
imaging systems will allow the analysis of (in particular the
viscoelastic) corneal biomechanical properties (viscoelastic prop-
erties in particular) in a clinical setting, facilitating diagnosis of
corneal pathologies, patient screening for refractive surgery and
evaluation of treatment efficacy such as after cross-linking. A potentially strong limitation of this study is that linear
material properties were assumed. Experimental diagrams from
uniaxial stress-strain testing suggest a linear stress-strain relation
up to about 5–6% strain [39]. Although most regions of the cornea
presented less than 6% strain (after IOP application and during
the air-puff event), maximal strains at the apex were up to 10%,
slightly over the linear range. This may have led to inaccuracies in
the regions of maximal bending resulting in a slightly different
overall deformation response. A simplification in the model is the
assumption of axis-symmetry, which does not capture potential
differences in the horizontal and vertical meridian. A 3D
expansion of the model would allow incorporating asymmetries
such as those occurring in keratoconus, but at the same time it
would require acquisition of spatial corneal deformation profiles in Author Contributions Conceived and designed the experiments: SK SM. Performed the
experiments: SK NB DP. Analyzed the data: SK NB CD SM. Contributed
reagents/materials/analysis tools: SM. Contributed to the writing of the
manuscript: SK NB SM. Designed and performed the simulations: SK NB. Designed and performed the experimental air-puff characterisation: CD
DP. Discussion and Conclusions Dynamic analysis hence will provide information on the instant In addition, by simulating different boundary conditions, the
movement of the cornea in response to the air puff could be
isolated from the additive effects of scleral deformation and
movement of the whole eye observed in the Scheimpflug images. The dynamic response [8,10] of a material can be very different
from its static [7] behavior, as viscoelastic materials typically
behave more rigid the faster a loading condition is applied [40]. Dynamic analysis hence will provide information on the instant Although the sensitivity analysis showed a correlation between
corneal rigidity and corneal deformation, geometrical parameters,
such as the central corneal thickness, as well as IOP, also showed a
large effect. This means that all factors need to be considered and August 2014 | Volume 9 | Issue 8 | e104904 PLOS ONE | www.plosone.org August 2014 | Volume 9 | Issue 8 | e104904 10 Corneal Biomechanical Evaluation by Air-Puff Finite Element Modelling 34. Bron AJ (2001) The architecture of the corneal stroma. Br J Ophthalmol 85:
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Thickness and surface area. Invest Ophthalmol Vis Sci 43: 2529–2532. August 2014 | Volume 9 | Issue 8 | e104904 PLOS ONE | www.plosone.org August 2014 | Volume 9 | Issue 8 | e104904 11 41. Tanter M, (2009) High-resolution quantitative imaging of cornea elasticity using
supersonic shear imaging. IEEE Trans Med Imag 28: 1881–1893. 38. Seiler T, Hafezi F (2006) Corneal cross-linking-induced stromal demarcation
line. Cornea 25: 10571059. p
36. Downs J, Suh JF, Thomas KA, Bellza AJ, Hart RT, et al (2005) Viscoelastic
material properties of the peripapillary sclera in normal and early-glaucoma
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37. Kampmeier J, Radt B, Birngruber R, Brinkmann R (2000) Thermal and
biomechanical parameters of porcine cornea. Cornea 19: 355–363. Corneal Biomechanical Evaluation by Air-Puff Finite Element Modelling 39. Wollensak G, Spoerl E, Seiler T (2003) Stress-strain measurements of human
and porcine corneas after riboflavin-ultraviolet-a-induced crosslinking. J Catar-
act Refract Surg 29: 1780–1785. 40. Knudson D, Fundamentals of Biomechanics (2nd ed), (2007) New York, NY,
Springer. p
36. Downs J, Suh JF, Thomas KA, Bellza AJ, Hart RT, et al (2005) Viscoelastic
material properties of the peripapillary sclera in normal and early-glaucoma
monkey eyes. Invest Ophthalmol Vis Sci 46: 540–546. . Tanter M, (2009) High-resolution quantitative imaging of cornea ela y y
p
37. Kampmeier J, Radt B, Birngruber R, Brinkmann R (2000) Thermal and
biomechanical parameters of porcine cornea. Cornea 19: 355–363. 38. Seiler T, Hafezi F (2006) Corneal cross-linking-induced stromal demarcation
line. Cornea 25: 10571059. PLOS ONE | www.plosone.org August 2014 | Volume 9 | Issue 8 | e104904 PLOS ONE | www.plosone.org 12
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PENGEMBANGAN ALAT PERAGA WIRELESS TURN SIGNAL SYSTEM PADA MATA KULIAH PRAKTIK KELISTRIKAN BODI TEKNIK MESIN UNIVERSITAS NEGERI SEMARANG
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JIPTEK : Jurnal Ilmiah Pendidikan Teknik dan Kejuruan/Jiptek
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ABSTRAK Media pembelajaran merupakan alat bantu untuk mempermudah proses pembelajaran, salah satunya
berupa peraga. Tujuan penelitian ini untuk mengetahui kelayakan dan keefektifan alat peraga wireless
turn signal system. Penelitian ini menggunakan metode penelitian pengembangan dengan metode
ADDIE. Dalam uji coba yang dilakukan, penelitian ini menggunakan desain one group pretest-
posttest. Subjek uji coba penelitian sejumlah 30 mahasiswa Prodi Pendidikan Teknik Otomotif
angkatan 2015. Instrumen yang digunakan untuk pengumpulan data yaitu instrumen tes dan angket. Alat peraga terbukti layak dilihat dari nilai hasil validasi ahli media sebesar 93 dengan presentase
kelayakan 83,04%, dan dari ahli materi sebesar 185 dengan persentase kelayakan 88,95%. Pengujian
hasil belajar mahasiswa terbukti efektif dilihat dari hasil pretest dengan nilai rata-rata 49,63 dan nilai
hasil posttest sebesar 80,89 Penghitungan signifikansi peningkatan hasil belajar juga dilakukan dengan
menggunkan uji t memperoleh hasil thitung = 3,19 dan ttabel = 2,01 pada db = 19 dengan ∝ = 5%
sehingga dapat disimpulkan terdapat peningkatan hasil belajar yang signifikan antara sebelum dan
sesudah pembelajaran dengan menggunakan alat peraga Wireless Turn Signal System. Hasil dari
penelitian didapatkan peraga Wireless Turn Signal System layak dan efektif dalam pelaksanaan
pembelajaran mata kuliah kelistrikan bodi. Kata kunci: Pengembangan, peraga, Wireless Turn Signal Sigit Bayu Aji1, Dwi Widjanarko2, Wahyudi3 Sigit Bayu Aji1, Dwi Widjanarko2, Wahyudi3 1,2,3Program Studi Pendidikan Teknik Otomotif, Universitas Negeri Semarang
Email: sigitbayuaji@ymail.com 1,2,3Program Studi Pendidikan Teknik Otomotif, Universitas Negeri Semarang
Email: sigitbayuaji@ymail.com DOI: https://doi.org/10.20961/jiptek.v13i1.21964 PENDAHULUAN pembelajaran. Alat peraga digunakan dengan
tujuan untuk memudahkan mahasiswa untuk
memahami sebuah materi yang disampaikan
oleh dosen. Prodi pendidikan teknik otomotif,
Universitas Negeri Semarang, dalam mata
kuliah praktik kelistrikan bodi, terdapat dua jenis
alat peraga sistem lampu tanda belok yang sudah
menyatu dengan stand engine dan alat peraga
berupa panel yang hanya terdapat sistem lampu
tanda belok saja. Dari kedua alat peraga tersebut
masih belum memenuhi syarat-syarat alat peraga
yang baik, selain itu ditemukan kekurangan pada
alat
peraga
sistem
lampu
tanda
belok,
kekurangan-kekurangan dari kedua alat tersebut
yaitu pemahaman tentang komponen, fungsi
tiap-tiap komponen dan perangkaian dari sistem
lampu tanda belok tidak disertakan pada alat
peraga. Kegiatan belajar mengajar merupakan
kegiatan utama dalam keseluruhan proses
pendidikan
di
universitas. Febriyono
&
Widjanarko (2014: 47) proses belajar mengajar
merupakan proses komunikasi antara guru
dengan siswa. Oleh karena itu, keberhasilan
pencapaian
tujuan
pendidikan
banyak
tergantung pada kualitas pelaksanaan proses
belajar mengajar. Universitas sebagai lembaga
pendidikan
berkewajiban
memberikan
kesempatan
belajar
seluas-luasnya
kepada
mahasiswa untuk mengembangkan potensi
dirinya seoptimal mungkin. y
p
g
Menurut Hariwung (1989: 22) kualitas
dalam suatu pengajaran dalam proses belajar
mengajar ditentukan oleh guru dan siswa yang
saling berinteraksi. Apabila para siswa mengerti
dan memahami apa yang disampaikan oleh guru
tersebut maka proses belajar mengajar berhasil
karena terdapat feedback yang baik dari
siswanya. Permono dkk (2013: 2) media yang
merupakan penyampai informasi dari pengirim
kepada penerima pesan, adalah salah satu faktor
penunjang keberhasilan pembelajaran. Proses
belajar menggunakan media pembelajaran dapat
membantu siswa memahami materi pelajaran. Hadinata dan Wijaya (2011: 40) membuktikan
terdapat peningkatan pemahaman mahasiswa
dalam mendiagnosis kelistrikan bodi sepeda
motor setelah menggunakan media peraga
dengan hasil nilai rata-rata pada tes sebelum
menggunakan alat sebesar 54,13 dan nilai rata-
rata pada tes setelah menggunakan alat sebesar
72,24 atau mengalami peningkatan sebesar
33,44%. Dari beberapa alat peraga yang ada, alat
peraga
lampu
tanda
belok
dapat
lebih
dimaksimalkan melalui pengembangan alat
peraga wireless turn signal system. Raharema
dan Salsabila (2016: 2) wireless technology
adalah teknologi elektronika yang beroprasi
tanpa kabel. Karena alat peraga ini bertujuan
untuk mengenalkan suatu sistem baru terhadap
mahasiswa, yaitu cara kerja sistem aliran
kelistrikan dengan pengendali tanpa kabel. Sehingga menghemat pengkabelan pada sistem
kelistrikan yang ada pada kendaraan dan
penambahan wiring diagram dapat berfungsi
membantu penyampaian suatu alat peraga untuk
menjelaskan aliran arus listrik agar komponen
bekerja sesuai dengan sistemnya. Wiring
diagram
juga
mempermudah
kita
dalam
mengatasi masalah pada rangkaian kelistrikan
apabila terjadi kerusakan pada sistem
kelistrikan. ABSTRACT Learning media is a tool to facilitate learning process, one of them in the form of visuals. The purpose
of thisresearch is to know the feasibility an effectiveness of a turn signal wireless system demonstrator. This research uses the development research method with ADDIE method. In the experiments
conducted, this study use the design of one group pretest- posttest. Subjects of research trial of 30
students course 2015 of Automotive Engineering Education Study Program. The instruments used for
data collection is instrument of the test and questionnaires. The demonstrator proved feasible of views
from the value of media expert validation results of 93 with the percentage of feasibility 83,04%, and
from the material expert of 185 with the percentage of eligibility 88,95%. The test result of student
learning proved effective seen from the value of the pretest with the value of the average 49,63 and
the value of the result posttest 80,89. The calculation of significance of the improvement of learning 149 DOI: https://doi.org/10.20961/jiptek.v13i1.21964 JIPTEK, Vol. 13 No.1 2020 outcomes is also done by using t test to obtain the result tcount = 3,19 and ttable = 2,01 on db = 19
with α = 5% so it can be concluded there is significant improvement of learning outcomes between
before and after learning using wireless turn signal system props. The result of the research obtained
that the wireless turn signal system props is feasible and effective in the implementation of electronical
subjects. outcomes is also done by using t test to obtain the result tcount = 3,19 and ttable = 2,01 on db = 19
with α = 5% so it can be concluded there is significant improvement of learning outcomes between
before and after learning using wireless turn signal system props. The result of the research obtained
that the wireless turn signal system props is feasible and effective in the implementation of electronical
subjects. Keywords : Development, props, Wireless Turn Signal Keywords : Development, props, Wireless Turn Signal PENDAHULUAN Dalam
bidang
pendidikan
teknik
otomotif
sangatlah
diperlukan
media
pembelajaran yang membantu meningkatkan
pemahaman mahasiswa, media yang digunakan
yaitu berupa alat peraga. Menurut Widiyatmoko
dkk (2012: 52), alat peraga merupakan alat bantu
yang dibuat oleh guru atau siswa supaya konsep
yang diajarkan guru mudah dimengerti oleh
siswa dan menjadi alat bantu dalam proses Widjanarko dkk (2014: 1) kendala yang
terjadi dalam memahami sistem kelistrikan
dalam bidang otomotif yaitu pada bagian
memahami rangkaian dari sistem kelistrikan dan
cara kerja dari rangkaian tersebut. Arsyad (2014:
9) alat peraga adalah media alat bantu
pembelajaran, dan segala macam benda yang 150 DOI: https://doi.org/10.20961/jiptek.v13i1.21964 JIPTEK, Vol. 13 No.1 2020 JIPTEK, Vol. 13 No.1 2020 digunakan
untuk
memperagakan
materi
pembelajaran. Oleh
karena
itu
siswa
membutuhkan
sebuah
alat
peraga
untuk
mempermudah
dalam
memahami
sistem
kelistrikan lampu tanda belok dan alat tersebut
juga dapat digunakan untuk latihan merangkai
sistem lampu tanda belok. Buntarto (2015: 25)
lampu tanda belok berfungsi untuk memberi
isyarat pada kendaraan yang ada didepan,
belakang dan sisi kendaraan bahwa pengendara
bermaksud untuk membelok atau pindah jalur. Berdasarkan masalah yang diuraikan di atas,
dikembangkanlah alat peraga wireless turn
signal system yaitu suatu sistem rangkaian
lampu
tanda
belok
tanpa
kabel
yang
memanfaatkan wireless sebagai pengganti kabel
sehingga menarik minat peserta didik dalam
mempelajari sistem lampu tanda belok dan
peserta didik dapat mempelajari sebuah sistem
baru, yaitu cara kerja sitem tanpa kabel pada
mata kuliah praktik kelistrikan bodi yang
nantinya berpengaruh terhadap hasil belajar
peserta didik. ahli media dan ahli materi selanjutnya dari
Jurusan Teknik Mesin Universitas Negeri
Semarang. Analisis
yang
digunakan
dalam
penelitian ini yaitu uji validitas alat peraga untuk
menghitung hasil akhir kelayakan alat peraga,
validitas tes untuk mengetahui validitas tiap-tiap
butir
soal,
reliabelitas
untuk
mengetahui
ketetapan soal jika digunakan berkali-kali,
normalitas untuk mengetahui bahwa hasil data
penelitian berdistribusi normal, dan uji t untuk
mengetahui perbedaan hasil belajar. Berdasarkan masalah yang diuraikan di atas,
dikembangkanlah alat peraga wireless turn
signal system yaitu suatu sistem rangkaian
lampu
tanda
belok
tanpa
kabel
yang
memanfaatkan wireless sebagai pengganti kabel
sehingga menarik minat peserta didik dalam
mempelajari sistem lampu tanda belok dan
peserta didik dapat mempelajari sebuah sistem
baru, yaitu cara kerja sitem tanpa kabel pada
mata kuliah praktik kelistrikan bodi yang
nantinya berpengaruh terhadap hasil belajar
peserta didik. HASIL PENELITIAN DAN PEMBAHASAN
Hasil Penelitian Alat
peraga
yang
dikembangkan
memiliki komponen yang sama dengan sistem
lampu tanda belok dan hazard dengan tambahan
komponen transmitter dan receiver serta sudah
disesuaikan dengan syarat media pembelajaran
yang baik. Arsyad (2013: 74-76) alat peraga
yang baik harus relevan dengan tujuan
pembelajaran,
selaras
dengan
kebutuhan
pembelajaran, praktis, mudah dipindahkan dan
mudah digunakan. METODE PENELITIAN Setelah disesua ikan dengan skala
tanggapan, skor tersebut masuk dalam kriteria
nilai 170-208 dengan kategori sangat layak. Dari
hasil penilaian kedua ahli media dan ahli materi
dapat disimpulkan bahwa alat peraga wireless
turn signal system dikatakan sangat layak. Gambar 3. nilai rata-rata hasil belajar Setelah diperoleh hasil data dari sebelum
menggunakan perga (pretest) dan sesudah
menggunakan peraga (posttest) dilakukan uji t
untuk mengetahui perbedaan hasil belajar. Tabel 1. Hasil Uji t
Pretest (Before)
Posttest (After)
Nilai rata-
rata
49,6
3
80,89
t hitung
3
,
1
9
t table
2
,
0
1
Keterangan
Efektif Sebelum soal tes digunakan untuk
mengukur keefektifan alat peraga maka soal tes
tersebut diujicobakan terlebih dahulu untuk
mengetahui validitas dan reliabelitas soal yang
telah dibuat. Hal ini sejalan dengan penelitian
Suh (2011: 60) Media harus diujicoba dan
divalidasi terlebih dahulu sebelum digunakan
dalam
proses
pembelajaran. Berdasarkan
pengujian yang telah dilakukan pada uji pretest
didapatkan nilai minimal sebesar 40,74 dan nilai
maksimal sebesar 59,26 dengan perolehan nilai
rata-rata sebesar 49,63 sedangkan pada uji coba
posttest didapat nilai minimal sebesar 66,67 dan
nilai maksimal sebesar 96,30 dengan perolehan
nilai rata-rata sebesar 80,89. setelah diperoleh
hasil dari uji pretest dan uji posttest maka
penelitian dilanjutkan dengan melakukan uji
normalitas, dan uji t. Berdasarkan tabel hasil uji t, thitung
dibandingkan dengan ttabel pada α=5% dengan
(1- α) (n1+n2-2), apabila thitung > ttabel maka alat
peraga yang dikembangkan dinyatakan efektif. Sehingga dari hasil pengujian dapat disimpulkan
bahwa pengembangan alat peraga wireless turn
signal systemyang digunakan dalam mata kuliah
praktik kelistrikan bodi dinyatakan efektif
dengan perbandingan hasil 3,19 > 2,01. METODE PENELITIAN Desain penelitian yang digunakan dalam
penelitian ini adalah model pengembangan
ADDIE. Model ADDIE terdiri dari lima tahapan
yaitu analyze, design, develop, implement, and
evaluate (Arkun and Akkoyunlu 2008: 4). Menurut Barokati dan Annas (2013: 355),
ADDIE adalah singkatan dari Analysis, Design,
Development,
Implementation,
Evaluation,
adalah salah satu dari berbagai model
pengembangan untuk mengembangkan suatu
pembelajaran
yang
efektif,
dinamis,
dan
mendukung pembelajaran itu sendiri Untuk
dapat menghasilkan produk yang optimal dan
tepat guna maka digunakan penelitian yang
bersifat analisis kebutuhan dengan ADDIE
model dan untuk menguji keefektifan produk
tersebut supaya berfungsi di masyarakat luas
(Cheung, 2016: 1-2). Gambar 1. Alat peraga wireless turn signal
system Metode
pengumpulan
data
pada
penelitian ini yaitu menggunakan metode tes
dengan tujuan untuk mengetahui keefektifan dari
alat peraga yang dikembangkan. Soal tes berupa
soal pilihan ganda dengan jumlah butir soal
sebanyak 30. Metode angket yang bertujuan
untuk mengetahui kelayakan dari alat peraga
yang dikembangkan. Subjek untuk menilai alat
peraga wireless turn signal system yaitu ahli
media dan ahli materi yang masing-masing 2
orang. Salah satu dari ahli media dan ahli materi
yaitu dari BPDIKJUR Semarang. Sedangkan Gambar 1. Alat peraga wireless turn signal
system 151 DOI: https://doi.org/10.20961/jiptek.v13i1.21964 JIPTEK, Vol. 13 No.1 2020 Gambar 2. Manual book Gambar 3. nilai rata-rata hasil belajar
Nilai rata-rata hasil belajar
100
80
80,89
60
49,63
40
20
Pretest
Posttest Gambar 2. Manual book Berdasarkan hasil penelitian dari kedua ahli
media dan ahli materi dapat ditarik kesimpulan
bahwa alat peraga wireless turn signal system
teruji sangat layak. Skor total yang diperoleh
dari kedua ahli media sebesar 93 dari skor
maksimal 112. Setelah disesuaikan dengan skala
tanggapan skor tersebut masuk dalam nilai 94-
112 dengan kategori sangat layak. Selanjutnya,
perolehan skor total dari kedua ahli materi
diperoleh skor sebesar 185 dari sekor maksimal
208. Setelah disesua ikan dengan skala
tanggapan, skor tersebut masuk dalam kriteria
nilai 170-208 dengan kategori sangat layak. Dari
hasil penilaian kedua ahli media dan ahli materi
dapat disimpulkan bahwa alat peraga wireless
turn signal system dikatakan sangat layak. Berdasarkan hasil penelitian dari kedua ahli
media dan ahli materi dapat ditarik kesimpulan
bahwa alat peraga wireless turn signal system
teruji sangat layak. Skor total yang diperoleh
dari kedua ahli media sebesar 93 dari skor
maksimal 112. Setelah disesuaikan dengan skala
tanggapan skor tersebut masuk dalam nilai 94-
112 dengan kategori sangat layak. Selanjutnya,
perolehan skor total dari kedua ahli materi
diperoleh skor sebesar 185 dari sekor maksimal
208. Pembahasan Penggunaan alat peraga Wireless Turn
Signal System dalam peneliti ini teruji layak
digunakan dalam pembelajaran dan efektif
digunakan untuk mendukung pelaksanaan kuliah
praktik kelistrikan bodi. Hasil dari penelitian
alat peraga yang sudah divalidasi didapatkan
nilai 93 dengan presentase kelayakan sebesar
83,04% dari ahli media dan 185 dengan DOI: https://doi.org/10.20961/jiptek.v13i1.21964 152 JIPTEK, Vol. 13 No.1 2020 persentase kelayakan sebesar 88,95% dari ahli
materi. dilakukan oleh Setiawan, dkk (2009: 29)
membuktikan bahwa dengan menggunakan
panel peraga sistem lampu kepala terjadi
peningkatan pemahaman mahasiswa. Hasil
pretest sebelum menggunakan panel peraga
sistem lampu kepala memperoleh hasil sebesar
52,33%. Panel peraga sistem lampu kepala
didesain dengan komponen-komponen yang
dapat dijalankan melalui saklar, sehingga panel
peraga sistem lampu kepala mendukung untuk
digunakan dalam pembelajaran praktik. Alat peraga wireless turn signal system
telah disesuaikan dengan rencana pembelajaran
semester
(RPS)
untuk
mencapai
tujuan
menjadikan alat peraga memiliki kelebihan
diantaranya: 1) Mencapai tujuan identifikasi
komponen sistem lampu tanda belok, alat peraga
ini
menampilkan
komponen
dan
nama
komponen sistem lampu tanda belok dengan
jelas sehingga mahasiswa dapat memahami
bentuk dan cara kerja komponen. 2) Untuk
mencapai tujuan merangkai sistem lampu tanda
belok, alat peraga ini menyediakan panel
perangkaian yang tertata dilengkapi dengan
wiring diagram dan keterangan nama terminal
pada
masing-masing
komponen
sehingga
perangkaian dapat dilakukan dengan mudah. Selain itu alat perga juga dilengkapi dengan
manual book untuk membantu mahasiswa
melakukan praktik sesuai prosedur yang baik
dan benar, dengan demikian alat peraga wireless
turn signal system dapat dinyatakan sudah
memenuhi syarat alat peraga yang efektif
digunakan untuk pembelajaran kuliah praktik
kelistrikan bodi. Simpulan Berdasarkan
pembahasan
dari
analisis
hasil
penelitian tentangpengembangan alat
peraga wireless turn signal system maka dapat
disimpulkan: p
1. Alat peraga wireless turn signal system
yang dikembangkan telah divalidasi oleh
ahli media yang memperoleh peresentase
sebesar 83,04% yaitu masuk dalam kriteria
sangat layak, dan oleh ahli materi yang
memperoleh
persentase
nilai
sebesar
88,95% yang masuk dalam kriteria sangat
layak. Sehingga dapat disimpulkan bahwa
alat peraga wireless turn signal system
dinyatakan valid dan dapat digunakan
sebagai media pembelajaran mata kuliah
prakktik kelistrikan bodi. Samsudduha dkk (2013: 5) penggunaan
manual book pembelajaran dapat meningkatkan
hasil
belajar
peserta
didik
yang
tidak
menggunakan manual book pembelajaran. Keefektifan alat peraga yang dikembangkan
setelah digunakan untuk praktik kelistrikan bodi,
dapat dilihat dari peningkatan hasil pretest dan
posttest yang selanjutnya diuji dengan uji t. Hasil
pengujian yang didapat dari pretest yaitu rata-
rata sebesar 49,63 dan nilai yang didapat dari
posttest sebesar 80,89. Dilihat dari rata-rata yang
didapat sudah terjadi peningkatan dengan nilai
selisih
31,25. Sejalan
dengan
penelitian
Widjanarko, dkk (2010: 10) membuktikan
dengan menggunakan panel peraga sistem
kelistrikan bodi multifungsi dapat meningkatkan
penguasaan sistem kelistrikan bodi mahasiswa
dengan
peningkatan
kompetensi
sebesar
31,87%. 2. Alat peraga wireless turn signal system
yang dikembangkan telah diuji cobakan
pada kegiatan pembelajaran. Hasil pada uji
before (pretest) memperoleh nilai rata-rata
sebesar 49,63 sedangkan pada uji after
(posttest)
memperoleh
nilai
rata-rata
sebesar 80,89. Hasil uji t memperoleh nilai
thitung 3,19 dan ttabel 2,01 (thitung>
ttabel), sehingga Ho ditolak dan Ha
diterima yang menyatakan alat peraga yang
dikembangkan efektif untuk digunakan
dalam pembelajaran. Saran Penghitungan signifikansi peningkatan
hasil belajar juga dilakukan dengan menggunkan
uji t dan memperoleh hasil thitung = 3,19 dan
ttabel = 2,01 pada db = 19 dengan ∝ = 5%
sehingga
dapat
disimpulkan
terdapat
peningkatan hasil belajar yang signifikan antara
sebelum dan sesudah pembelajaran dengan
menggunakan alat peraga Wireless Turn Signal
System dilihat dari thitung > ttabel. Penelitian ini
juga memperkuat penelitian sebelumnya yang Berdasarkan simpulan penelitian maka dapat
diberikan saran-saran ditinjau dari segi manfaat
yaitu sebagai berikut: 1. Bagi
pendidik
diharapkan
dapat
menerapkan alat peraga wireless turn signal
system dalam pembelajaran mata kuliah
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Sistem Kelistrikan Bodi Sepeda Motor 154 DOI: https://doi.org/10.20961/jiptek.v13i1.21964 JIPTEK, Vol. 13 No.1 2020
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English
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Involvement of GluD2 in Fear-Conditioned Bradycardia in Mice
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PloS one
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cc-by
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Hiroko Kotajima-Murakami1¤a¤b, Sakae Narumi2, Michisuke Yuzaki2, Dai Yanagihara1*
1 Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Meguro-ku
Tokyo, Japan, 2 Department of Physiology, School of Medicine, Keio University, Shinjuku-ku, Tokyo, Japan Hiroko Kotajima-Murakami1¤a¤b, Sakae Narumi2, Michisuke Yuzaki2, Dai Yanagihara1*
1 Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Meguro-ku
Tokyo, Japan, 2 Department of Physiology, School of Medicine, Keio University, Shinjuku-ku, Tokyo, Japan 1 Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Meguro-ku,
Tokyo, Japan, 2 Department of Physiology, School of Medicine, Keio University, Shinjuku-ku, Tokyo, Japan ¤a Current address: Department of Biosciences, School of Science and Engineering, Utsunomiya-shi,
Tochigi, Japan
¤b Current address: Addictive Substance Project, Tokyo Metropolitan Institute of Medical Science,
Setagaya-ku, Tokyo, Japan
* dai-y@idaten.c.u-tokyo.ac.jp g ,
p
¤b Current address: Addictive Substance Project, Tokyo Metropolitan Institute of Medical Science,
Setagaya ku Tokyo Japan a1111 OPEN ACCESS Citation: Kotajima-Murakami H, Narumi S, Yuzaki
M, Yanagihara D (2016) Involvement of GluD2 in
Fear-Conditioned Bradycardia in Mice. PLoS ONE
11(11): e0166144. doi:10.1371/journal. pone.0166144 Citation: Kotajima-Murakami H, Narumi S, Yuzaki
M, Yanagihara D (2016) Involvement of GluD2 in
Fear-Conditioned Bradycardia in Mice. PLoS ONE
11(11): e0166144. doi:10.1371/journal. pone.0166144 Editor: Manabu Sakakibara, Tokai University,
JAPAN Copyright: © 2016 Kotajima-Murakami et al. This is
an open access article distributed under the terms
of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. Abstract Lesions in the cerebellar vermis abolish acquisition of fear-conditioned bradycardia in ani-
mals and human patients. The δ2 glutamate receptor (GluD2) is predominantly expressed
in cerebellar Purkinje cells. The mouse mutant ho15J carries a spontaneous mutation in
GluD2 and these mice show a primary deficiency in parallel fiber-Purkinje cell synapses,
multiple innervations of Purkinje cells by climbing fibers, and impairment of long-term
depression. In the present study, we used ho15J mice to investigate the role of the cerebel-
lum in fear-conditioned bradycardia. We recorded changes in heart rate of ho15J mice
induced by repeated pairing of an acoustic (conditioned) stimulus (CS) with an aversive
(unconditioned) stimulus (US). The mice acquired conditioned bradycardia on Day 1 of the
CS-US phase, similarly to wild-type mice. However, the magnitude of the conditioned bra-
dycardia was not stable in the mutant mice, but rather was exaggerated on Days 2–5 of the
CS-US phase. We examined the effects of reversibly inactivating the cerebellum by injec-
tion of an antagonist against the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate
receptor (AMPAR). The antagonist abolished expression of conditioned responses in both
wild-type and ho15J mice. We conclude that the GluD2 mutation in the ho15J mice affects
stable retention of the acquired conditioned bradycardia. RESEARCH ARTICLE Involvement of GluD2 in Fear-Conditioned
Bradycardia in Mice Hiroko Kotajima-Murakami1¤a¤b, Sakae Narumi2, Michisuke Yuzaki2, Dai Yanagihara1*
1 D
f Lif S i
G
d
S h
l f A
d S i
Th U i
i
f T k
M Hiroko Kotajima-Murakami1¤a¤b, Sakae Narumi2, Michisuke Yuzaki2, Dai Yanagihara1* Data Availability Statement: All relevant data are
within the paper. Data Availability Statement: All relevant data are
within the paper. The cerebellum plays an important role in motor control and is also involved in the control of
cardiovascular functions [1]. The latter role is exemplified by the marked changes in blood
pressure and heart rate produced by electricalstimulation of the anterior or posterior cerebellar
vermis [2, 3]. The linkage of the cerebellum with heart rate control has been investigated using
a classical conditioning paradigm, so-called fear-conditioned bradycardia. Classical condition-
ing is achieved by pairing a conditioning stimulus (CS) with an unconditioned stimulus (US);
this pairing elicits defensive behavioral outcomes such as bradycardia, freezing, and eyeblink-
ing in response to a CS [4]. It has been shown that lesions of the cerebellar vermis severely Funding: This work was supported by: Grants in
Aid for Scientific Research (C), https://www.jsps. go.jp/english/e-grants/, Sasakawa Scientific
Research, Grant from The Japan Science Society,
www.jss.or.jp/ikusei/sasakawa/. Competing Interests: The authors have declared
that no competing interests exist. 1 / 18 PLOS ONE | DOI:10.1371/journal.pone.0166144
November 7, 2016 Involvement of GluD2 in Fear-Conditioned Bradycardia in Mice limit the acquisition and/or expression of conditioned bradycardia in restrained rats and rab-
bits, without disrupting the baseline heart rate or heart rate responses to the US [5, 6]. Condi-
tioned bradycardia is impaired in patients with medial cerebellar lesions [7]. The Purkinje cells
show robust short-latency responses during the CS in conditioned rabbits [8]. In goldfish, cere-
bellar Purkinje cells show simple spike responses to the CS and complex spike responses to the
US during acquisition of fear-conditioned bradycardia [9]. These studies indicate that the cere-
bellum is involved in fear-conditioned bradycardia. It has been suggested that parallel fibers
(PFs) convey the CS and climbing fibers (CFs) transmit the US to the cerebellum during eye-
blink conditioning [10]. In an earlier study, we found that a lesion in the inferior olive nuclei of
mice disrupted the acquisition and/or expression of conditioned bradycardia and the attenua-
tion of tachycardiac responses to the US [11]. These results raise the question of whether
reduction in PF inputs to Purkinje cells and impairment of synaptic plasticity at the PF-Pur-
kinje cell synapses in the cerebellar cortex affect fear-conditioned bradycardia. The δ2 glutamate receptor (GluD2) is a unique receptor that is selectively expressed in Pur-
kinje cells in the cerebellum and localizes at postsynaptic densities in PF-Purkinje cell synapses,
but not at CF-Purkinje cell synapses [12, 13]. Animals Twenty-three wild-type and fourteen ho15J mice (8–10 weeks of age) were used in the experi-
ments. Animals were housed individually and maintained under a 12 hr light/ 12 hr dark cycle
with food and water available ad libitum. The well-being of the mice was carefully monitored,
and all efforts were made to minimize the number of animals used and any suffering in the
course of the experiments. The present study was approved by the Ethics Committee for Animal
Experiments at the University of Tokyo and the Animal Resource Committee of Keio University,
and was performed according to the Guidelines for Research with Experimental Animals of the
University of Tokyo, the Guidelines set by the Animal Resource Committee of Keio University,
and the Guide for the Care and Use of Laboratory Animals (NIH Guide), revised in 1996. Data Availability Statement: All relevant data are
within the paper. GluD2 regulates formation and maintenance of
PF-Purkinje cell synapses by binding Cbln1 released from PFs [14]. GluD2-/- mice show
approximately 50% fewer PF-Purkinje cell synapses than wild-type mice [15]. In addition,
GluD2 is thought to serve as a gatekeeper for long-term depression (LTD) of PF-Purkinje cell
synaptic transmission by regulating endocytosis of postsynaptic α-amino-3-hydroxy-
5-methyl-4-isoxazole propionate receptors (AMPARs) [16, 17]. GluD2-/- mice also display
impaired LTD and ataxic gait [15]. Hotfoot 4J (ho4J) is a spontaneous mutant in which GluD2
is retained in the endoplasmic reticulum [18]. Interestingly, although ho4J mice show normal
acquisition of sound-cued fear conditioned responses (freezing), they do not retain the
responses at 10 min and 24 h following conditioning [19]. However, it is unclear whether fear-
conditioned bradycardia can occur in GluD2 mutants. Ho15J is another spontaneous mutant mouse in which a deletion of exon 2 of the Grid2
gene results in a 52 amino acid deletion in the leucine/isoleucine/valinebinding protein-like
domain of the mature protein [20, 21]. Recently, several human families with a similar deletion
of the GRID2 gene have been reported. These patients show ataxia, abnormal eye movements
and cognitive delay [22–24]. Thus, in the present study, we aimed to clarify the role of the cere-
bellum in fear-conditioned bradycardia through use of ho15J mice. Surgery After anesthetizing with 2% isoflurane, mice were restrained in a stereotaxic instrument and
the scalp was incised. Chronic electrodes for heart rate recording were anchored with dental 2 / 18 PLOS ONE | DOI:10.1371/journal.pone.0166144
November 7, 2016 Involvement of GluD2 in Fear-Conditioned Bradycardia in Mice acrylic.Fine stainless steel wires were inserted subcutaneously and sewn into the sides of the
mice. At the end of surgery, the mice were allowed to recover in a 22°C chamber to avoid hypo-
thermia. The animals were given at least 2 days of recovery before conditioning began. Conditioning procedures Each mouse was habituated to restraint by placing it in a mouse restrainer and attaching elec-
trodes for heart rate recording and tail-shock electrodes; this was carried out twice daily for 60
min. Heart rate was sampled at 90 s intervals throughout each habituation. After habituation,
each mouse received 2 consecutive days of unreinforced CS with a fixed 180 s inter-stimulus
interval. The CS was a 5 s, 80 dB, 2.5 kHz tone. The conditioning phase was carried out for 5
consecutive days and consisted of 50 trials each day. Each presentation of the CS was followed
at offset by a 500 ms, 0.3 mA tail shock US; the trials were performed at 180 s inter-stimulus
intervals. The extinction phase was carried out for 5 consecutive days after the conditioning
phase, followed by 20 presentations of the CS without the US. In all procedures, except for
habituation, the first daily trial was initiated 10 min after the mouse was placed into the cham-
ber without the CS and the US. After the conditioning procedures, the mice underwent a US-
alone phase and the tail-flick test to confirm their response to pain stimuli. The US-alone
phase consisted of 20 trials in a single day in which only the US was presented. In the tail-flick
test, mice received two levels of thermal radiation (80 and 110°C) to the tail and their response
latencies were measured. Conditioning apparatus The conditioning apparatus consisted of a restraining device enclosed within a darkened sound-
attenuating chamber. The chamber contained two speakers mounted on a two-tier rack. The
heart rates of the mice being conditioned were amplified using the implanted chronic electrode
connected to an amplifier (MEG-2100; Nihon Kohden, Tokyo, Japan). The output signal from
the amplifier was divided into two: one part was monitored on an oscilloscope(VC-6725; Hita-
chi, Tokyo, Japan) and the other was digitized using an analog to digital converter (MacLab 8s;
AD Instruments, Dunedin, New Zealand) and stored on a computer at a 1 kHz sampling fre-
quency. A conditioning (tone) stimulus (CS) and unconditioned (electricalshock) stimulus were
delivered using a programmable pulse generator (Master 8; A.M.P.I., Jerusalem, Israel). The tone
stimulus was generated by a synthesizer (1941-Wave-Factory; NF Corporation,Yokohama,
Japan), amplified by a two-channel power amplifier (SRP-P150; Sony, Tokyo, Japan), and deliv-
ered to the mice through two speakers. The sound intensity was measured with a sound-level
meter. The unconditioned stimulus was delivered using an electricalstimulator (SEN-2201;
Nihon Kohden) connected to two shock electrodessecured around the tail of each mouse. Data analysis Changes in heart rate were assessed by measuring the interbeat interval, defined as the time
between successive R-waves (R–R interval) of the cardiac cycle. R-waves of the cardiac cycle
were analyzed using LabChart Software (v.3.6.1/s; AD Instruments). The topography of the
response to the CS was determined by comparing the mean pre-CS heart rate with each 1 s
interval during the 5 s tone. The topography of the response to the US was determined by com-
paring the mean pre-US heart rate with each 1 s interval of the 6 s following the offset of the
US. The occurrence of conditioned bradycardia was defined as a heart rate response during the
fifth second of CS that was lower by 1 standard deviation than the mean heart rate responses
for each of the 10 trials. 3 / 18 PLOS ONE | DOI:10.1371/journal.pone.0166144
November 7, 2016 Involvement of GluD2 in Fear-Conditioned Bradycardia in Mice Histology After injection of 5 μl 0.5% pontamine sky blue into the cerebellar cortex (lobule IV-V), the
mice were intra-cardially perfusedwith saline, followed by 4% paraformaldehyde in 0.1 M
phosphate buffer. The brains were removed and stored in 4% paraformaldehyde in 0.1 M phos-
phate buffer for 1 day. They were then washed sequentially in 5, 10 and 20% sucrose in phos-
phate buffer for 7 days at each concentration. Sagittal sections (80 μm-thickness) were then cut
from each brain using a cryostat. Sections were cut through the entire injection site and stained
with Neutral Red. The sections were then mounted on glass microscope slides and covered
with a coverglass. The extent of diffusion of the pontamine sky blue from the injection site was
measured under a microscope. Rotarod Rotarod tests were performed to measure the effects of NBQX injection. Mice were subjected
to a rotarod test on Day 2 of the CS-alone phase and on Days 2, 3, and 4 of the CS-US phase. The rotarod test on each day was conducted 10 min after conditioning. The mice were placed
on a 5 cm (diameter) rod that was rotated at 8 rpm. Maximum retention time was set as 120 s. Each mouse was used for 10 rotarod trials except on Day 2 of the CS-US phase, when each
mouse was used in 5 trials. Prior to the rotated trials, the mice were examined on the static rod
for up to 120 s. Injection of an antagonist of AMPAR into the cerebellar cortex We investigated whether reversible blocking of excitatory synaptic transmissions in the cerebellar
cortex contributed to the expression of fear-conditioned bradycardia in ho15J mice during the
CS-US phase. To this end, we injected NBQX (2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]qui-
noxaline-7-sulfonamide) into the cerebellar vermis on Day 3 of the CS-US phase in wild-type
and ho15J mice. Seven wild-type mice (termed wild-type (NBQX) mice) and five ho15J mice
(termed ho15J (NBQX) mice) were used. Surgery and conditioning procedures were performed
as describedabove. Under anesthesia (1.5% isoflurane), 5 μl NBQX sodium solution (100 μM)
containing 0.5% pontamine sky blue were injected into the subarachnoid space of the cerebellar
vermis (lobules IV-V) at a rate of 1 μl/min using a 33-gauge micro-syringe needle. Six wild-type
mice (termed wild-type(saline) mice) received 5 μl of saline/ 0.5% pontamine sky blue delivered
in the same manner as in the treated animals. The fear-conditioning experiment was initiated
within 30 min after the injection. Statistical Analyses All data were analyzed using the Statistical Package for Social Sciences (SPSS, Japan, Inc.,
Tokyo, Japan) version 14.0 using Student’s t-test, one-way ANOVA, two-way ANOVA, and
three-way ANOVA for repeated measures and the Bonferroni post hoc test. Results are pre-
sented as means ± standard error of the mean (SEM) and statistically significant differences are
defined as p < 0.05. Conditioned bradycardia in wild-type and ho15J mice during CS-US Conditionedbradycardia was investigated for five consecutive days of the CS-US phase in wild-
type and ho15J mice. The topography of heart rate responses during CS on Day 1 of the CS-US
phase was compared in wild-typeand ho15J mice; the aggregate patterns from 50 trials are shown
in Fig 2. In both wild-typeand ho15J mice, the dominant responses were a decrease in heart rate,
i.e., bradycardia during the five seconds of the CS. There were no significant differences in the
conditioned bradycardia during CS between wild-typeand ho15J mice [F(4, 68) = 0.595, p = 0.514
by two-way repeated measure ANOVA]. There were no significant differences between wild-type
and ho15J mice at the 5th second of CS [wild-typemice, -80 ± 15; ho15J mice, -105 ± 8.4; t (17) =
0.565, p = 0.578 by two-tailed t-test]. Baseline heart rates during the 5 days of the CS-US phase
did not differ between wild-typeand ho15J mice [F(4, 68) = 1.008, p = 0.382 by two-way repeated
measure ANOVA; Fig 3A]. The probabilities of occurrenceof conditioned bradycardia on each of
the five days of the CS-US phase are shown in Fig 3B. Both wild-typeand ho15J mice showed con-
ditioning with no significant difference between the two groups [F(4, 32) = 0.828, p = 0.517 by
two-way repeated measure ANOVA]. These results indicate that the ho15J mice acquired condi-
tioned bradycardia at the same rate as wild-type mice. We compared conditioned bradycardia
during Days 2–5 of the CS-US phase after acquisition in the two groups of mice; conditioned bra-
dycardia in each group over the 5 days of the CS-US phase is shown in Fig 3C and 3D. The data
points in the Figures represent the means from 10 trials collectedat the 5th second of the CS in
each trial. On Days 2–5 of the CS-US phase, the ho15J mice had significantly different response
curvesto the wild-type mice [F(4, 345) = 3.104, p = 0.016 by three-way repeated measure
ANOVA; Fig 3C]. In daily trials, conditioned bradycardia in ho15J mice occurredat similar levels
to that in wild-type mice. Conditioned bradycardia in wild-type and ho15J mice during CS-US Wild-type mice exhibited stable conditioned bradycardia when the last
block on one day was compared to the first block on the next [Day 1–2 of CS-US, -97 ± 12,
-102 ± 9, t(18) = 0.324, p = 0.75; Day 2–3 of CS-US, -79 ± 12, -101 ± 12, t(18) = 1.237, p = 0.232;
Day 3–4 of CS-US, -81 ± 14, -109 ± 8, t(18) = 1.667, p = 0.113; Day 4–5 of CS-US, -92 ± 7,
-102 ± 13, t(18) = 0.684, p = 0.503 by two-tailed t-test; Fig 3D]. By contrast, ho15J mice showed
rather enhanced bradycardia on the first block of each day compared to the last block on the pre-
vious day [Day 1–2 of CS-US, -107± 10, -131 ± 10, t(16) = 1.604, p = 0.128; Day 2–3 of CS-US,
-84 ± 10, -141 ± 19, t(16) = 2.616, p = 0.019; Day 3–4 of CS-US, -99 ± 10, -150 ± 14, t(15) = 2.799,
p = 0.013; Day 4–5 of CS-US, -99 ± 11, -161 ± 14, t(16) = 3.411, p = 0.04 by two-tailed t-test; Fig
3D]. These results indicate that conditioned bradycardia in ho15J mice was not retained appropri-
ately from the last block of each day to the first block on the following day. Heart rate responses in the sound-attenuating chamber and to the CS Ten wild-type and nine ho15J mice were used in the experiments. Wild-type and ho15J mice
showed similar and consistent patterns of baseline heart rates on the second day of habituation
(Fig 1A). The mean heart rate of wild-type mice did not differ significantly from that of ho15J
mice [wild-type, 738 ± 14 beats/min; ho15J, 727 ± 10 beats/min; t (17) = 0.565, p = 0.578 by 4 / 18 PLOS ONE | DOI:10.1371/journal.pone.0166144
November 7, 2016 Involvement of GluD2 in Fear-Conditioned Bradycardia in Mice two-tailed t-test]. On the second day of CS-alone testing, the topography of heart rate
responses was examined at 1 s intervals during the 5 s CS in 50 trials. The aggregate patterns of
heart rate responses for the two mouse groups are shown in Fig 1B. Both wild-type and ho15J
mice showed similar heart rate responses [F(4, 68) = 1.015, p = 0.406 by two-way repeated mea-
sure ANOVA] indicating that the ho15J mutant mice had stable heart rate responses to the
sound-attenuating chamber and the tone-conditioning stimulus. PLOS ONE | DOI:10.1371/journal.pone.0166144
November 7, 2016 Extinction of conditioned bradycardia in wild-type and ho15J mice After the CS-US phase, the mice were allowed five consecutive days of extinction. Both wild-
type and ho15J mice showed gradual extinction of their conditioned bradycardia over this five
day period with no significant differences between genotypes [F(4, 68) = 2.394, p = 0.059 by
two-way repeated measure ANOVA; Fig 4]. Thus, the mutation of GluD2 in the ho15J mice
did not affect the rate of extinction of conditioned bradycardia. 5 / 18 PLOS ONE | DOI:10.1371/journal.pone.0166144
November 7, 2016 Involvement of GluD2 in Fear-Conditioned Bradycardia in Mice PLOS ONE | DOI:10.1371/journal.pone.0166144
November 7, 2016
6 / 18 ne 0166144
November 7 2016
6 / 18 PLOS ONE | DOI:10.1371/journal.pone.0166144
November 7, 2016 PLOS ONE | DOI:10.1371/journal.pone.0166144
November 7, 2016 6 / 18 Involvement of GluD2 in Fear-Conditioned Bradycardia in Mice Fig 1. Heart rate responses to the conditioning chamber and to the tone stimulus. (A) Bars show the mean
heart rate (n = 50 trials) on the second day of habituation for the wild-type (open bar) and ho15J mice (filled bar). (B) Mean change in heart rate (beats/min) during each 1 s period of the 5 s CS collapsed across 50 trials on the
second day of CS-alone testing in wild-type (open circles) and ho15J mice (filled circles). The horizontal line
indicates the final mean heart rate before the 5 s CS collapsed across 50 trials. doi:10.1371/journal.pone.0166144.g001 Involvement of GluD2 in Fear-Conditioned Bradycardia in Mice Effect of NBQX injection into the cerebellum on Day 3 of the CS-US phase
Fig 3. Conditioned bradycardia during the CS-US phase. (A) Mean heart rate for the wild-type and ho15J mice during 5 consecutive days of the
CS-US phase. (B) Probabilities of conditioned bradycardia for wild-type and ho15J mice during the 5 days of the CS-US phase. (C) The conditioned
bradycardia for each second-block on each of the 5 consecutive days of the CS-US phase is shown for the wild type and the ho15J mice. The mean
heart rate change relative to the pre-CS level is shown. (D) The mean heart rate change of the last 10 trials (trial 41–50) on each day and that of the
first 10 trials (trial 1–10) on the following day for each type of mouse during the 5 days of the CS-US phase is indicated by the bars. The mean heart
rate change relative to the pre-CS level is shown. *p < 0.05. doi:10.1371/journal.pone.0166144.g003 Fig 3. Conditioned bradycardia during the CS-US phase. (A) Mean heart rate for the wild-type and ho15J mice during 5 consecutive days of the
CS-US phase. (B) Probabilities of conditioned bradycardia for wild-type and ho15J mice during the 5 days of the CS-US phase. (C) The conditioned
bradycardia for each second-block on each of the 5 consecutive days of the CS-US phase is shown for the wild type and the ho15J mice. The mean
heart rate change relative to the pre-CS level is shown. (D) The mean heart rate change of the last 10 trials (trial 41–50) on each day and that of the
first 10 trials (trial 1–10) on the following day for each type of mouse during the 5 days of the CS-US phase is indicated by the bars. The mean heart
rate change relative to the pre-CS level is shown. *p < 0.05. Response to pain stimuli After completion of the conditioning procedure, mice were subjected to US-alone and tail flick
tests to confirm their responsiveness to pain stimuli. The wild-type and ho15J mice showed no
differences in tachycardia responses to US-alone [F(5, 65) = 0.226, p = 0.950 by two-way
repeated measure ANOVA; Fig 5A] or to tail-flick tests [80°C: wild-type, 7.6 ± 0.2, ho15J,
7.3 ± 0.2, t(17) = 0.848, p = 0.408 by two-tailed t-test, 110°C: wild-type, 4.1 ± 0.1, ho15J,
3.9 ± 0.1, t(17) = 0.805, p = 0.432 by two-tailed t-test; Fig 5B]. The ho15J mice therefore did not
impair heart rate responses to the electricalshock used as the US in fear-conditioning or the
responses to a tail shock. (
)
p
y
yp
J
3.9 ± 0.1, t(17) = 0.805, p = 0.432 by two-tailed t-test; Fig 5B]. The ho15J mice therefore did not
impair heart rate responses to the electricalshock used as the US in fear-conditioning or the
responses to a tail shock. Fig 2. The acquisition of conditioned bradycardia on Day 1 of the CS-US phase. Shown is the mean heart rate change from the pre-CS
baseline through the 5 s CS collapsed across 50 trials on Day 1 of the CS-US phase. The wild-type (open circles) and ho15J mice (filled circles)
demonstrated progressively increasing bradycardia throughout the CS period. doi:10.1371/journal.pone.0166144.g002 Fig 2. The acquisition of conditioned bradycardia on Day 1 of the CS-US phase. Shown is the mean heart rate change from the pre-CS
baseline through the 5 s CS collapsed across 50 trials on Day 1 of the CS-US phase. The wild-type (open circles) and ho15J mice (filled circles)
demonstrated progressively increasing bradycardia throughout the CS period. doi:10.1371/journal.pone.0166144.g002 Fig 2. The acquisition of conditioned bradycardia on Day 1 of the CS-US phase. Shown is the mean heart rate change from the pre-CS
baseline through the 5 s CS collapsed across 50 trials on Day 1 of the CS-US phase. The wild-type (open circles) and ho15J mice (filled circles)
demonstrated progressively increasing bradycardia throughout the CS period. doi:10.1371/journal.pone.0166144.g002 PLOS ONE | DOI:10.1371/journal.pone.0166144
November 7, 2016 7 / 18 E | DOI:10.1371/journal.pone.0166144
November 7, 20 Effect of NBQX injection into the cerebellum on Day 3 of the CS-US phase
The ho15J mice acquired conditioned bradycardia on Day 1 of the CS-US phase and showed The ho15J mice acquired conditioned bradycardia on Day 1 of the CS-US phase and showed
abnormal conditioned bradycardia on Days 2–5 of the CS-US phase. 8 / 18 PLOS ONE | DOI:10.1371/journal.pone.0166144
November 7, 2016 Involvement of GluD2 in Fear-Conditioned Bradycardia in Mice Fig 4. Extinction of conditioned bradycardia. Shown is the daily mean change in heart rate (beats/min) during the 5 s CS
collapsed across daily 20 trials during the five days of the extinction phase. doi:10 1371/journal pone 0166144 g004 Fig 4. Extinction of conditioned bradycardia. Shown is the daily mean change in heart rate (beats/min) during the 5 s CS
collapsed across daily 20 trials during the five days of the extinction phase. doi:10.1371/journal.pone.0166144.g004 doi:10.1371/journal.pone.0166144.g004 To investigate whether conditioned bradycardia in ho15J mice was derived from the cerebel-
lum, we next injected an AMPAR inhibitor NBQX on Day 3 of the CS-US phase; injection of
the AMPAR inhibitor into conditioned rabbits temporarily attenuates the expression of condi-
tioned responses in the eyeblink conditioning paradigm [25]. Mean heart rates during Days
2–4 of the CS-US phase did not differ among wild-type (saline), wild-type (NBQX) and ho15J
(NBQX) mice (see Methods for explanation of the treatment groups) [CS-US 2; F(2, 15) =
0.368, p = 0.698, CS-US 3; F(2, 15) = 2.913, p = 0.085, CS-US 4; F(2, 15) = 0.532, p = 0.598 by
one-way ANOVA; Fig 6A]. We analyzed baseline heart rates in each block on Day 3 of the
CS-US phase (NBQX injection day) (Fig 6B). The data points represent the means from 10 tri-
als collected at one second prior to the CS in each trial. There were no significant differences
among wild-type (saline), wild-type (NBQX) and ho15J (NBQX) mice [F(2, 15) = 2.781,
p = 0.094 by two-way repeated measure ANOVA; Fig 6B]. These results suggest that surgery,
anesthesia and NBQX injection did not influence the baseline heart rate in wild-type (saline),
wild-type (NBQX) and ho15J (NBQX) mice. We showed that conditioned bradycardia
occurredin each group during Days 2–4 of the CS-US phase (compare Fig 7A and 7B to Fig 3C
and 3D). On Day 2 of the CS-US phase (prior to NBQX injection), conditioned bradycardia in
ho15J (NBQX) mice occurredat similar levels to that in wild-type (saline) and wild-type
(NBQX) mice in daily trials, similar to that shown in Fig 3C. PLOS ONE | DOI:10.1371/journal.pone.0166144
November 7, 2016 PLOS ONE | DOI:10.1371/journal.pone.0166144
November 7, 2016 Fig 5. Heart rate responses following a tail shock and responses to pain stimuli. (A) Topography of
heart rate responses following application of a tail-shock as the US, during US-alone testing. Mean changes
in heart rate (beats/min) during each 1 s period of the 6 s following the offset of the US collapsed across 20
trials. (B) Tail-flick test. Wild-type and ho15J mice received 2 levels of thermal stimulus (80˚C or 110˚C). Involvement of GluD2 in Fear-Conditioned Bradycardia in Mice Effect of NBQX injection into the cerebellum on Day 3 of the CS-US phase
The ho15J mice acquired conditioned bradycardia on Day 1 of the CS-US phase and showed However, on Day 3 of the CS-US
phase (NBQX injection day), wild-type (NBQX) mice showed attenuated conditioned brady-
cardia compared to wild-type (saline) mice [F(2, 12) = 11.348, p = 0.002 by two-way repeated
measure ANOVA; Fig 7A]. There were no significant differences in conditioned bradycardia 9 / 18 PLOS ONE | DOI:10.1371/journal.pone.0166144
November 7, 2016 Involvement of GluD2 in Fear-Conditioned Bradycardia in Mice 1/journal.pone.0166144
November 7, 2016
10 / 18 10 / 18 PLOS ONE | DOI:10.1371/journal.pone.0166144
November 7, 2016 Involvement of GluD2 in Fear-Conditioned Bradycardia in Mice Involvement of GluD2 in Fear-Conditioned Bradycardia in Mice Fig 5. Heart rate responses following a tail shock and responses to pain stimuli. (A) Topography of
heart rate responses following application of a tail-shock as the US, during US-alone testing. Mean changes
in heart rate (beats/min) during each 1 s period of the 6 s following the offset of the US collapsed across 20
trials. (B) Tail-flick test. Wild-type and ho15J mice received 2 levels of thermal stimulus (80˚C or 110˚C). Fig 5. Heart rate responses following a tail shock and responses to pain stimuli. (A) Topography of
heart rate responses following application of a tail-shock as the US, during US-alone testing. Mean changes
in heart rate (beats/min) during each 1 s period of the 6 s following the offset of the US collapsed across 20
trials. (B) Tail-flick test. Wild-type and ho15J mice received 2 levels of thermal stimulus (80˚C or 110˚C). in heart rate (beats/min) during each 1 s period of the 6 s following the offset of the US collapsed across 20
trials. (B) Tail-flick test. Wild-type and ho15J mice received 2 levels of thermal stimulus (80˚C or 110˚C). doi:10.1371/journal.pone.0166144.g005
Fig 6. The effects of NBQX injection on mean heart rate. (A) Mean heart rate for the wild-type (saline),
wild-type (NBQX) and ho15J (NBQX) mice during Days 2–4 of the CS-US phase. NBQX was injected into
the wild-type (NBQX) and ho15J (NBQX) mice on Day 3 of the CS-US phase. The wild-type (saline) mice
were injected with saline. (B) Baseline heart rate for the wild-type (saline), wild-type (NBQX) and ho15J
(NBQX) mice during Day 3 of the CS-US phase. doi:10.1371/journal.pone.0166144.g006 doi:10.1371/journal.pone.0166144.g005 Fig 6. The effects of NBQX injection on mean heart rate. (A) Mean heart rate for the wild-type (saline),
wild-type (NBQX) and ho15J (NBQX) mice during Days 2–4 of the CS-US phase. Effect of NBQX injection into the cerebellum on Day 3 of the CS-US phase
The ho15J mice acquired conditioned bradycardia on Day 1 of the CS-US phase and showed However, wild-type (NBQX) mice showed a reduction in conditioned bradycardia on the
first block on Day 3 of the CS-US phase compared to that of the last block on Day 2 of the
CS-US phase [t(12) = -5.431, p < 0.0001; Fig 7B]. There were no significant differences Fig 7. The effects of NBQX injection on Day 3 of the CS-US phase. (A) The conditioned bradycardia for the second-block on each interval
at Days 2–4 of the CS-US phase is shown for the wild-type (saline), wild-type (NBQX) and the ho15J (NBQX) mice. The mean heart rate
change relative to the pre-CS level is shown. (B) The mean heart rate change of the last 10 trials (trial 41–50) on each day and that of the first
10 trials (trial 1–10) on the following day for each type of mouse during Days 2–4 of the CS-US phase is indicated by the bars. The mean
heart rate change relative to the pre-CS level is shown. **p < 0.01, ***p < 0.0001. (C) Probability of the occurrence of conditioned
bradycardia for the wild-type (saline), wild-type (NBQX) and ho15J (NBQX) mice during Days 2–4 of the CS-US phase. ***p < 0.0001. doi:10.1371/journal.pone.0166144.g007 Fig 7. The effects of NBQX injection on Day 3 of the CS-US phase. (A) The conditioned bradycardia for the second-block on each interval
at Days 2–4 of the CS-US phase is shown for the wild-type (saline), wild-type (NBQX) and the ho15J (NBQX) mice. The mean heart rate
change relative to the pre-CS level is shown. (B) The mean heart rate change of the last 10 trials (trial 41–50) on each day and that of the first
10 trials (trial 1–10) on the following day for each type of mouse during Days 2–4 of the CS-US phase is indicated by the bars. The mean
heart rate change relative to the pre-CS level is shown. **p < 0.01, ***p < 0.0001. (C) Probability of the occurrence of conditioned
bradycardia for the wild-type (saline), wild-type (NBQX) and ho15J (NBQX) mice during Days 2–4 of the CS-US phase. ***p < 0.0001. doi:10.1371/journal.pone.0166144.g007 Fig 7. The effects of NBQX injection on Day 3 of the CS-US phase. (A) The conditioned bradycardia for the second-block on each interval
at Days 2–4 of the CS-US phase is shown for the wild-type (saline), wild-type (NBQX) and the ho15J (NBQX) mice. Effect of NBQX injection into the cerebellum on Day 3 of the CS-US phase
The ho15J mice acquired conditioned bradycardia on Day 1 of the CS-US phase and showed NBQX was injected into
the wild-type (NBQX) and ho15J (NBQX) mice on Day 3 of the CS-US phase. The wild-type (saline) mice
were injected with saline. (B) Baseline heart rate for the wild-type (saline), wild-type (NBQX) and ho15J
(NBQX) mice during Day 3 of the CS-US phase. d i 10 1371/j
l
0166144 006 Fig 6. The effects of NBQX injection on mean heart rate. (A) Mean heart rate for the wild-type (saline),
wild-type (NBQX) and ho15J (NBQX) mice during Days 2–4 of the CS-US phase. NBQX was injected into
the wild-type (NBQX) and ho15J (NBQX) mice on Day 3 of the CS-US phase. The wild-type (saline) mice
were injected with saline. (B) Baseline heart rate for the wild-type (saline), wild-type (NBQX) and ho15J
(NBQX) mice during Day 3 of the CS-US phase. doi:10.1371/journal.pone.0166144.g006 Fig 6. The effects of NBQX injection on mean heart rate. (A) Mean heart rate for the wild-type (saline),
wild-type (NBQX) and ho15J (NBQX) mice during Days 2–4 of the CS-US phase. NBQX was injected into
the wild-type (NBQX) and ho15J (NBQX) mice on Day 3 of the CS-US phase. The wild-type (saline) mice
were injected with saline. (B) Baseline heart rate for the wild-type (saline), wild-type (NBQX) and ho15J
(NBQX) mice during Day 3 of the CS-US phase. doi:10.1371/journal.pone.0166144.g006 11 / 18 PLOS ONE | DOI:10.1371/journal.pone.0166144
November 7, 2016 Involvement of GluD2 in Fear-Conditioned Bradycardia in Mice between wild-type (NBQX) and ho15J (NBQX) mice [p = 0.392 by two-way repeated measure
ANOVA; Fig 7A]. On Day 4 of CS-US phase (the Day following NBQX injection), there were
no significant differences in conditioned bradycardia among the mice [F(2, 13) = 1.367,
p = 0.289 by two-way repeated measure ANOVA; Fig 7A]. On both Day 2 and Day 4 of the
CS-US phase, wild-type (NBQX) mice showed similar conditioned bradycardia responses
[F = 0.266, p = 0.616 by two-way repeated measure ANOVA; Fig 7A]. Similar results were also
shown in the ho15J (NBQX) mice [F = 0.025, p = 0.880 by two-way repeated measure ANOVA;
Fig 7A]. Wild-type (saline) mice exhibited stable conditioned bradycardia when the last block
on one day was compared to the first block on the next [Day 2–3 of CS-US, -56 ± 10, -71 ± 11,
t(10) = 1.004, p = 0.339; Day 3–4 of CS-US, -91 ± 10, -109 ± 10, t(10) = 1.207, p = 0.255; Fig
7B]. Involvement of GluD2 in Fear-Conditioned Bradycardia in Mice between the last block on Day 2 of the CS-US phase and the first block on Day 3 of the CS-US
phase in ho15J (NBQX) mice [t(6) = -1.941, p = 0.100; Fig 7B]. Both wild-type (NBQX) mice
and ho15J (NBQX) mice showed an increased conditioned bradycardia on the first block on
Day 4 of the CS-US phase compared to the last block on Day 3 of the CS-US phase [wild-type
(NBQX); t(11) = 3.414, p = 0.006, ho15J (NBQX); t(8) = 3.479, p = 0.008; Fig 7B]. Fig 7C shows
the probability of occurrence of conditioned bradycardia for each type of mouse during Days
2–4 of the CS-US phase. There were no significant differences in the probability of occurrence
of conditioned bradycardia between the three groups on Day 2 of the CS-US phase [F(2, 15) =
0.447, p = 0.647 by one-way ANOVA; Fig 7C]. On Day 3 of the CS-US phase (the NBQX injec-
tion day), the wild-type (NBQX) mice showed a reduction in the probability of occurrenceof
conditioned bradycardia compared to that of the wild-type (saline) mice [p < 0.0001 by Bon-
ferroni post-hoc test]. There were no significant differences in the probability of occurrence of
conditioned bradycardia between the wild-type (NBQX) mice and the ho15J (NBQX) mice
[p = 0.286 by Bonferroni post-hoc test]. On the following day, there were no significant differ-
ences in the probability of occurrence of conditioned bradycardia between the wild-type
(saline) and the wild-type (NBQX) mice [p = 0.866 by Bonferroni post-hoc test]. The levels of
fear-conditioned bradycardia in the wild-type mice (NBQX) were not significantly different
from those in the ho15J (NBQX) mice [p = 0.1000 by Bonferroni post-hoc test]. We confirmed
the effect of NBQX injection on locomotor performance of the wild-type (saline) mice and the
wild-type (NBQX) mice using a rotarod test. The results are shown in Fig 8, in which the first
plotted point in each phase reflects data using the static rod. The wild-type (NBQX) mice
showed a worse performance on Day 3 of the CS-US phase than the wild-type (saline) mice
[F (1, 7) = 6.912, p = 0.034 by two-way repeated measure ANOVA, static rod t(5) = -3.132, Fig 8. The effects of NBQX injection on performance in a rotarod test. Effect of NBQX injection into the cerebellum on Day 3 of the CS-US phase
The ho15J mice acquired conditioned bradycardia on Day 1 of the CS-US phase and showed The mean heart rate
change relative to the pre-CS level is shown. (B) The mean heart rate change of the last 10 trials (trial 41–50) on each day and that of the first
10 trials (trial 1–10) on the following day for each type of mouse during Days 2–4 of the CS-US phase is indicated by the bars. The mean
heart rate change relative to the pre-CS level is shown. **p < 0.01, ***p < 0.0001. (C) Probability of the occurrence of conditioned
bradycardia for the wild-type (saline), wild-type (NBQX) and ho15J (NBQX) mice during Days 2–4 of the CS-US phase. ***p < 0.0001. doi:10.1371/journal.pone.0166144.g007 12 / 18 PLOS ONE | DOI:10.1371/journal.pone.0166144
November 7, 2016 Results of rotarod tests at a
speed of 8 rpm for wild-type (NBQX) (open circles) and control wild-type (saline) mice (gray circles). The
mice were allowed a maximum retention time of 120 s per trial. The first plotted point in each phase
represents data of the static rod. doi:10.1371/journal.pone.0166144.g008 Fig 8. The effects of NBQX injection on performance in a rotarod test. Results of rotarod tests at a
speed of 8 rpm for wild-type (NBQX) (open circles) and control wild-type (saline) mice (gray circles). The
mice were allowed a maximum retention time of 120 s per trial. The first plotted point in each phase
represents data of the static rod. Fig 8. The effects of NBQX injection on performance in a rotarod test. Results of rotarod tests at a
speed of 8 rpm for wild-type (NBQX) (open circles) and control wild-type (saline) mice (gray circles). The
mice were allowed a maximum retention time of 120 s per trial. The first plotted point in each phase
represents data of the static rod. doi:10.1371/journal.pone.0166144.g008 13 / 18 PLOS ONE | DOI:10.1371/journal.pone.0166144
November 7, 2016 Involvement of GluD2 in Fear-Conditioned Bradycardia in Mice p = 0.026 by two-tailed t-test, Fig 8], however, the performance of the wild-type (NBQX) mice
recovered to a level that was similar to that of the wild-type (saline) mice on the following day. A histological analysis confirmed that pontamine sky blue was present in a diffuse pattern
from the cerebellar vermis to the intermediate cerebellum following injection (Fig 9A). In a
sagittal section, the pontamine sky blue was present from lobules III to VI (Fig 9B and 9C). These results indicate that conditioned bradycardia in the ho15J mice was determined by the
cerebellum just as in the wild-type mice. Discussion In the present study, we show that fear-conditioned bradycardia responses could be acquired,
expressed and extinguished in ho15J mice, a GluD2 mutant mouse similar to certain human expressed and extinguished in ho15J mice, a GluD2 mutant mouse similar to certain human
Fig 9. Injection site and diffusion of NBQX in the cerebellum. (A) Schematic showing the injection site and diffusion of pontamine
sky blue in a dorsal view of the cerebellum. The black dot in the blue area indicates the injection site and the blue area shows the
extent of diffusion of pontamine sky blue. The pontamine sky blue was confirmed to extend from the cerebellar vermis to the
intermediate cerebellum. (B) Schematic showing the injection site and diffusion of pontamine sky blue in a sagittal section of the
cerebellum. Arrow shows the injection site. Pontamine sky blue was confirmed to extend from cerebellar lobules III to VI. (C) Sagittal
section was selected at 0.48 mm from midline. Blue dye is pontamine sky blue and red dye is Neutral Red. doi:10.1371/journal.pone.0166144.g009 Fig 9. Injection site and diffusion of NBQX in the cerebellum. (A) Schematic showing the injection site and diffusion of pontamine
sky blue in a dorsal view of the cerebellum. The black dot in the blue area indicates the injection site and the blue area shows the
extent of diffusion of pontamine sky blue. The pontamine sky blue was confirmed to extend from the cerebellar vermis to the
intermediate cerebellum. (B) Schematic showing the injection site and diffusion of pontamine sky blue in a sagittal section of the
cerebellum. Arrow shows the injection site. Pontamine sky blue was confirmed to extend from cerebellar lobules III to VI. (C) Sagittal
section was selected at 0.48 mm from midline. Blue dye is pontamine sky blue and red dye is Neutral Red. doi:10.1371/journal.pone.0166144.g009 Fig 9. Injection site and diffusion of NBQX in the cerebellum. (A) Schematic showing the injection site and diffusion of pontamine
sky blue in a dorsal view of the cerebellum. The black dot in the blue area indicates the injection site and the blue area shows the
extent of diffusion of pontamine sky blue. The pontamine sky blue was confirmed to extend from the cerebellar vermis to the
intermediate cerebellum. (B) Schematic showing the injection site and diffusion of pontamine sky blue in a sagittal section of the
cerebellum. Arrow shows the injection site. Involvement of GluD2 in Fear-Conditioned Bradycardia in Mice patients. Unlike wild-type mice, however, ho15J mice showed enhanced bradycardia on the
first block of trials during Days 2–5 of CS-US phase. In contrast, in the fear-conditioned freez-
ing, although ho4J mice could acquire the conditioned freezing response, they could not retain
it 10 min and 24 h after the conditioning [19]. Interestingly, mature wild-type mice which have
been injected with an anti-GluD2 antibody (anti-δH2) have a similar ability to remain on a
rotarod as control mice; however, 24 h after injection, their performance was poorer than that
of control mice [26]. Similarly, immature TgR/K mice, in which D-Ser-dependent LTD is
impaired, performed more poorly on the first block every day during 5-day sessions [27]. The
results reported in the previous studies are similar to those obtained here, but it remains
unclear why fear-conditioned bradycardia is enhanced, rather than impaired, as in the case for
fear-conditioned freezing and motor learning using rotarod tests. CCAAT/enhancer binding
protein δ (CyEBPd) is a transcriptional regulator and widely expressed in the central nervous
systems [28]. Mice with a targeted deletion of the CyEBPd gene show enhanced contextual fear
responses 24 hr after training [29], suggesting that enhanced fear responses are produced by
deletion of a transcriptional regulator. Although no specific explanation can yet be provided, it
is interesting to speculate that signaling pathways involving GluD2 may be required for mice to
stably retain the fear-memory. We showed that fear-conditioned bradycardia was attenuated by injection of NBQX into
the cerebellum of wild-type and ho15J mice on Day 3 of the CS-US phase. It has been reported
that injection of AMPAR inhibitor into a conditioned rabbit temporarily attenuates the expres-
sion of conditioned responses in eye-blink conditioning [25]. Here, we found that on Day 4 of
the CS-US phase, the ho15J mice showed conditioned bradycardia similar to that prior to the
NBQX injection day (Day 2 of the CS-US phase). This finding indicated that there were no
effects such as irreversible damage of the retention process of fear-conditioned bradycardia in
ho15J mice during Days 2–4 of the CS-US phase. Thus, we suggest that reversible blocking of
AMPAR by injection of NBQX in ho15J mice affected the expression of acquired conditioned
bradycardia, but did not affect the retention of acquired conditioned bradycardia. Discussion Pontamine sky blue was confirmed to extend from cerebellar lobules III to VI. (C) Sagittal
section was selected at 0.48 mm from midline. Blue dye is pontamine sky blue and red dye is Neutral Red. doi:10.1371/journal.pone.0166144.g009 PLOS ONE | DOI:10.1371/journal.pone.0166144
November 7, 2016 14 / 18 14 / 18 PLOS ONE | DOI:10.1371/journal.pone.0166144
November 7, 2016 We are thankful to Ms. Matsuoka for technical support.
Author Contributions
Conceptualization: DY HK.
Formal analysis: HK.
Funding acquisition: DY HK.
Investigation: DY HK SN.
Methodology:DY HK.
Project administration: DY.
Resources: DY HK MY SN.
Software: DY.
Supervision:DY.
Validation: DY HK.
Visualization: HK.
Writing – original draft: HK.
Writing – review& editing: DY MY HK.
References
1.
Nisimaru N. Cardiovascular modules in the cerebellum. Jpn J of Physiol. 2004; 54(5):431–48. doi: 10.
2170/jjphysiol.54.431 PMID: 15667667. We are thankful to Ms. Matsuoka for technical support. Involvement of GluD2 in Fear-Conditioned Bradycardia in Mice eyeblink conditioning was severely impaired in Cbln1-null mice, in which the Cbln1-GluD2
signaling pathway is disrupted, extinction of learned eyeblink responses occurrednormally in
these mice [35]. In the current study, we also found that the absence of GluD2 in ho15J mice
did not affect the extinction of conditioned bradycardia responses, indicating that
Cbln1-GluD2 signaling might be dispensable for extinction of conditioned responses in mice. In eyeblink conditioning, it has been shown that the CS is conveyed to the cerebellum via
PFs, and that the CFs relay the US to the cerebellum [10]. Purkinje cells show simple spike
responses to the CS and complex spike responses to the US during acquisition of fear-condi-
tioned bradycardia in goldfish [9]. Although PF-Purkinje cell synapses are reduced to about
50% and LTD is impaired at PF-Purkinje cell synapses [20, 21], the heart rate responses of
ho15J mice were associated with the CS tone stimulus. These results indicate that cerebellar cir-
cuits might differentially mediate various forms of learning. Further studies are warranted to
better understand physiological and pathological mechanisms by which the cerebellar circuit
mediates different aspects of learning and motor coordination. In the present study, we found that ho15J mice showed acquisition of conditioned bradycar-
dia on Day 1 of the CS-US phase and that they showed aberrant retention of conditioned bra-
dycardia during Days 2–5 of the CS-US phase. Conditioned bradycardia was attenuated by the
injection of NBQX into the cerebellum of ho15J as well as in wild-type mice. Therefore, we con-
clude that the GluD2 signaling pathway plays a crucial role in stable retention of the acquired
conditioned bradycardia. Conceptualization: DY HK. Formal analysis: HK. Funding acquisition: DY HK. Investigation: DY HK SN. Methodology:DY HK. Project administration: DY. Resources: DY HK MY SN. Resources: DY HK MY SN. Software: DY. Supervision:DY. Supervision:DY. Validation: DY HK. Validation: DY HK. Visualization: HK. Writing – original draft: HK. Writing – review& editing: DY MY HK. Author Contributions Conceptualization: DY HK. The cerebel-
lum has previously been shown to be important for fear-conditioned freezing responses and
post-retrievalprocesses in conditioned rats, while synthesis of proteins in the cerebellum is
required for retrieved fear memory [30]. It is believed that the cerebellum may have an impor-
tant role in the retention of fear memory in fear conditioned bradycardia. The role of synaptic plasticity (e.g., long-term potentiation (LTP) vs. LTD) at PF-Purkinje
cell synapses in cerebellum-dependentlearning is contentious. Many genetically engineered
mice, in which molecules required for LTD induction are modified,display impaired motor
learning, such as eyeblink conditioning and adaption of the optokinetic and vestibulo-ocular
reflexes [31]. Recently, GluA2-K882A knock-in mice, in which LTD is abolished, were found
to display normal motor learning [32]. Conversely, Purkinje cell-specificcalcineurin knockout
mice, which have a normal LTD but disrupted LTP, show impaired motor learning [33]. One
of the confounding factors is compensation by parallel backup pathways for motor learning in
genetically modifiedmice. However, we showed that fear-conditioned bradycardia was attenu-
ated similarly by the injection of NBQX in the cerebellum in both wild-type and ho15J mice. We confirmed that the injected NBQX solution spread from the site of injection into the cere-
bellum. The effect of NBQX injection was also confirmed by its effect on the motor perfor-
mance in a rotarod test. These results indicate that fear-conditioned bradycardia during the
CS-US phase was caused by intrinsic mechanisms within the cerebellum in ho15J as well as in
wild-type mice. Because LTD is impaired in ho15J mice [20], LTD may not play a crucial role
in acquisition of fear-conditioned bradycardia. Acquisition and extinction of conditioned freezing responses are reported to be mediated by
LTP and depotentiation in the lateral amygdala, respectively [34]. Although acquisition of 15 / 18 PLOS ONE | DOI:10.1371/journal.pone.0166144
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Nisimaru N. Cardiovascular modules in the cerebellum. Jpn J of Physiol. 2004; 54(5):431–48. doi: 10.
2170/jjphysiol.54.431 PMID: 15667667. References 1. Nisimaru N. Cardiovascular modules in the cerebellum. Jpn J of Physiol. 2004; 54(5):431–48. doi: 10. 2170/jjphysiol.54.431 PMID: 15667667. 16 / 18 PLOS ONE | DOI:10.1371/journal.pone.0166144
November 7, 2016 Involvement of GluD2 in Fear-Conditioned Bradycardia in Mice 2. Nisimaru N, Yamamoto M, Shimoyama I. Inhibitory effects of cerebellar cortical stimulation on sympa-
thetic nerve activity in rabbits. Jpn J of Physiol. 1984; 34(3):539–51. doi: 10.2170/jjphysiol.34.539
PMID: MEDLINE:6492498. 3. Bradley DJ, Ghelarducci B, Spyer KM. The role of the posterior cerebellar vermis in cardiovascular
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MEDLINE:1660994. 4. Sacchetti B, Scelfo B, Strata P. The cerebellum: Synaptic changes and fear conditioning. Neurosci-
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tioned bradycardia in the rabbit. J Neurosci. 1993; 13(9):3705–11. PMID: MEDLINE:8366342. 7. Maschke M, Schugens M, Kindsvater K, Drepper J, Kolb FP, Diener HC, et al. Fear conditioned
changes of heart rate in patients with medial cerebellar lesions. J Neurol Neurosurg Psychiatry. 2002;
72(1):116–8. doi: 10.1136/jnnp.72.1.116 PMID: 11784838. 8. Supple WF Jr., Sebastiani L, Kapp BS. Purkinje cell responses in the anterior cerebellar vermis during
Pavlovian fear conditioning in the rabbit. Neuroreport. 1993; 4(7):975–8. doi: 10.1097/00001756-
199307000-00035 PMID: MEDLINE:8369493. 9. Yoshida M, Kondo H. Fear conditioning-related changes in cerebellar Purkinje cell activities in goldfish. Behav Brain Funct. 2012; 8:52. doi: 10.1186/1744-9081-8-52 PMID: 23114007; PubMed Central
PMCID: PMCPMC3505750. 10. Thompson RF, Steinmetz JE. The role of the cerebellum in classical conditioning of discrete behavioral
responses. Neuroscience. 2009; 162(3):732–55. doi: 10.1016/j.neuroscience.2009.01.041 PMID:
19409234. 11. Kotajima H, Sakai K, Hashikawa T, Yanagihara D. Effects of inferior olive lesion on fear-conditioned
bradycardia. Neuroreport. 2014; 25(8):556–61. doi: 10.1097/WNR.0000000000000135 PMID:
24784584; PubMed Central PMCID: PMCPMC4004639. 12. Araki K, Meguro H, Kushiya E, Takayama C, Inoue Y, Mishina M. Selective expression of the gluta-
mate receptor channel delta 2 subunit in cerebellar Purkinje cells. Biochem Biophys Res Commun. 1993; 197(3):1267–76. doi: 10.1006/bbrc.1993.2614 PMID: MEDLINE:7506541. 13. Takayama C, Nakagawa S, Watanabe M, Mishina M, Inoue Y. Developmental changes in expression
and distribution of the glutamate receptor channel delta 2 subunit according to the Purkinje cell matura-
tion. Brain Res Dev Brain Res. 1996; 92(2):147–55. PMID: 8738121. 14. PLOS ONE | DOI:10.1371/journal.pone.0166144
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glutamate receptor delta2, a bidirectional synapse organizer. Science. 2010; 328(5976):363–8. doi:
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nation, Purkinje cell synapse formation, and cerebellar long-term depression in GluR delta 2 mutant
mice. Cell. 1995; 81(2):245–52. doi: 10.1016/0092-8674(95)90334-8 PMID: 7736576. 16. Kohda K, Kakegawa W, Matsuda S, Yamamoto T, Hirano H, Yuzaki M. The δ2 glutamate receptor
gates long-term depression by coordinating interactions between two AMPA receptor phosphorylation
sites. Proc Natl Acad Sci U S A. 2013; 110(10):E948–57. doi: 10.1073/pnas.1218380110 PMID:
23431139; PubMed Central PMCID: PMCPMC3593918. 17. Hirai H, Launey T, Mikawa S, Torashima T, Yanagihara D, Kasaura T, et al. New role of delta 2-gluta-
mate receptors in AMPA receptor trafficking and cerebellar function. Nat Neurosci. 2003; 6(8):869–76. doi: 10.1038/nn1086 PMID: 12833050. 18. Matsuda S, Yuzaki M. Mutation in hotfoot-4J mice results in retention of delta2 glutamate receptors in
ER. Eur J Neurosci. 2002; 16(8):1507–16. PMID: 12405964. 19. Sacchetti B, Scelfo B, Tempia F, Strata P. Long-term synaptic changes induced in the cerebellar cortex
by fear conditioning. Neuron. 2004; 42(6):973–82. doi: 10.1016/j.neuron.2004.05.012 PMID:
15207241. 20. Motohashi J, Kakegawa W, Yuzaki M. Ho15J—A new hotfoot allele in a hot spot in the gene encoding
the delta 2 glutamate receptor. Brain Res. 2007; 1140:153–60. doi: 10.1016/j.brainres.2006.03.068
PMID: 16647695. 21. Takeuchi E, Sato Y, Miura E, Yamaura H, Yuzaki M, Yanagihara D. Characteristics of gait ataxia in δ2
glutamate receptor mutant mice, ho15J. PLoS One. 2012; 7(10):e47553. doi: 10.1371/journal.pone. 0047553 PMID: 23077633; PubMed Central PMCID: PMCPMC3471872. 22. Hills LB, Masri A, Konno K, Kakegawa W, Lam AT, Lim-Melia E, et al. Deletions in GRID2 lead to a
recessive syndrome of cerebellar ataxia and tonic upgaze in humans. Neurology. 2013; 81(16):1378– PLOS ONE | DOI:10.1371/journal.pone.0166144
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PMCPMC3806907. 86. doi: 10.1212/WNL.0b013e3182a841a3 PMID: 24078737; PubMed Central PMCID:
PMCPMC3806907. 23. 23. Utine GE, Haliloğlu G, Salanci B, C¸ etinkaya A, Kiper P, Alanay Y, et al. A homozygous deletion in
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vents performance of classically conditioned nictitating membrane responses. J Neurosci. 1999; 19
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90386-7 PMID: 8137425. 29. Sterneck E, Paylor R, Jackson-Lewis V, Libbey M, Przedborski S, Tessarollo L et al. Selectively
enhanced contextual fear conditioning in mice lacking the transcriptional regulator CCAAT/enhancer
binding protein delta. Proc Natl Acad Sci U S A. 1998; 95(18):10908–13. PMID: 9724803. 30. Sacchetti B, Sacco T, Strata P. Reversible inactivation of amygdala and cerebellum but not perirhinal
cortex impairs reactivated fear memories. Eur J Neurosci. 2007; 25(9):2875–84. PMID: 17466022. doi: 10.1111/j.1460-9568.2007.05508.x 31. Yuzaki M. Cerebellar LTD vs. motor learning-lessons learned from studying GluD2. Neural Netw. 2013; 47:36–41. doi: 10.1016/j.neunet.2012.07.001 PMID: 22840919. 32. Schonewille M, Gao Z, Boele HJ, Veloz MF, Amerika WE, Simek AA, et al. Reevaluating the role of
LTD in cerebellar motor learning. Neuron. 2011; 70(1):43–50. doi: 10.1016/j.neuron.2011.02.044
PMID: 21482355; PubMed Central PMCID: PMCPMC3104468. 33. Schonewille M, Belmeguenai A, Koekkoek SK, Houtman SH, Boele HJ, van Beugen BJ, et al. Purkinje
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Central PMCID: PMCPMC2941980. 34. Kim J, Lee S, Park K, Hong I, Song B, Son G, et al. Amygdala depotentiation and fear extinction. Proc
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PMCPMC3828671. 18 / 18
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https://www.researchsquare.com/article/rs-764300/latest.pdf
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English
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Effects of Extreme High Temperatures on Proliferation, Cell Cycle, Cell Differentiation and ROS of Adipose-Derived Mesenchymal Stromal Cells
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Research Square (Research Square)
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Hui Cao Haikou Municipal People's Hospital and Central South University Xiangya Medical College A¨liated
Hospital Yuanhui Gao Yuanhui Gao
Haikou Municipal People's Hospital and Xiangya Medical College A¨liated Hospital: Haikou Municipa
People's Hospital and Central South University Xiangya Medical College A¨liated Hospital
Hui Cao
Haikou Municipal People's Hospital and Central South University Xiangya Medical College A¨liated
Hospital
Shunlan Wang
Haikou Municipal People's Hospital and Central South University Xiangya Medical College A¨liated
Hospital
Linlin Zheng
Haikou Municipal People's Hospital and Central South University Xiangya Medical College A¨liated
Hospital
Haowei He
Haikou Municipal People's Hospital and Central South University Xiangya Medical College A¨liated
Hospital
Zhenyu Nie
Haikou Municipal People's Hospital and Central South University Xiangya Medical College A¨liated
Hospital
Mei Chen
Haikou Municipal People's Hospital and Central South University Xiangya Medical College A¨liated
Hospital
Rong Jiang
Fujian Medical University
Denggao Huang ( hdg_qx@163.com )
Haikou Municipal People's Hospital and Xiangya Medical College A¨liated Hospital: Haikou Municipa
People's Hospital and Central South University Xiangya Medical College A¨liated Hospital
https://orcid.org/0000-0002-9927-1622
Shufang Zhang
Haikou Municipal People's Hospital and Central South University Xiangya Medical College A¨liated
Hospital Haikou Municipal People's Hospital and Xiangya Medical College A¨liated Hospital: Haikou Municipal
People's Hospital and Central South University Xiangya Medical College A¨liated Hospital
Hui Cao Research Keywords: high temperature, adipose-derived mesenchymal stromal cells, proliferation, cell cycle, ROS
Posted Date: August 9th, 2021 Effects of extreme high temperatures on proliferation, cell cycle, cell
1
differentiation and ROS of adipose-derived mesenchymal stromal
2
cells
3
Yuanhui Gao1, Hui Cao1, Shunlan Wang1, Linlin Zheng1, Haowei He1, Zhenyu Nie1, Mei Chen1,
4 Backgrounds: Global warming has led to extreme temperatures in different
17
latitudinal regions, resulting in the extinction of a large number of species. This study
18
focuses on the effects of extreme high temperatures on cell proliferation, cell cycle,
19
cell differentiation and mitochondria activity in human adipose-derived mesenchymal
20
stromal cells (hADSCs). 21 stromal cells (hADSCs). 21
Methods: hADSCs were divided into three groups and incubated in 37℃, 39℃ and
22
40℃ environment for 5 hours of exposure each day, and then to 37℃ circumstances
23
for further incubation. Cell surface markers, cell cycle, cell proliferation activity,
24
mitochondrial activity and cell polarization were detected and analyzed by flow
25
cytometry, CCK-8 assay, ROS and JC-1 staining respectively on the 1st and 3rd day of
26
cell culture; osteogenic and adipogenic differentiation ability of hASCs was analyzed
27
by staining after 21 days of osteogenic and adipogenic differentiation induction
28
culture. 29
Results: The results of this study showed that hASCs grown under high temperature
30
conditions had restricted growth activity, blocked S and G2 phases of the cell cycle,
31
reduced cytokinesis and impaired mitochondrial activity, while their osteogenic
32
differentiation ability and membrane potential depolarization were enhanced. 33
Conclusions: hADSCs were subjected to high temperature stimulation with restricted
34 Methods: hADSCs were divided into three groups and incubated in 37℃, 39℃ and
22
40℃ environment for 5 hours of exposure each day, and then to 37℃ circumstances
23
for further incubation. Cell surface markers, cell cycle, cell proliferation activity,
24
mitochondrial activity and cell polarization were detected and analyzed by flow
25
cytometry, CCK-8 assay, ROS and JC-1 staining respectively on the 1st and 3rd day of
26
cell culture; osteogenic and adipogenic differentiation ability of hASCs was analyzed
27
by staining after 21 days of osteogenic and adipogenic differentiation induction
28
culture. 29 Results: The results of this study showed that hASCs grown under high temperature
30
conditions had restricted growth activity, blocked S and G2 phases of the cell cycle,
31
reduced cytokinesis and impaired mitochondrial activity, while their osteogenic
32
differentiation ability and membrane potential depolarization were enhanced. DOI: https://doi.org/10.21203/rs.3.rs-764300/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Effects of extreme high temperatures on proliferation, cell cycle, cell
1
differentiation and ROS of adipose-derived mesenchymal stromal
2
cells
3
Yuanhui Gao1, Hui Cao1, Shunlan Wang1, Linlin Zheng1, Haowei He1, Zhenyu Nie1, Mei Chen1,
4
Rong Jiang2, Denggao Huang1#, Shufang Zhang1#
5
6
#Correspondence:
7
hdg_qx@163.com (Denggao Huang)
8
haikuoyiyuan@163.com (Shufang Zhang)
9
1Department of Central Laboratory, Afliated Haikou Hospital of Xiangya Medical College,
10
Central South University, Haikou 570208, Hainan, China
11
2Department of General Surgery, 900th Hospital of the Joint Logistics Team/Clinical Institute of
12
Fuzhou General Hospital, Fujian Medical University/Dongfang Hospital Affiliated of Xiamen
13
University, Fuzhou 350025, Fujian, China
14
15
Abstract
16
Backgrounds: Global warming has led to extreme temperatures in different
17
latitudinal regions, resulting in the extinction of a large number of species. This study
18
focuses on the effects of extreme high temperatures on cell proliferation, cell cycle,
19
cell differentiation and mitochondria activity in human adipose-derived mesenchymal
20
stromal cells (hADSCs). 21
M h d
hADSC
di id d i
h
d i
b
d i
37℃39℃
d Effects of extreme high temperatures on proliferation, cell cycle, cell
1
differentiation and ROS of adipose-derived mesenchymal stromal
2
cells
3
Yuanhui Gao1, Hui Cao1, Shunlan Wang1, Linlin Zheng1, Haowei He1, Zhenyu Nie1, Mei Chen1,
4
Rong Jiang2, Denggao Huang1#, Shufang Zhang1#
5
6
#Correspondence:
7
hdg_qx@163.com (Denggao Huang)
8
haikuoyiyuan@163.com (Shufang Zhang)
9
1Department of Central Laboratory, Afliated Haikou Hospital of Xiangya Medical College,
10
Central South University, Haikou 570208, Hainan, China
11
2Department of General Surgery, 900th Hospital of the Joint Logistics Team/Clinical Institute of
12
Fuzhou General Hospital, Fujian Medical University/Dongfang Hospital Affiliated of Xiamen
13
University, Fuzhou 350025, Fujian, China
14
15
Abstract
16
Backgrounds: Global warming has led to extreme temperatures in different
17
latitudinal regions, resulting in the extinction of a large number of species. This study
18
focuses on the effects of extreme high temperatures on cell proliferation, cell cycle,
19
cell differentiation and mitochondria activity in human adipose-derived mesenchymal
20
stromal cells (hADSCs). DOI: https://doi.org/10.21203/rs.3.rs-764300/v1 21
Methods: hADSCs were divided into three groups and incubated in 37℃, 39℃ and
22
40℃ environment for 5 hours of exposure each day, and then to 37℃ circumstances
23
for further incubation. Cell surface markers, cell cycle, cell proliferation activity,
24
mitochondrial activity and cell polarization were detected and analyzed by flow
25
cytometry, CCK-8 assay, ROS and JC-1 staining respectively on the 1st and 3rd day of
26
cell culture; osteogenic and adipogenic differentiation ability of hASCs was analyzed
27
by staining after 21 days of osteogenic and adipogenic differentiation induction
28
culture. 29
Results: The results of this study showed that hASCs grown under high temperature
30
conditions had restricted growth activity, blocked S and G2 phases of the cell cycle,
31 Effects of extreme high temperatures on proliferation, cell cycle, cell
1
differentiation and ROS of adipose-derived mesenchymal stromal
2
cells
3
Yuanhui Gao1, Hui Cao1, Shunlan Wang1, Linlin Zheng1, Haowei He1, Zhenyu Nie1, Mei Chen1,
4 33 Results: The results of this study showed that hASCs grown under high temperature
30
conditions had restricted growth activity, blocked S and G2 phases of the cell cycle,
31
reduced cytokinesis and impaired mitochondrial activity, while their osteogenic
32
differentiation ability and membrane potential depolarization were enhanced. 33 Conclusions: hADSCs were subjected to high temperature stimulation with restricted
34 Conclusions: hADSCs were subjected to high temperature stimulation with restricted
34 Conclusions: hADSCs were subjected to high temperature stimulation with restricted
34 growth activity, reduced cell division, impaired mitochondrial activity, significant cell
35
depolarization and enhanced osteogenic differentiation, and these results were closely
36
related to the pathogenic mechanisms of skin aging and heat stroke due to outdoor sun
37
exposure. 38
Keywords: high temperature; adipose-derived mesenchymal stromal cells;
39
proliferation; cell cycle; ROS
40
41 growth activity, reduced cell division, impaired mitochondrial activity, significant cell
35
depolarization and enhanced osteogenic differentiation, and these results were closely
36
related to the pathogenic mechanisms of skin aging and heat stroke due to outdoor sun
37
exposure. 38 growth activity, reduced cell division, impaired mitochondrial activity, significant cell
35
depolarization and enhanced osteogenic differentiation, and these results were closely
36
related to the pathogenic mechanisms of skin aging and heat stroke due to outdoor sun
37
exposure. 38 Keywords: high temperature; adipose-derived mesenchymal stromal cells;
39
proliferation; cell cycle; ROS
40
41 Introduction
42 Recently, global warming has led to extreme temperatures in different latitudinal
43
regions. Some studies have reported that high temperatures from temperate and
44
tropical regions may result in wildlife experiencing a dramatic increase in disease risk
45
[1]. As anthropogenic climate change continues to worsen, the risks to biodiversity will
46
increase over time, and future projections suggest that a potentially catastrophic
47
decline in global biodiversity is imminent [2], and these studies suggest that high
48
temperature environments pose a serious threat to both animals and people. 49 Previous reports have shown that high temperature radiation can cause skin aging
50
and slow down lipid metabolism. Ken Kobayashi et al [3] showed that lactating mice,
51
under moderately high temperature conditions at 39℃, induced higher lactation
52
capacity of mammary epithelial cells (MECs) through control of STAT5 and STAT3
53
signaling. In contrast, prolonged exposure to 41℃ gave rise to a decrease in milk
54
production capacity through inactivation of STAT5 and a decrease in the total number
55
of MECs. It has also been shown that when rats are exposed to high temperature
56
(50℃), lipolysis in adipose tissue is inhibited due to their high body temperature,
57
while intravascular lipolysis is activated [4]. Therefore, understanding the response of
58
human cells to high temperature environment can help us to make positive responses
59
to future environmental changes. 60 With climate change, the increasing incidence of heat-related deaths has been a
61
frequent concern. The 2019 Global Burden of Disease, Injury, and Risk Factors Study
62
demonstrates the non-optimal temperature as one of the top 10 causes of death
63
globally. Globally, 5 million deaths from 2000 to 2019 are associated with abnormal
64
temperatures [6]. For example, a common illness, heat stroke (HS) is a life- threatening
65
disease defined as exposure to excessive hyperthermia at core temperatures above
66
40°C and resulting in a systemic inflammatory response syndrome [7]. HS occurs
67 when multiple tissues and organs are damaged, allowing the secretion of
68
pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α) and
69
interleukin-6 (IL-6), and ultimately systemic inflammation [8]. However, the specific
70
pathogenic mechanisms need to be further investigated. 71 Adipose tissue is a highly metabolically active endocrine organ, which plays an
72
important role in energy storage, energy balance and metabolic regulation [9]. 1 Materials and Methods
86
1.1 Materials
87 Human adipose-derived mesenchymal stromal cells (#HUXMD-01001) and
88
human adipose-derived mesenchymal stromal cell medium (#HUXMD-90031) were
89
purchased from Cyagen Biosciences (Guangzhou). Dulbecco's Phosphate Buffer
90
Solution (D-PBS, #8119217) and Trypsin (#2177715) purchased from Gibco;
91
Antibodies of CD105-PE (#560839), CD73-PE (#550257), CD34-PE (#348057),
92
CD90-PE (#561970), CD45-PE (#557059) purchased from Becton, Dickinson and
93
Company (Guangzhou, China). Cell Counting Kit-8 kit (#SA616) was purchased from
94
Dojindo; ROS fluorescent probe Dihydroethidium-Hydroethidine (DHE, #D1008) and
95
Mitochondrial membrane-potential dye (JC-1, #J4001) were purchased from US
96
EVERBRIGHT, Inc. 97 Introduction
42 73
However, many statistics in recent years have shown that body temperature in healthy
74
adults is gradually decreasing [10-11], suggesting that some changes in adipose tissue in
75
the body are occurring with environmental changes or induced. Therefore, studying
76
the changes in cellular properties in adipose tissue in abnormal environments will help
77
us better understand the effects of environmental changes on the human body. 78
Currently, there are fewer reports on the effects of high temperature environments on
79
adipocytes. The aim of this experiment is to analyze the changes in cell morphology,
80
cell phenotype, cell proliferation, cell differentiation, cell cycle, ROS and membrane
81
potential depolarization of hADSCs at 37℃ - 40℃. The purpose of this study was to
82
analyze the effect of high temperature environment on the biological properties of
83
hADSCs. 84 1.2.1 Cell culture and temperature exposure experiments
99 To understood the effect of different temperatures on hADSCs cells. For the experiment, Passage 4(P4) of hADSCs cells were adjusted to the appropriate cell
101
density and inoculated in 6-well plates. The cells were incubated in different
102
temperature (37℃, 39℃, 40℃) incubators for 5h every day, and the cells were
103
uniformly placed in the same temperature incubator at 37℃ after the end of
104
stimulation, then the cell morphology was then observed and photographed. The
105
duration of continuous treatment ranged from 1-28d depending on the design of the
106
different experiments. 107 1.2.2 Cell surface marker determination assay
108 The cells treated with different temperatures were used to determine the
109
expression of hADSCs surface markers CD105-PE, CD90-PE, CD73-PE, CD34-PE
110
and CD45-PE by flow cytometry. The cells were first digested with 0.25% trypsin and
111
collected in suspension, the cell density was adjusted (1×105 cells/mL) and washed
112
twice with Dulbecco's Phosphate Buffer Solution (D-PBS); the cells were then
113
resuspended in D-PBS, centrifuged at 3000 rpm for 5 min, the supernatant was
114
discarded, the appropriate amount of fluorescently labeled monoclonal antibody was
115
added and incubated for 30 min at room temperature and protected from light. The
116
cells were resuspended with 300 μL D-PBS and analyzed by BD FACSDIVA software,
117
and the surface antigen expression was expressed as a percentage (%). 118 1.2.4 Cell cycle assay
125 The P4 cells were adjusted to 106 cells per tube and centrifuged at 250g for 5 min. 126
The supernatant was discarded, and the cells were fixed in 70% ethanol at 4°C for
127
more than 2 h and centrifuged at 250g for 5 min. 100 μL of RNaseA was added to the
128
supernatant for 30 min in a water bath at 37°C. 400 μL of PI stain was added and
129
mixed at 4°C for 30 min, protected from light. The cells were detected by FACScanto
130
6 flow cytometer and the percentage of cells in G0/G1, S and G2/M phases of the cell
131
cycle (%) was calculated. 132 1.2.3 Cell Counting Kit-8 (CCK-8) assay
119 When the stimulated cells reach the time point of the assay, aspirate all the
120
medium and add 200 μL of fresh medium and 20 μL of CCK-8 reagent (10%), then
121
incubate in the incubator for 1 to 4 h. At the time point, 100 μL of supernatant
122
medium was taken into a 96-well plate and the optical density (OD) was read at 450
123
nm using an enzyme marker. 124 1.2.5 Osteogenic and lipogenic differentiation induction assay
133 The hADSCs of P4 were inoculated into gelatinized 6-well plates. When the
134 cells were confluent to 60-70%, the osteogenic differentiation induction medium was
135
changed every 3 days, and after 2-4 weeks of induction, alizarin red staining was
136
performed; When the cells were confluent to 100%, they were replaced with lipogenic
137
differentiation induction medium solution A and after 3 days with lipogenic
138
differentiation induction medium solution B. After 24 h, solution B was aspirated and
139
solution A was added, and solution A and B were alternately induced 3-5 times, and
140
finally the culture was maintained with solution B for 4-7 days until the lipid droplets
141
became large and round enough for oil red O staining. After induction of
142
differentiation staining by both methods, 10 randomly selected fields of view were
143
photographed under high magnification using uniform photographic conditions. The
144
area of cells stained with red calcified nodules (osteogenic differentiation) or red lipid
145
droplets (lipogenic differentiation) and the area of all cells in the whole area
146
photographed were measured using Image J software, and the ratio of the area of
147
differentiated cells to the total area was calculated as the result and expressed as a
148
percentage (%). 149 1.2.6 Reactive oxygen species (ROS) and JC-1 staining assay
150 The hADSCs of P4 were stained with JC-1 dye and ROS probe to determine the
151
mitochondrial membrane potential and intracellular ROS content, respectively. 152
Staining was performed according to the instructions of the ROS Fluorescent
153
Probes-DHE and JC-1 kits. Diluted ROS probe solution (1μM) and JC-1 dye
154
(10μg/ml) were added to the cell well plates and incubated in an incubator at 37℃ for
155
10-30min protected from light. After being washed twice with D-PBS, they were
156
observed under a fluorescent microscope and photographed. Cells with weak
157
mitochondrial activity stained red for ROS, while cells stained with JC-1 went from
158
red to green fluorescence, indicating a decrease in mitochondrial membrane potential. 159 1.2.7 Statistical Analysis
160 Data were reported as mean and standard error, and SPSS 22.0 and Data were
161
expressed as mean and standard error, and data were analyzed using IBM SPSS
162
Statistics 22.0 (IBM, USA) and plotted using Adobe Photoshop Cs5 (Adobe Systems
163
Inc, USA) and GraphPad prism 7 (GraphPad Software, USA). Differences and
164
significance were verified by one-way ANOVA. A p value <0.05 was considered as
165
statistically significant. 166 2.1 Cell morphology of hADSCs
169 The cell morphology of hADSCs was observed under different temperature
170
gradient treatments, and no morphological differences were observed in hADSCs on
171
the 1st day (Fig 1). When the cells grew to the 3rd day, the cells in the 37℃ group
172
showed a gradual swirling growth trend, while the cells in the 39℃ group showed a
173
scattered growth. vacuole-like changes were seen in the cytoplasm of the cells in the
174
40℃ group. 175 176
Fig1. Cell morphological characteristics of hADSCs in different temperature treatment groups. 177
A-C were the cell morphology of the 1st day in the 37°C, 39°C and 40°C groups, respectively, and
178
D-F were the cell morphology of the 3rd day in each group. 179 76 176
Fig1. Cell morphological characteristics of hADSCs in different temperature treatment groups. 177
A-C were the cell morphology of the 1st day in the 37°C, 39°C and 40°C groups, respectively, and
178
D-F were the cell morphology of the 3rd day in each group. 179
2.2 Cell surface markers of hADSCs
180
To understood whether the phenotype of hADSCs changed after different
181
temperature exposures, we performed flow cytometric assays on cell surface markers
182
of hADSCs treated at 37°C, 39°C and 40°C groups, respectively. The results showed
183
that CD90, CD73 and CD105 expression rates were above 90%, while CD34 and
184
CD45 expression rates were <3% (Fig 2). Statistical analysis showed that CD90 and
185
CD73 were not statistically different between groups on the 1st day and the 3rd day,
186
CD105 expression increased with increasing temperature on the 3rd day (P<0.5), and
187
CD34 and CD45 expression decreased with increasing temperature on the 3rd day
188
(P<0.05). 189 Fig1. Cell morphological characteristics of hADSCs in different temperature treatment groups. Fig1. Cell morphological characteristics of hADSCs in different temperature treatment groups. 177
A-C were the cell morphology of the 1st day in the 37°C, 39°C and 40°C groups, respectively, and
178
D-F were the cell morphology of the 3rd day in each group. 179 g
p
g
p
g
p
A-C were the cell morphology of the 1st day in the 37°C, 39°C and 40°C groups, respectively, and
178
D-F were the cell morphology of the 3rd day in each group. 179 2.2 Cell surface markers of hADSCs
180 To understood whether the phenotype of hADSCs changed after different
181
temperature exposures, we performed flow cytometric assays on cell surface markers
182
of hADSCs treated at 37°C, 39°C and 40°C groups, respectively. The results showed
183
that CD90, CD73 and CD105 expression rates were above 90%, while CD34 and
184
CD45 expression rates were <3% (Fig 2). Statistical analysis showed that CD90 and
185
CD73 were not statistically different between groups on the 1st day and the 3rd day,
186
CD105 expression increased with increasing temperature on the 3rd day (P<0.5), and
187
CD34 and CD45 expression decreased with increasing temperature on the 3rd day
188
(P<0.05). 189 190
Fig2. Flow cytometry analysis of cell surface markers. hADSCs were positive for CD90, CD73,
191
CD10 expression and negative for CD45 and CD34 expression. A-E indicates the percentage of
192
each marker on the 1st day and the 3rd day after hADSCs were treated with different temperature
193
groups, respectively. Values bar were expressed as mean±SEM, *P < 0.05, **P < 0.01. 194
2 3 C ll
lif
i
f hADSC Fig2. Flow cytometry analysis of cell surface markers. hADSCs were positive for CD90, CD73,
191
CD10 expression and negative for CD45 and CD34 expression. A-E indicates the percentage of
192
each marker on the 1st day and the 3rd day after hADSCs were treated with different temperature
193
groups, respectively. Values bar were expressed as mean±SEM, *P < 0.05, **P < 0.01. 194 Fig2. Flow cytometry analysis of cell surface markers. hADSCs were positive for CD90, CD73,
191
CD10 expression and negative for CD45 and CD34 expression. A-E indicates the percentage of
192
each marker on the 1st day and the 3rd day after hADSCs were treated with different temperature
193
groups, respectively. Values bar were expressed as mean±SEM, *P < 0.05, **P < 0.01. 194 2.3 Cell proliferation of hADSCs
195 This experiment used CCK-8 assay to analyze the proliferation viability of
196
hADSCs at different temperatures (37°C, 39°C and 40°C group) by measuring the OD
197
values of the cells. The results were shown in Figure 3: on the 1st day, hADSCs were
198
significantly increased in the 39°C group compared with the other two groups
199
(P<0.05); on the 3rd day, there was no statistical difference between the three groups. 200 201
Fig3. Comparison of different temperature treatment groups on the proliferation of hADSCs. 202 Fig3. Comparison of different temperature treatment groups on the proliferation of hADSCs
202 2.4 Cell cycle variation in hADSCs
204 C and D represent histograms of the proportion of each phase of the cell cycle of
218
hADSCs on the 1st day and the 3rd day after treatment with different temperature groups,
219
respectively. Values bar were expressed as mean±SEM, *P < 0.05, **P < 0.01, ***P < 0.001. 220 2.4 Cell cycle variation in hADSCs
204 The cell cycle of hADSCs in each group was measured by flow cytometry, and
205
the results showed (Fig 4) that at the 1st day, the proportion of cells in G1 phase was
206
greater in the 37℃ group (50.01%±0.40%) than in the 39℃ group (48.53%±
207
0.96%) and less than in the 40℃ group (54.71%±0.61%), and the difference was
208
statistically significant; the proportion of cells in G2 phase was highest in the 39℃
209
group, which was significantly different from the 40°C group; the proportion of
210
S-phase of cells in both the 37℃ and 39°C groups was greater than that in the 40°C
211
group. At the 3rd day, the G1 phase of all three groups increased compared with the 1st
212
day, and the proportion of G1 phase of cells gradually decreased with the increase of
213
temperature, while the proportion of G2 and S phases of cells gradually increased. 214 215
Fig4. Flow cytometry analysis of cell cycle. A and B represent scatter plots of the cell cycle of
216
hADSCs on the 1st day and the 3rd day after treatment with different temperature groups,
217
respectively. C and D represent histograms of the proportion of each phase of the cell cycle of
218
hADSCs on the 1st day and the 3rd day after treatment with different temperature groups,
219 Fig4. Flow cytometry analysis of cell cycle. A and B represent scatter plots of the cell cycle of
216 Fig4. Flow cytometry analysis of cell cycle. A and B represent scatter plots of the cell cycle of
216
hADSCs on the 1st day and the 3rd day after treatment with different temperature groups
217 Fig4. Flow cytometry analysis of cell cycle. A and B represent scatter plots of the cell cycle of
216
hADSCs on the 1st day and the 3rd day after treatment with different temperature groups,
217
respectively. C and D represent histograms of the proportion of each phase of the cell cycle of
218
hADSCs on the 1st day and the 3rd day after treatment with different temperature groups,
219 Fig4. Flow cytometry analysis of cell cycle. A and B represent scatter plots of the cell cycle of
216
hADSCs on the 1st day and the 3rd day after treatment with different temperature groups,
217
respectively. 2.5 Osteogenic and lipogenic differentiation ability of hADSCs
221 After osteogenesis-induced differentiation of hADSCs from three different
222
temperature-stimulated groups, the formation of red calcified nodules was detected by
223
alizarin red staining, and microscopic observation showed that all three groups of cells
224 could form red calcified nodules to different degrees, and the rate of cellular calcium
225
nodule formation increased with the increase of temperature (Fig5.A-F); as shown in
226
the bar graph (Fig5.G), the trend of cellular calcium nodule formation was 37℃ group
227
( 18.71±1.73) < 39 ℃ group (91.03±8.03) < 40 ℃ group (103.58±7.32) (P<0.01). 228
After lipogenesis-induced differentiation of hADSCs from three different temperature
229
stimulation groups, they were stained with oil red O. Microscopic observation showed
230
that all three groups of cells showed lipid droplets of different sizes (Fig6.A-F), the
231
saturation of cellular lipid droplets decreased with increasing temperature, and the
232
size of cellular lipid droplets showed 37°C group ( 32.90±4.32) > 39°C group
233
(28.19±1.32 ) > 40°C group ( 26.69±2.55), with a statistical difference between the
234
37°C and 40°C groups. 235 could form red calcified nodules to different degrees, and the rate of cellular calcium
225
nodule formation increased with the increase of temperature (Fig5.A-F); as shown in
226
the bar graph (Fig5.G), the trend of cellular calcium nodule formation was 37℃ group
227
( 18.71±1.73) < 39 ℃ group (91.03±8.03) < 40 ℃ group (103.58±7.32) (P<0.01). 228
After lipogenesis-induced differentiation of hADSCs from three different temperature
229
stimulation groups, they were stained with oil red O. Microscopic observation showed
230
that all three groups of cells showed lipid droplets of different sizes (Fig6.A-F), the
231
saturation of cellular lipid droplets decreased with increasing temperature, and the
232
size of cellular lipid droplets showed 37°C group ( 32.90±4.32) > 39°C group
233
(28.19±1.32 ) > 40°C group ( 26.69±2.55), with a statistical difference between the
234
37°C and 40°C groups. 235 236 236
Fig5. Osteogenic differentiation potential of hADSCs after different temperature treatment. A-C
237
were the results of alizarin red staining in the 37 °C group, 39 °C group and 40 °C group observed
238
at low magnification, while D-F represent their results at high magnification, respectively. G was
239
the quantification of alizarin red staining score. Values bar were expressed as mean±SEM, **P <
240
0.01, ***P < 0.001. 241 Fig5. Osteogenic differentiation potential of hADSCs after different temperature treatment. 2.5 Osteogenic and lipogenic differentiation ability of hADSCs
221 A-C
237
were the results of alizarin red staining in the 37 °C group, 39 °C group and 40 °C group observed
238
at low magnification, while D-F represent their results at high magnification, respectively. G was
239
the quantification of alizarin red staining score. Values bar were expressed as mean±SEM, **P <
240
0.01, ***P < 0.001. 241 Fig5. Osteogenic differentiation potential of hADSCs after different temperature treatment. A-C Fig5. Osteogenic differentiation potential of hADSCs after different temperature treatment. A-C
237
were the results of alizarin red staining in the 37 °C group, 39 °C group and 40 °C group observed
238
at low magnification, while D-F represent their results at high magnification, respectively. G was
239
the quantification of alizarin red staining score. Values bar were expressed as mean±SEM, **P <
240
0.01, ***P < 0.001. 241 242
Fig6. Lipogenic differentiation potential of hADSCs after different temperature treatment. A-C
243
were the results of oil-red O staining observed at low magnification for the 37 °C group, 39 °C
244
group and 40 °C group, while D-F represent their results at high magnification, respectively. G
245
was the quantification of oil-red O staining scores. Values bar were expressed as mean±SEM, *P <
246
0.05. 247 Fig6. Lipogenic differentiation potential of hADSCs after different temperature treatment. A-C
243
were the results of oil-red O staining observed at low magnification for the 37 °C group, 39 °C
244
group and 40 °C group, while D-F represent their results at high magnification, respectively. G
245
was the quantification of oil-red O staining scores. Values bar were expressed as mean±SEM, *P <
246
0.05. 247 Fig6. Lipogenic differentiation potential of hADSCs after different temperature treatment. A-C
243
were the results of oil-red O staining observed at low magnification for the 37 °C group, 39 °C
244
group and 40 °C group, while D-F represent their results at high magnification, respectively. G
245
was the quantification of oil-red O staining scores. Values bar were expressed as mean±SEM, *P <
246
0.05. 247 2.6 Results of ROS and JC-1 assay
248 The ROS content of hADSCs on the 1st day and the 3rd day after different
249
temperature treatments in the three groups was measured by ROS probes. As shown in
250
Fig 7, at the 1st day, the ROS fluorescence intensity was weak and not significantly
251
different between the 37°C and 39°C groups, while the ROS fluorescence intensity
252
was significantly enhanced in the high temperature (40°C) group; at the 3rd day, the
253
fluorescence was still weak in the 37°C group, while the ROS fluorescence expression
254
intensity was significantly enhanced in the 39°C and 40°C groups. The results of
255
mitochondrial membrane potential measurement using JC-1 dye were shown in Figure
256
8, which showed that the trend of mitochondrial membrane potential change was a
257
gradual increase in membrane potential depolarization with the increase in ambient
258
temperature. The effect of JC-1 membrane potential sensitive dye on mitochondrial
259
depolarization was observed by fluorescence microscopy, with high potentials
260
characterized by red fluorescence, while low potentials showed green fluorescence. 261
The results showed that the depolarization was more pronounced at higher
262
temperatures. 263 The ROS content of hADSCs on the 1st day and the 3rd day after different
249
temperature treatments in the three groups was measured by ROS probes. As shown in
250
Fig 7, at the 1st day, the ROS fluorescence intensity was weak and not significantly
251
different between the 37°C and 39°C groups, while the ROS fluorescence intensity
252
was significantly enhanced in the high temperature (40°C) group; at the 3rd day, the
253
fluorescence was still weak in the 37°C group, while the ROS fluorescence expression
254
intensity was significantly enhanced in the 39°C and 40°C groups. The results of
255
mitochondrial membrane potential measurement using JC-1 dye were shown in Figure
256
8, which showed that the trend of mitochondrial membrane potential change was a
257
gradual increase in membrane potential depolarization with the increase in ambient
258
temperature. The effect of JC-1 membrane potential sensitive dye on mitochondrial
259
depolarization was observed by fluorescence microscopy, with high potentials
260
characterized by red fluorescence, while low potentials showed green fluorescence. 261
The results showed that the depolarization was more pronounced at higher
262
temperatures. 263 264
Fig7. Effect of different temperature groups on ROS content of hADSCs. A-C were the expression
265 264
Fig7. 2.6 Results of ROS and JC-1 assay
248 Effect of different temperature groups on ROS content of hADSCs. A-C were the expression
265
of ROS of hADSCs at the 1st day; D-F were the expression of ROS of hADSCs at the 3rd day. 266 Fig7. Effect of different temperature groups on ROS content of hADSCs. A-C were the
265 Fig7. Effect of different temperature groups on ROS content of hADSCs. A-C were the expression
265
of ROS of hADSCs at the 1st day; D-F were the expression of ROS of hADSCs at the 3rd day. 266 of hADSCs at the 3 day. F were the expression of ROS 267 267 267
Fig 8. Effect of different temperature groups on mitochondrial depolarization of hADSCs. A-C
268
were the mitochondrial membrane potential changes of hADSCs at the 1st day; D-F were the
269
mitochondrial membrane potential changes of hADSCs at the 3rd day. 270
271 Fig 8. Effect of different temperature groups on mitochondrial depolarization of hADSCs. A-C
268
were the mitochondrial membrane potential changes of hADSCs at the 1st day; D-F were the
269
mitochondrial membrane potential changes of hADSCs at the 3rd day. 270
271 Discussion
272 Global warming will affect ecosystems as well as human health in multiple ways,
273
and these impacts are expected to rise dramatically with increasing warming, with
274
estimates that the number of people at climate-related risk will increase by hundreds
275
of millions by 2050. However, it remains difficult to predict the human impacts of the
276
complex interplay of mechanisms driven by warming [12]. At present, the impact of
277
climate warming on human survival and in animals is still poorly understood. 278 Yellow adipose tissue is mainly distributed in subcutaneous tissues, omentum
279
and mesentery, and is the largest energy reservoir in the body. It has the function of
280
storing fat, maintaining and regulating body temperature and participating in fat
281
metabolism [13]. There are more reports on how adipose tissue responds to changes in
282
the external environment at low temperatures, but few studies have examined the
283
changes in adipose tissue at high temperatures. In this study, we chose adipose-
284
derived human adipose mesenchymal stromal cells as the subject to explore the
285
biological properties of the cells and the changes in oxidative stress under high
286
temperature environment. 287 Available reports show that positive expression of CD73, CD105 and CD90, and
288
negative expression of CD34 and CD45 are cell surface markers of ADSCs [14,15]. To
289
clarify whether cell surface markers change at different temperatures exposure. This
290
study showed no significant changes from the cell morphology, but subtle differences
291
in their cell surface markers have been observed. At the 3rd day of treatment at
292
different temperatures, the expression of CD105 was increased at 39°C and 40°C
293
compared to 37°C, indicating an enhanced chondrogenic capacity [16]. In contrast, the
294
expression of CD34 and CD45 decreased and was <5%, suggesting that the cells still
295
have the characteristics of mesenchymal stromal cells [17]. Analysis of cell
296
proliferation viability showed that at 24 h after passaging, the proliferation capacity of
297
hADSCs in the 39°C environment was significantly higher than the other two groups,
298
and by 72 h, the proliferation capacity of cells in the 37°C group was slightly higher
299
than the others, showing that the change in environmental high temperature caused a
300
short-term rapid proliferation of hADSCs, while the opposite proliferation trend
301
occurred by 72 h. Discussion
272 Further analysis in terms of cell cycle showed that at the 3rd day of
302
cell growth, the G1 phase (pre-DNA synthesis phase) was reduced and the S phase
303
(DNA replication phase) and G2 phase (late DNA synthesis phase) were significantly
304
higher in the 39°C and 40°C environments compared to the 37°C group, illustrating
305
that the S and G2 phases of the cells were blocked and the cell division ability was
306
reduced, which were consistent with the formation of a gradual decrease in cell
307
proliferation activity at the 3rd day. 308 The multidirectional differentiation potential of stromal cells is one of the
309
functions of stromal cells measured. This experiment showed that the rate of cellular
310
calcium nodule formation increased with increasing temperature after osteogenesis-
311
induced differentiation of hADSCs in each group, and the saturation of cellular lipid
312 droplets
decreased
with
increasing
temperature
after
lipogenesis-induced
313
differentiation. This indicates that temperature exerts an important influence on the
314
differentiation of stromal cells. Jang et al [18] compared the differences in indoor and
315
outdoor summer temperatures and humidity in Korea and Southeast Asia to assess
316
changes in skin studies showed that repeated exposure to high temperatures and high
317
humidity increased sebum content and hemoglobin index in human skin. However,
318
repeated temperature differences can relax skin blood vessels and reduce skin
319
elasticity, which can contribute to skin aging. These results were associated with a
320
greater tendency for cells to become chondrogenic and differentiated at elevated
321
temperatures. Kim et al [19] recruited 20 women aged 20 to 40 years. After exposing
322
them to outdoor and indoor environments for 90 minutes, skin assessments were
323
performed on the face (forehead and cheeks) and forearms to determine the degree of
324
skin hydration, sebum secretion, transepidermal water loss, pH, and oil content. The
325
study showed that hot environments produce more sweat, increase hydration, sebum
326
secretion, water loss, and lower skin pH. These results suggest that hot environments
327
produce varying degrees of disruption to human skin and to the function and
328
metabolism of epidermal cells. These results are relatively similar to the tendency of
329
hADSCs to osteogenic differentiation by high-temperature environments. 330 Proton pumps are present in the inner mitochondrial membrane and can pump
331
matrix protons into the membrane gap. Discussion
272 Proton translocation across the membrane
332
allows the mitochondrial membrane gap to accumulate large internal amounts of
333
protons, described as a proton gradient: the mitochondrial membrane gap generates a
334
large positive charge while the mitochondrial matrix generates a large negative charge,
335
resulting in a transmembrane potential (ΔΨ) across the inner mitochondrial membrane,
336
referred to as the mitochondrial membrane potential. Normal mitochondrial
337
membrane potential is a prerequisite for maintaining oxidative phosphorylation and
338
ATP production in mitochondria and is necessary for maintaining mitochondrial
339
function, while an important change during apoptosis is the collapse of mitochondrial
340
membrane potential [20]. JC-1 is an ideal fluorescent probe widely used to detect
341
mitochondrial membrane electrical ΔΨm sites. JC-1 dye accumulates within
342
mitochondria in a potential- dependent manner and can be used to detect cellular,
343
tissue or purified mitochondrial membrane potentials. In normal mitochondria, JC-1
344
aggregates in the mitochondrial matrix to form polymers, which emit intense red
345
fluorescence (Ex=585 nm, Em=590 nm); in unhealthy mitochondria, due to a decrease
346 or loss of membrane potential, JC-1 can only exist as a monomer in the cytoplasm,
347
producing green fluorescence (Ex=514 nm, Em=529 nm). JC-1 is not only used for
348
qualitative detection, because the change of color can reflect the change of
349
mitochondrial membrane potential very directly, but also for quantitative detection,
350
because the degree of mitochondrial depolarization can be measured by the ratio of
351
red/green fluorescence intensity. From the experimental results, it was observed that
352
the JC-1 staining changed from red to green with the increase of temperature,
353
suggesting that the mitochondrial membrane potential depolarization was serious at
354
high temperature, and the membrane potential decreased and apoptosis occurred in the
355
cells. 356 ROS is a class of single-electron reduction products of oxygen in the body,
357
which are generated by leaking electrons out of the oxidative respiratory chain and
358
consuming about 2% of the oxygen before it is passed to the terminal oxidase,
359
including the one-electron reduction product of oxygen, superoxide anion, two-
360
electron reduction product, hydrogen peroxide, three-electron reduction product,
361
hydroxyl radical, and nitric oxide. Discussion
272 391 hypoxic state, ATP is reduced, Ca2+ concentration is increased, followed by elevated
381
ROS and oxidative stress, leading to apoptosis, tissue necrosis and autophagy, and
382
eventually multi-organ dysfunction and individual death [23-24]. Interestingly, the
383
results in this experiment showed that ROS was elevated and mitochondrial activity
384
was reduced when hADSCs cells were in a high temperature environment; the cell
385
membrane depolarization was severe during JC-1 staining, which may also lead to
386
dysregulation of Ca2+ flow. It may further explain that when people are under high
387
temperature all the time, the growth of hADSCs cells is restricted and cellular
388
oxidative stress is severe, and then apoptosis or cell death occurs, resulting in the loss
389
of the ability of adipocytes to maintain body temperature, and the final trend is the
390
loss of organismal function and death. 391 ADSCs are widely distributed in vivo, including subcutaneous tissues. The
392
changes in cellular activity and function of hADSCs induced by high temperature are
393
closely related to explain the aging of epidermal cells, dysregulation of lipid
394
metabolism and maintenance of body temperature. Therefore, we hypothesize that
395
humans and animals growing near the equator or in hot regions are affected by high
396
temperatures, which may cause aging of surface cells such as skin in mild cases and
397
impairment of metabolism of cells, tissues and organs in the body in severe cases,
398
thus causing various diseases. 399 In conclusion, hADSCs grown under high temperature conditions have restricted
400
growth activity, blocked S and G2 phases resulting in reduced cell division, enhanced
401
osteogenic differentiation, impaired mitochondrial activity, and severe cell
402
depolarization. These phenomena are directly related to skin aging, apoptosis and heat
403
stroke diseases caused by outdoor exposure to sunlight in summer as reported by
404
previous researchers. These results may provide a further explanation for global
405
warming to localize high temperatures, causing the cause of species extinction. 406
407
Abbreviations
408
hADSCs: human adipose-derived mesenchymal stromal cells; ROS: Reactive oxygen
409
species. 410
411
Acknowledgments
412
The authors thank all students and technicians in the laboratory for their cooperation. 413
414 In conclusion, hADSCs grown under high temperature conditions have restricted
400
growth activity, blocked S and G2 phases resulting in reduced cell division, enhanced
401
osteogenic differentiation, impaired mitochondrial activity, and severe cell
402
depolarization. Discussion
272 ROS production is mainly formed by the high
362
oxygen environment and the high reduced state of the respiratory chain in the
363
mitochondrial transition from state III to state IV, which causes a large number of
364
electrons to leak out and reduce oxygen molecules. Under pathological conditions, the
365
loss of the normal balance between the production and removal of ROS often results
366
in damage to the body by ROS. Oxidative damage mainly includes: first, oxidative
367
damage to nucleic acids, after oxidative damage to the DNA may occur breaks,
368
mutations and changes in thermal stability, which seriously affects the normal
369
transcription and translation of genetic information; second, modification of amino
370
acids, peptide chain breaks, the formation of cross-linked polymers of proteins,
371
changes in conformation and immunogenicity, and other five aspects. Most of the
372
hydroxyl radicals in the organism are produced in the organelles, especially in the
373
mitochondria, causing damage to the mitochondrial membrane and leading to
374
impaired energy metabolism in the cells and the organism [21-22]. The present study
375
showed that with increasing temperature, mitochondrial oxidative stress ROS content
376
increases, demonstrating increased production and release of mitochondrial oxides
377
and inhibited activity. 378 The results of ROS and JC- 1 assays were in line with the mechanistic studies in
379
humans when heat stroke occurs, such as in heat stroke, cells in the body are in a
380 hypoxic state, ATP is reduced, Ca2+ concentration is increased, followed by elevated
381
ROS and oxidative stress, leading to apoptosis, tissue necrosis and autophagy, and
382
eventually multi-organ dysfunction and individual death [23-24]. Interestingly, the
383
results in this experiment showed that ROS was elevated and mitochondrial activity
384
was reduced when hADSCs cells were in a high temperature environment; the cell
385
membrane depolarization was severe during JC-1 staining, which may also lead to
386
dysregulation of Ca2+ flow. It may further explain that when people are under high
387
temperature all the time, the growth of hADSCs cells is restricted and cellular
388
oxidative stress is severe, and then apoptosis or cell death occurs, resulting in the loss
389
of the ability of adipocytes to maintain body temperature, and the final trend is the
390
loss of organismal function and death. Discussion
272 These phenomena are directly related to skin aging, apoptosis and heat
403
stroke diseases caused by outdoor exposure to sunlight in summer as reported by
404
previous researchers. These results may provide a further explanation for global
405
warming to localize high temperatures, causing the cause of species extinction. 406
407 The authors thank all students and technicians in the laboratory for their cooperation. 413
414 Author’s Contributions
415
Yuanhui Gao
performed experiments, provided funding supported
Hui Cao
performed experiments
Shunlan Wang
performed experiments
Linlin Zheng
performed experiments
Haowei He
performed experiments
Zhenyu Nie
helped with the experiments
Mei Chen
helped with the experiments
Rong Jiang
discussed with clinical disease
Denggao Huang
provided funding supported, provided the expert opinion in
temperature design, co-wrote and finalized the manuscript text,
monitored the research design and progress
Shufang Zhang
provided funding supported, monitored the research design and
progress
416
Funding
417
This research was supported by the Hainan Provincial Natural Science Foundation of
418
China (819QN386, 819QN390, ZDKJ2017007)
419
420
Availability of data and materials
421
The data that support the findings of this study are available from the corresponding
422
author upon request. 423
424
Ethics approval and consent to participate
425
Not applicable. 426
427
Consent for publication
428
Not applicable. 429
430
Competing interests
431
The authors declare that they have no competing interests. 432
433
Author information
434 Author’s Contributions
415 Author s Contributions
415
Yuanhui Gao
performed experiments, provided funding supported
Hui Cao
performed experiments
Shunlan Wang
performed experiments
Linlin Zheng
performed experiments
Haowei He
performed experiments
Zhenyu Nie
helped with the experiments
Mei Chen
helped with the experiments
Rong Jiang
discussed with clinical disease
Denggao Huang
provided funding supported, provided the expert opinion in
temperature design, co-wrote and finalized the manuscript text,
monitored the research design and progress
Shufang Zhang
provided funding supported, monitored the research design and
progress
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2019, 2019: 8187529. 496
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Gestão de carreira de psicólogos organizacionais.
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Revista de Carreiras e Pessoas
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Resumo Esta pesquisa tem como objetivo analisar como os psicólogos que realizaram formação
em um Instituto de Especialização em Psicologia Organizacional estão percebendo o sucesso
e a satisfação profissional em suas carreiras. Participaram 42 psicólogos que responderam
a um questionário sociodemográfico, a perguntas de planejamento de carreira e satisfação
profissional e a uma escala de percepção de sucesso na carreira. Os dados foram analisados
descritivamente. Os psicólogos que mais se percebem satisfeitos com suas carreiras
caracterizam-se com idade entre 33 e 34 anos, são casados e conseguem cobrir suas
despesas e se sustentar economicamente. Palavras chave: Sucesso e Satisfação Profissional, Gestão de Carreira, Psicologia Orga-
nizacional. Edemar Zardo 1
Lívia Maria Bedin 2 2 Pós-doutorado em Psicologia (Universitat de Girona), Doutora em Psicologia (UFRGS), Professora do curso
de Especialização em Psicologia Organizacional do IDG-FACCAT, Professora pesquisadora do núcleo stricto
sensu em Psicologia da Faculdade Meridional - IMED, Rio Grande do Sul. liviabedin@gmail.com Artigo recebido 14/09/2015 aprovado 30/03/2016 1 Mestrando em Psicologia pela Unisinos, Pós-Graduação em Psicologia Organizacional pelo IDG Instituto de
Desenvolvimento Global (2015), Graduação em Psicologia pela Universidade Luterana do Brasil (2009).
edezardo@hotmail.com Palavras chave: Sucesso e Satisfação Profissional, Gestão de Carreira, Psicologia Orga-
nizacional. Abstract This research aims to analyze how psychologists who underwent training in a Organizational
Psychology Specialization Institute are perceiving success and job satisfaction in their careers. Forty-two psychologists have attended a sociodemographic survey, a questionnaire on career
planning and job satisfaction, and a career success perception scale. The data was analyzed
descriptively. Psychologists who have most perceived themselves satisfied with their careers
were characterized as aged 33 and 34, were married and could cover their expenses and
sustain themselves economically. Keywords: Success and Career Satisfaction, Career Management, Organizational Psychol-
ogy. a 36
1 Mestrando em Psicologia pela Unisinos, Pós-Graduação em Psicologia Organizacional pelo IDG Instituto de
Desenvolvimento Global (2015), Graduação em Psicologia pela Universidade Luterana do Brasil (2009). edezardo@hotmail.com Página 36 Págin
2 Pós-doutorado em Psicologia (Universitat de Girona), Doutora em Psicologia (UFRGS), Professora do curso
de Especialização em Psicologia Organizacional do IDG-FACCAT, Professora pesquisadora do núcleo stricto
sensu em Psicologia da Faculdade Meridional - IMED, Rio Grande do Sul. liviabedin@gmail.com Artigo recebido 14/09/2015 aprovado 30/03/2016 Zardo, Bedin,
Páginas 36-52 Zardo, Bedin,
Páginas 36-52 Introdução A configuração do mercado de trabalho e das relações de trabalho do século XXI exigem
novas atitudes na postura profissional dos trabalhadores frente às suas carreiras (LASSANCE
& SARRIERA,2012). As trajetórias profissionais antes lineares estão, gradualmente, tornando-
se uma sucessão de transições, e o vínculo com o trabalho torna-se cada vez mais flexível,
provisório e precário (LASSANCE & SARRIERA,2012). Para os profissionais da psicologia
organizacional, o cenário não é diferente. Os psicólogos organizacionais defrontam-se com
uma realidade complexa: uma política salarial defasada que não valoriza os anos de formação
e investimento. A preocupação com o exercício profissional torna-se elemento de reflexão
acerca das necessidades a serem preenchidas no que diz respeito ao conhecimento técnico,
às competências comportamentais e à defasagem entre o conhecimento teórico, a prática
vivenciada e as constantes exigências do mercado de trabalho (COELHO-LIMA, FREIRE
COSTA & YAMAMOTO, 2011). O psicólogo organizacional encontra-se em um contexto incerto na gestão da sua carreira,
tornando-a um desafio e um enfrentamento perante as possibilidades oferecidas e as ações
a serem criadas e desenvolvidas diante de tantas circunstâncias profissionais e laborais
(BEDIN, PARADISO e SARRIERA, 2013). Algumas empresas de grande porte já inseriram o
psicólogo na gestão em todos os níveis hierárquicos de sua estrutura organizacional, todavia
isso se restringe a uma minoria de organizações no Brasil. Em contrapartida, de acordo com
nosso cenário econômico, composto essencialmente por empresas de pequeno e médio porte
que contam com psicólogos em seus quadros funcionais, esses profissionais acabam se
restringindo a tarefas operacionais e cotidianas de administração de pessoal. Isso se verifica
no estudo de Zaneli (2002) o qual afirma que o fazer do psicólogo está estritamente ligado ao
fazer operacional e técnico. Assim, o presente estudo teve como objetivo levantar e analisar de forma descritiva
como os psicólogos organizacionais, ex-alunos de uma Instituição de Formação na área de
Psicologia Organizacional, em nível de Especialização estão gerenciando suas carreiras e
como estão percebendo o sucesso e a satisfação profissional. Psicologia Organizacional A profissão do psicólogo é regulamentada pela lei nº 4.119 de 27 de agosto de 1962
que dispõe sobre essa profissão no território brasileiro quanto ao exercício profissional,
funções legais do psicólogo, formação, diplomação e vida escolar do psicólogo. Essa lei
federal regulamenta a profissão e estabelece os critérios legais e civis para desempenhá-la. A
regulamentação da profissão garante seu exercício, delimitando sua prática e competências
a graduados em curso superior em Psicologia (CFP, 2014). Ao realizar seu exercício, o psicólogo esbarra-se com inúmeras casualidades como,
por exemplo, os desafios da qualificação profissional e as defasagens entre o que é
necessário para um exercício adequado da profissão e o que lhe é instruído. No contexto
de empregabilidade, um estudo de Bedin, Sarriera e Paradiso (2013) avaliou psicólogos e
relatou que esses profissionais, além das dificuldades do mundo profissional, têm que manter
vários vínculos de trabalho, combinam empregos de tempo parcial e, ademais, conciliam o
exercício da psicologia com trabalhos em outras áreas fora do campo. Além disso, no que diz
respeito ao vínculo empregatício, os autores afirmam que apenas 28% dos psicólogos atuam
exclusivamente como autônomos, sendo que 73% possuem algum vínculo assalariado. Para
a maioria, o trabalho autônomo parece ser complementar ao trabalho assalariado (BEDIN,
SARRIERA & PARADISO, 2013). Já no contexto técnico formativo, os pontos mais críticos no âmbito do ensino superior
são a formação científica e as competências para trabalhar com unidades de análise mais
complexas que não o indivíduo, tais como grupos e organizações. As competências nas áreas
clássicas de avaliação, psicodiagnóstico e clínica são, em geral, reconhecidas como as mais
desenvolvidas nos cursos; o que ainda revela o viés clínico presente em grande parte do
nosso sistema de ensino em psicologia (BASTOS, GONDIM, BORGES-ANDRADE, 2010). Do mesmo modo, no âmbito da psicologia organizacional, nota-se uma enorme amplitude,
pois esta almeja entender o comportamento das pessoas que trabalham tanto em determinantes
e suas consequências como nas possibilidades da construção produtiva das ações de
trabalho, satisfação e bem estar e da promoção de qualidade de vida no trabalho (BASTOS &
ZANELLI, 2004). Psicologia Organizacional A atuação prática do psicólogo organizacional direciona-se para o processo
de gestão de pessoas que abrange: perfil de cargo, recrutamento e seleção, treinamento e
desenvolvimento, educação corporativa, avaliação de desempenho, planejamento de carreira,
desenvolvimento gerencial, motivação, saúde do trabalhador, desenvolvimento de lideranças,
gestão, cultura e clima organizacional e processo de mudança, assim como, a questão de
indivíduo-grupo-organização e ambiente. Em síntese, conforme Bastos e Zanelli (2004), o
comportamento humano transposto para a Psicologia Organizacional e do Trabalho pode ser
compreendido como o fazer humano no ambiente de trabalho. Gestão de Carreira O entendimento do cenário de trabalho e o próprio conceito de carreira transformaram-se
durante as últimas três décadas, assumindo as diretrizes do desenvolvimento de carreira a
mesma importância, sejam de natureza individual ou organizacional, valorizando-se a interação
existente entre eles (DUTRA, 2004). O contexto social no qual as experiências de trabalho
e a carreira estão inseridas mudou drasticamente nas últimas décadas. Magalhães (2011)
afirma que, nos últimos 40 anos, permanecer no mesmo trabalho não é mais o modelo de
carreira. Segundo ele, os profissionais devem estar preparados para um padrão de mudanças
periódicas e rápidas em suas carreiras. O autor afirma ainda que o controle da carreira está
se transferindo da organização para o trabalhador e que, a cada dia, torna-se mais importante
para o profissional a condução de sua própria carreira. Pesquisadores e práticos em orientação de carreiras constataram que existem diferenças
individuais importantes relacionadas à adaptabilidade de carreira. Enquanto alguns indivíduos
mostram-se capazes de lidar com as incertezas, outros apresentaram dificuldades para
responder às demandas de autonomia, comportamento exploratório, tomada de decisão e
planejamento profissional (LASSANCE, 2005). Greenhaus (1999) define a gestão de carreira
como um processo pelo qual indivíduos desenvolvem, implementam e monitoram metas e
estratégias de carreira. O autor pressupõe que a otimização desses processos, através de
uma gestão estruturada de carreira, resulta em indivíduos mais produtivos e auto realizados. Martins (2001) complementa essa definição, demonstrando a importância da continuidade
desse processo ao longo do tempo. aPe Revista de Carreiras e Pessoas São Paulo. Volume VI - Número 01 - Jan/Fev/Mar/Abr 201 Gestão de Carreira e Satisfação Profissional 38
Ferramenta, de acordo com Houaiss (2001), é qualquer instrumento necessário à
prática profissional, é um meio para alcançar um fim. A planificação da carreira é a utilização
de métodos, ferramentas e instrumentos intelectuais na facilitação do processo de gerir a
carreira profissional vinculado à vida pessoal. As narrativas são ferramentas usadas em um
primeiro momento no trabalho de orientação, já que engloba quatro conceitos de construção
de carreira: estrutura de vida e de papéis desempenhados, estratégias de adaptação às
situações, motivações e interesses/estilos pessoais. Esses conceitos são retratados nas
histórias de vida de cada sujeito (ARAÚJO, PARADISO, LASSANCE & SARRIERA, 2013). Página 3
O autor Donald E. Super dedicou sua vida ao estudo do desenvolvimento vocacional e de carreira,
criando diversos conceitos, instrumento e ferramentas, entre eles, o conceito de autoconceito
vocacional, a saliência de papéis e o conceito de Maturidade Vocacional (MV) (SUPER, 1980). A Zardo, Bedin,
Páginas 36-52 Zardo, Bedin,
Páginas 36-52 MV foi definida como uma prontidão para realizar escolhas vocacionais alinhadas às demandas
relativas ao desenvolvimento biológico e social, bem como, pelas expectativas sociais de outras
pessoas que alcançaram essa etapa de desenvolvimento (SUPER, SAVICKAS, & SUPER. M,
1996). De acordo com Super, na carreira adulta, estas tarefas se apresentam como exploração,
estabelecimento, manutenção e desengajamento. (LASSANCE, & SARRIERA, 2012). O sucesso
e o reconhecimento na adaptação de cada tarefa evolutiva resultam, em maior efetividade, no
desempenho dos papéis sociais e nas capacidades e estabelecem as bases para o progresso na
sequência de tarefas (LASSANCE, & SARRIERA, 2012). Costa (2010) também contribuiu com o seu conceito de carreira, pontuando em seus
estudos que para entender as carreiras contemporâneas é preciso atribuir maior importância
à carreira subjetiva. Segundo ele, na ausência de uma estabilidade das posições hierárquicas,
as pessoas tendem a focar na carreira subjetiva como parâmetro para o crescimento na
carreira, dedicando-se mais aos processos do que aos resultados, às competências ao invés
de títulos, à realização antes da promoção e preferindo papéis a posições. Somando-se a isso, Weiner (1986), em seu modelo de crenças atribucionais, analisa as
explicações referidas por indivíduos sobre os fatores de sucesso ou de fracasso em contextos
de realização e permite a identificação de um reduzido número de causas particularmente
salientes. Gestão de Carreira e Satisfação Profissional Janeiro (2011), por exemplo, afirma que o esforço é considerado uma atribuição
interna controlável, já as aptidões ou capacidades são percebidas como não controláveis,
uma vez que estão para além da vontade pessoal. De acordo com Janeiro (2011), as crenças
atribucionais estão na base de uma rede de interações salientes para o desenvolvimento
vocacional. A noção de controle pessoal sobre o sucesso na carreira influencia diretamente
sobre o modo como os jovens perspectivam o futuro. Percepção de Sucesso na Carreira. ReCaPe Revista de Carreiras e Pessoas São Paulo. Volume VI - Número 01 - Jan/Fev/Mar/Abr 2016 Procedimentos para coleta de dados Procedimentos para coleta de dados Os participantes foram contatados por um e-mail padrão no qual constavam os objetivos
do estudo, os procedimentos éticos para a pesquisa online com seres humanos e um link com
o qual poderiam acessar os instrumentos. Na abertura da página, apresentou-se o Termo de
Consentimento Livre e Esclarecido (ANEXO A) e, após a indicação de estarem cientes do
conteúdo da pesquisa e aceitarem participar, os participantes tinham acesso aos instrumentos
para preenchimento. Porém, devido ao tempo de existência do Instituto de Formação de mais
de 15 anos, muitas informações dos e-mails dos alunos não estavam atualizadas. Assim, optou-
se por fazer uso de uma rede social (Facebook) e, por meio da formação indicada pelos ex-
alunos, foi possível localizar os participantes. Dessa forma, seguiu-se o mesmo procedimento
via e-mail, encaminhando convite com o link da pesquisa e, após o consentimento, os alunos
poderiam responder aos questionários. Esta pesquisa foi aprovada pelo Comitê de Ética em
Pesquisa da Faccat. Dos alunos contatados na rede social, foi possível notar que 164 foram
alcançados: 85 receberam a mensagem de convite, mas não o visualizaram e 79 receberam
o convite e visualizaram. Destes, houve um número de 45 respondentes, sendo que 3 foram
excluídos da pesquisa por ocorrerem ausências de respostas em algumas perguntas, sendo
válidos para análise 42. Procedimento Metodológico Esta pesquisa é quantitativa, caracterizando-se por um estudo descritivo transversal,
realizada por meio do levantamento de dados de uma amostra de psicólogos (as)
organizacionais. Participantes Participaram deste estudo 42 psicólogos organizacionais, 39 mulheres (92,9%) e 3
homens (7,1%), com idade entre 30 anos e 68 anos (M = 37 anos e DP = 0,261). Todos
possuem nível superior e renda entre R$1.500,00 e R$ 13.000,00 mensal. Foram incluídos
na pesquisa os psicólogos que responderam aos questionários e que concluíram a formação
ou Pós-Graduação em Psicologia Organizacional. A atuação profissional se caracterizou
por profissionais autônomos, com vínculo CLT efetivos, prestadores de serviço e psicólogos
que, além de trabalharem com psicologia organizacional, também atuam como psicólogos
clínicos, escolares e docentes, bem como, em outras áreas da psicologia ou atividades para
complementar renda. Percepção de Sucesso na Carreira. O sucesso na carreira é definido como o acúmulo de resultados positivos, psicológicos e
profissionais reflexos do trabalho. O sucesso é um conceito avaliativo, por isso, julgamentos
de sucesso na carreira dependem de quem está julgando. Quando julgado por outros, o
sucesso é determinado com bases relativamente objetivas e com critérios visíveis, o que
muitos pesquisadores chamam de sucesso objetivo na carreira, sendo mensurado por
métricas observáveis, tais como, salário e número de promoções (JUDGE, et al, 1995). Percepção de sucesso na carreira é a interpretação da pessoa sobre as suas realizações
em relação à sequência de posições ocupadas, às ações, aos comportamentos, aos trabalhos
e aos estudos. Além disso, a percepção de sucesso está relacionada às experiências de
vida que refletem o desenvolvimento de competências para lidar com situações de maior
complexidade. Essas situações estão em constante transformação e são influenciadas e
negociadas em função de motivos e aspirações individuais e de expectativas e imposições
organizacionais e sociais (DRIES, et al, 2008). Dries, et al (2008) pontua algumas:
desempenho, promoção, contribuição, desenvolvimento, criatividade, segurança, satisfação,
reconhecimento e cooperação. Pá i
39
Percepção é o processo pelo qual os indivíduos organizam e interpretam suas impressões
sensoriais com o objetivo de dar sentido ao seu ambiente. Embora a percepção tenha uma
definição voltada para os órgãos dos sentidos, ou seja, para a interpretação de fatos concretos,
a psicologia considera este termo para os estudos do comportamento, pois o comportamento
das pessoas é baseado na interpretação que elas fazem da realidade e não na realidade em
si (ROBBINS, 2005). Assim, indivíduos percebem o sucesso na carreira como a realização
de metas desejáveis em relação ao trabalho ao longo das experiências profissionais e de
acordo com o contexto e o momento profissional em que se encontram ao longo da trajetória
profissional. Essa definição enfoca o alcance de metas pessoais, ou seja, a carreira deve ser
avaliada em função das expectativas da pessoa e de sua percepção. (ARTHUR, et al, 2005) ReCaPe Revista de Carreiras e Pessoas São Paulo. Volume VI - Número 01 - Jan/Fev/Mar/Abr 2016 Instrumentos Para este estudo, foi aplicado um questionário composto por duas partes: Parte 1 – Questionário sociodemográfico: idade, sexo, nível de formação, renda, estado civil,
número de filhos e questões relacionadas ao planejamento de carreira. As perguntas sobre
planejamento de carreira foram respondidas numa escala likert: 1 discordo totalmente e 5
concordo totalmente. Parte 2 - Escala de percepção de Sucesso na Carreira (COSTA, 2010): essa Escala foi validada
por Luciano Venelli Costa (2010) e é composta por 48 itens divididos em 11 dimensões e uma
questão específica referente à percepção de sucesso na carreira. Todas as perguntas foram
respondidas numa escala likert: 1 - discordo totalmente - e 10 - concordo totalmente. Na
Tabela 1, pode-se observar a definição de cada dimensão, bem como, a consistência interna
medida pelo alfa de Cronbach da escala original e da amostra deste estudo. Página 40 Zardo, Bedin,
Páginas 36-52 i
41
Tabela 1: Alfa de Cronbach da escala original e da amostra deste estudo. Fator
Definição
Itens
Alfa
(Costa,
2010)
Alfa
(Presente
estudo)
Identidade
Revela o quanto a carreira faz senti-
do para a pessoa como expressão do
seu ser, trazendo-lhe felicidade e re-
conhecimento das pessoas. 13, 15,
22, 25 e
34
0,81
0,89
Competência
Revela o quanto a pessoa sente-se
competente no que faz, o quanto se
diferencia em termos profissionais. 17 ,23,
24, 35,
38 e 39
0,82
0,97
Desenvolvimento
Revela o quanto a pessoa percebe
que seu trabalho é cada vez mais
desafiante, desenvolvendo-se como
profissional. 7, 8, 26,
27 e 43
0,78
0,97
Contribuição
Revela o quanto a pessoa percebe
que sua carreira contribui com a so-
ciedade. 14, 41,
46, 49 e
52
0,83
0,98
Cooperação
Revela o quanto a pessoa se consi-
dera bem sucedida no trabalho em
equipe. 16, 21,
33, 47 e
51
0,74
0,94
Criatividade
Revela o quanto a pessoa se percebe
criativa profissionalmente. 1, 3 e 28
0,67
0,92
Empregabilidade
Revela o quanto a pessoa se sente
segura em relação a ter oportunidade
de trabalho. 30, 32 e
50
0,71
0,87
Valores
Revela o quanto a pessoa percebe
que respeita seus valores enquanto
desenvolve sua carreira, trabalhando
de forma ética e com orgulho. 10, 12 e
29
0,57
0,97
Hierarquia e Pro-
moção
Revela o quanto a pessoa está satis-
feita com seu sucesso em termos de
posição hierárquica e promoções ob-
tidas na carreira. Dados Sociodemográficos. O estudo foi composto 42 psicólogos organizacionais, 39 mulheres (92,9%) e 3 homens
(7,1%), com idade entre 30 anos e 68 anos (M = 37 anos e DP = 0,261). A idade enquadrou-
se em faixas entearias: até 32 anos 8 participantes = 19,0%, 33 até 34 anos 15 participantes =
35,7%, de 35 a 38 anos 8 participantes = 19,0% e 39 anos ou mais 11 participantes = 26,2%. O estado civil caracterizou-se em solteiro (a) / separados (as) 15 participantes = 35,7% e
casado(a) / relação estável 27 participantes = 64,3%. Os participantes que possuem filhos
são 17 = 40,5% e que não possuem são 25 = 59,5%. O número de filhos deu-se que 1 filho =
8 participantes = 19,0%, 2 filhos 6 participantes e 3 filhos = 2 participantes = 4,8%. A moradia
descreve-se com 6 participantes = 14,3% que moram sozinhos(as) e 36 participantes = 85,7%
moram com família ou parceiro(a). O grau de instrução temos 31 participantes = 73,8% com
especialização e outros cursos, 7 participantes = 16,7% com mestrado e 4 participantes =
9,5% com doutorada e pós-doutorado. A renda ficou em quatro faixas, sendo a primeira faixa
até R$ 2.5000,00 tendo 9 participantes = 21,4%, a segunda faixa de R$ 2.500,00 até R$
4.000,00 com 12 participantes = 28,6%, a terceira faixa de R$ 4.000,00 até R$ 5.000,00
com 9 participantes = 21,4% e a quarta faixa acima de R$ 7.000,00 com 12 participantes =
28,6%. Dos participantes que consegue cobri suas despesas temos 32 participantes = 76,2%
que sim e 10 participantes = 23,8% que não. Aos participantes que recebem auxilio temos
15 participantes = 35,7% e os que não recebem são 27 participantes = 64,3%. O vínculo
empregatício caracteriza-se por CLT efetivo com 19 participantes = 45,2%, profissional
autônomo / consultor / proprietário de negócio 16 participantes = 38,1%, servidor público
3 participantes = 7,1% e outros 4 participantes = 9,5%. O tempo de atuação profissional
ficou dentro de 4 faixas, a primeira faixa até 4 anos com 9 participantes = 21,4%, a segunda
faixa de 4 anos a 8 anos com 5 participantes = 11,9%, a terceira faixa 8 anos a 10 anos com
9 participantes = 21,4% e a quarta faixa mais de 10 anos com 19 participantes = 45,2%. Análise de dados Análise de dados Os dados foram analisados no software SPSS (Statistical Package for the Social Science)
– versão 20. Foram realizadas análises descritivas como média, desvio padrão e frequências
das variáveis estudadas. Para verificar como os psicólogos organizacionais estão percebendo
o sucesso e a satisfação profissional, foram realizadas correlações entre as 11 dimensões
de sucesso e a percepção de satisfação na carreira. Também são realizadas análises de
variância (ANOVA) para verificar se existem diferenças entre as médias de satisfação e as
do sucesso na carreira entre os grupos, considerando-se: estado civil (Solteiro/Separado ou
Casado), ter filhos (Sim ou Não), idade (Até 32 anos, 33 e 34 anos, 35 a 38 anos e 39
anos ou mais), tipo de vínculo empregatício (CLT, Autônomo, Servidor Público ou Outros),
se consegue cobrir as despesas com o seu salário (sim ou não), renda (Até R$ 2.500,00,
De R$ 2.500,00 Até R$ 4.000,00, De R$ 4.000,00 Até 5.000,00 e Acima de R$ 7.,000,00),
escolaridade (Especialização e Outros, Mestrado e Doutorado e Pós Doutorado) e tempo de
atuação profissional (Até 4 anos, De 4 a 8 anos, De 8 a 10 anos e Mais de 10 anos). Instrumentos 19, 36,
42, 45 e
48
0,84
0,93
Remuneração
Revela o quanto a pessoa se percebe
bem-sucedida em termos de recom-
pensas financeiras obtidas na carrei-
ra. 2, 4, 20
e 53
0,78
0,93
Equilíbrio
Vida-
Trabalho
Revela o quanto a pessoa está satis-
feita com sua capacidade em lidar
com os desafios de desenvolver a
carreira e aproveitar a vida pessoal. 5, 11, 37
e 44
0,72
0,87 ReCaPe Revista de Carreiras e Pessoas São Paulo. Volume VI - Número 01 - Jan/Fev/Mar/Abr 2016 ReCaPe Revista de Carreiras e Pessoas São Paulo. Volume VI - Número 01 - Jan/Fev/Mar/Abr 2016 Dados Sociodemográficos. A área de atuação abrange psicologia organizacional 23 participantes = 54,8%, psicologia
organizacional e clínica 5 participantes = 11,9%, psicologia organizacional de docência 2
participantes = 4,8%, psicologia organizacional e outros trabalhos para complementar renda
2 participantes = 4,8%, psicologia organizacional, clínica e outros 1 participante = 2,4%,
psicologia clínica 2 participantes = 4,8%, psicologia clínica, docência e outros 1 participante
= 2,4%, docência 1 participante = 4,2%, docência e outros 1 participante = 2,4% e outros 4
participantes = 9,5%. Página 42 Zardo, Bedin,
Páginas 36-52 Zardo, Bedin,
Páginas 36-52 Zardo, Bedin,
Páginas 36-52 Variáveis de Planejamento de Carreira Variáveis de Planejamento de Carreira A Tabela 2 exibe os resultados descritivos das questões relacionadas com o planejamento
de carreira dos psicólogos organizacionais. Nela apresenta-se o mínimo, o máximo, a média
e o desvio-padrão (DP). Nota-se que a variável que apresentou a média mais alta foi: “Planejo
e faço a gestão de minha carreira por meio do conhecimento adquirido como profissional
e da experiência de vida” (M = 4,24 e DP = 0,72) e a que teve a média mais baixa foi: “No
planejamento de minha carreira, sigo as orientações de um profissional especialista em
planejamento de carreira” (M = 1,76 e DP = 1,07). Tabela 2 - Variáveis de Planejamento de Carreira
Variáveis de Planejamento de Carreira
Min. Máx. Média
DP
15. No planejamento de minha carreira, sigo as
orientações de um profissional especialista em plane-
jamento de carreira. 1
4
1,76
1,07
16. Planejo e faço a gestão de minha carreira por
meio do conhecimento adquirido como profissional e
da experiência de vida. 2
5
4,24
0,72
17. No planejamento de minha carreira, sigo o fluxo
da vida e do mercado de trabalho. 1
5
3,55
0,96
18. No planejamento de minha carreira, recebi ajuda
de colegas para as realizações profissionais da minha
vida. 1
5
3,02
1,25
19. No planejamento de minha carreira, a ajuda/apoio
de colegas foi de fundamental importância para a
minha atuação como profissional. 1
5
3,21
1,20
20. O quanto você está satisfeito com sua carreira
profissional. 2
5
3,95
0,88
Dimensões de Percepção de Sucesso Na Tabela 3, são descritas as 11 dimensões de percepção de sucesso com respectivas
mínimo, máximo, média, desvio-padrão (DP). Na Tabela 3, são descritas as 11 dimensões de percepção de sucesso com respectivas
mínimo, máximo, média, desvio-padrão (DP). Dados Sociodemográficos. Página 43 ReCaPe Revista de Carreiras e Pessoas São Paulo. Volume VI - Número 01 - Jan/Fev/Mar/Abr 2016 Tabela 3 - 11 Dimensões de Percepção de Sucesso. 11 Dimensões de Percepção de Sucesso
Míni
Máxi
Média
DP
Média Identidade
2
10
7,19
2,12
Média Competência
1,5
10
7,61
2,26
Média Desenvolvimento
2
10
7,46
2,35
Média Contribuição
1,2
10
7,81
2,35
Média Cooperação
1,6
10
7,64
2,27
Média Criatividade
2,33
10
6,76
2,22
Média Empregabilidade
1,67
10
6,57
2,48
Média Valores
1,33
10
8,30
2,32
Média Hierarquia e Promoção
2,4
10
6,92
2,36
Média Remuneração
1,5
9
5,73
2,21
Média Equilíbrio Vida Trabalho
2
9,75
6,71
2,18 Tabela 3 - 11 Dimensões de Percepção de Sucesso. Com relação às 11 dimensões de percepção de sucesso na careira, observa-se que os
psicólogos entrevistados apresentam médias mais baixas para as dimensões remuneração
(M = 5,73; DP = 2,21) e empregabilidade (M = 6,57; DP = 2,48). As médias mais altas foram
para as dimensões de valores (M = 8,30; DP = 2,32), contribuição (M = 7,81; DP = 2,35) e
cooperação (M = 7,64; DP = 2,27). Correlação. A Tabela 4 apresenta as correlações entre as 11 dimensões de percepção de sucesso e a
percepção de satisfação na carreira. A Tabela 4 apresenta as correlações entre as 11 dimensões de percepção de sucesso e a
percepção de satisfação na carreira. Tabela 4 - Correlação
O quanto você está satisfeito com sua carreira profis-
sional? (r de Pearson)
Média Identidade
0,518**
Média Competência
0,432**
Média Desenvolvimento
0,449**
Média Contribuição
0,416**
Média Cooperação
0,385**
Média Criatividade
0,433**
Média Empregabilidade
0,533**
Média Valores
0,312*
Média Hierarquia Promoção
0,583**
Média Remuneração
0,615**
Média Equilíbrio Vida-Trabalho
0,468**
Nota. ** p<0,01 *p<0,05. O quanto você está satisfeito com sua carreira profis-
sional? Página 44 Zardo, Bedin,
Páginas 36-52 Zardo, Bedin,
Páginas 36-52 Observa-se que as 11 dimensões se correlacionam significativamente e positivamente
com a satisfação dos profissionais. A média da dimensão remuneração mostra que quanto
maior a percepção de recompensas financeiras, maior a satisfação do profissional com a
carreira, sendo esta a variável com maior correlação. A dimensão valores foi a que apresentou
menor correlação com a satisfação, mas mesmo assim se correlacionam significativamente. Escolaridade Especialização e
Outros
3,90 (0,94)
p = 0,208
6,84 (2,11)
p = 0,383
Mestrado
4,43 (0,53)
7,86 (2,34)
Doutorado e
Pós-doutorado
3,50 (0,57)
6,00 (2,22)
Tempo
de
atuação Pro-
fissional
Até 4 anos
3,89 (1,05)
p = 0,769
6,67 (2,34)
p = 0,910
De 4 a 8 anos
3,60 (0,54)
6,80 (1,30)
De 8 a10 anos
4,11 (0,78)
6,67 (2,55)
Mais de 10 anos
4,00 (0,94)
7,21 (2,32) Observa-se, conforme a Tabela 5, que não há diferenças significativas entre as médias
de percepção de sucesso na carreira considerando-se os grupos demográficos. Já para a
satisfação com a carreira, observa-se que existem diferenças significativas entre as médias
considerando-se o estado civil (com média mais alta para os casados), a idade (com média
mais alta para os participantes de 33 a 34 anos), a renda (com média mais alta para os
participantes com renda acima de R$ 4.000,00) e se consegue cobrir as despesas (com média
mais alta para os participantes que conseguem cobrir). Comparações de Médias da Satisfação, Percepção de Sucesso e Dados Sociodemográfico A Tabela 5 apresenta as diferenças de médias da satisfação e do sucesso entre os grupos
considerando: estado civil (solteiro/separado ou casado), ter filhos (sim ou não), idade (até
32 anos, 33 e 34 anos, 35 a 38 anos e 39 anos ou mais), tipo de vínculo empregatício (CLT,
autônomo, servidor público ou outros), se consegue cobrir as despesas com o seu salário (sim
ou não), renda (até R$ 2.500,00, de R$ 2.500,00 até R$ 4.000,00, de R$ 4.000,00 até 5.000,00
e acima de R$ 7.000,00), Escolaridade (especialização e outros, mestrado e doutorado e pós-
doutorado) e tempo de atuação profissional (até 4 anos, de 4 a 8 anos, de 8 a 10 anos e mais
de 10 anos). Médias, desvio padrão e significância da ANOVA entre os grupos demográficos,
e percepção de sucesso carreira. Tabela 5 - Médias, desvio padrão e significância da ANOVA entre os grupos demográficos,
satisfação e percepção de sucesso carreira. Satisfação
M (DP)
P
Sucesso
M (DP)
P
Estado Civil
Solteiro
3,53 (1,06)
p = 0,044
7,00 (2,17)
p = 0,879
Casado
4,19 (0,68)
6,89 (2,29)
Filhos
Sim
4,18 (0,80)
p = 0,178
6,41 (2,18)
p = 0,218
Não
3,80 (0,91)
7,28 (2,23)
Idade
Até 32 anos
3,88 (0,64)
p = 0,008
7,75 (1,58)
p = 0,191
33 e 34 anos
4,07 (0,59)
6,53 (2,50)
35 a 38 anos
3,88 (1,12)
7,38 (2,20)
39 anos ou mais
3,91 (1,22)
6,55 (2,29)
Tipo de vín-
culo empre-
gatício
CLT
4,00 (0,81)
p = 0,428
6,58 (2,09)
p = 0,230
Autônomo
4,05 (0,92)
6,75 (2,45)
Servidor Público
4,00 (1,00)
9,33 (1,15)
Outros
3,25 (0,95)
7,50 (1,73)
Consegue
cobrir as des-
pesas com o
seu salário? Sim
4,16 (0,76)
p = 0,006
6,97 (2,37)
p = 0,837
Não
3,30 (0,94)
6,80 (1,75)
Renda
Até
R$ 2.500,00
3,44 (0,72)
p = 0,001
6,57 (1,87)
p = 0,629
De R$ 2,500,00
até R$ 4,000,00
3,42 (0,90)
6,42 (2,31)
De R$ 4,000,00
até R$ 5,000,00
4,67 (0,50)
7,67 (2,29)
Acima de R$
7,000,00
4,33 (0,65)
7,08 (2,42) Tabela 5 - Médias, desvio padrão e significância da ANOVA entre os grupos demog
satisfação e percepção de sucesso carreira. ReCaPe Revista de Carreiras e Pessoas São Paulo. Volume VI - Número 01 - Jan/Fev/Mar/Abr 2016 Discussão dos Resultados O objetivo deste trabalho foi levantar e analisar de forma descritiva como os psicólogos
organizacionais em nível de Formação e Especialização, ex-alunos de uma Instituição
de Formação na área de Psicologia Organizacional, estão gerenciando suas carreiras e
como estão percebendo o sucesso e a satisfação profissional. Nas questões referentes ao
planejamento de carreira, a variável que obteve a maior média foi: “Planejo e faço a gestão
de minha carreira por meio do conhecimento adquirido como profissional e da experiência
de vida” (M = 4,24 e DP = 0,72). Nota-se que o psicólogo organizacional se apropria de suas
habilidades e competências, alicerçadas nos conhecimentos adquiridos com as trajetórias
profissional e pessoal, que direcionam o seu planejamento, suas ações e suas decisões
profissionais. Neste contexto, um senso de identidade e de resiliência pessoal torna-se mais
importante, pois, se a estrutura ocupacional estável do passado não exigia a iniciativa e a
criatividade em alto grau, o cenário emergente demanda o planejamento independente da
vida e da carreira (MAGALHÃES, 2011). Em sua pesquisa, Sorji (2000) postula que o cenário contemporâneo do mundo do
trabalho requer profissionais que sejam capazes de gerenciar suas próprias carreiras e suas
vidas. O enfraquecimento dos sindicatos e da relativa erosão das identidades ocupacionais ou
de classe gera isolamento no indivíduo numa trajetória particular de construção das próprias
condições de empregabilidade. Assim, a importância que tem a adequação contínua da
pessoa ao emprego que ela pretende conquistar ou manter é indispensável. Enquanto alguns
indivíduos mostram-se capazes de lidar com as incertezas e direcionam suas carreiras,
outros apresentam dificuldades em responder às demandas de autonomia, comportamento
exploratório, tomada de decisão e planejamento profissional (LASSANCE, 2005). A variável de planejamento que teve a média mais baixa foi: “No planejamento de minha
carreira sigo as orientações de um profissional especialista em planejamento de carreira” (M
= 1,76 e DP = 1,07). Nota-se que essa variável direciona o profissional e reforça a anterior
para uma gestão de carreira dirigida por ele e por suas questões internas de autoestima e
conhecimento. Hall, em sua teoria proteana, ratifica a ideia de que a pessoa reconhece o
sucesso como uma questão interna, como sucesso psicológico, e que a carreira é desenhada
pelo indivíduo e pode ser redirecionada para atender às necessidades e realizações da
pessoa (HALL, 1996). Discussão dos Resultados Página 46 Zardo, Bedin,
Páginas 36-52 Zardo, Bedin,
Páginas 36-52 Em estudo de Bedin (2010), os participantes salientam que a desvalorização do psicólogo
e a baixa remuneração são fatores que exercem influência em suas carreiras, mas foram
unânimes em suas escolhas de seguir investindo na profissão por esta ser o que lhes dava
realização e satisfação. A realização está relacionada ao profissional fazer aquilo de que gosta,
aquilo que é interessante e aquilo que optou fazer. E Bedin (2010) afirma que o profissional
assume a responsabilidade pelo gerenciamento de sua carreira que é visualizada como uma
relação estabelecida entre indivíduo com ele mesmo quando atua como profissional autônomo. Ademais, quando atua em uma organização na qual os interesses das partes precisam ser
contemplados e trabalhados em conjunto, a carreira passa a ser concebida como um processo
de negociação entre interesses pessoais e profissionais. Assim, o sentimento de satisfação
acaba sendo percebido tanto pelo equilíbrio vida-trabalho quanto pela autorrealização sentida
em função das conquistas pessoais, felicidade com as possibilidades e orgulho de si mesmo
e da independência conquistada (DRIES et al, 2008). Os resultados neste estudo evidenciam que a remuneração (M = 5,73 e DP = 2,21) e a
empregabilidade (M = 6,57 e DP = 2,48) tiveram as médias mais baixas, demostrando que
esse grupo de participantes não está satisfeito com essas duas dimensões. A satisfação está
nos fatores internos, tais como, valores (M = 8,30 e DP = 2,32), contribuição (M = 7,81 e DP
= 2,35) e cooperação (M = 7,64 e DP = 2,27), reforçando que a percepção de sucesso tem
mais a ver com questões pessoais internas do que com questões externas. Judge, et al (1995)
reforça esses resultados, afirmando que o sucesso na carreira é definido com as realizações
percebidas ou reais que os indivíduos têm acumulado como resultado de suas experiências
de trabalho. Nesta definição, as realizações percebidas são percepções do próprio indivíduo,
enquanto as reais seriam mensuráveis e observáveis externamente. Em seu estudo, Clarke (2009) postula que não se trata de não desejar mais o progresso
na hierarquia ou as recompensas financeiras, mas de buscar uma carreira mais adaptativa,
sem fronteiras, com forte ênfase na satisfação pessoal e no sucesso subjetivo. ReCaPe Revista de Carreiras e Pessoas São Paulo. Volume VI - Número 01 - Jan/Fev/Mar/Abr 2016 experiências do indivíduo ligadas ao percurso profissional, definindo o sucesso como a
realização de desejáveis resultados relativos ao trabalho em algum ponto, seja financeiramente
ou emocionalmente ao longo das suas experiências profissionais. Esta definição enfoca a
carreira subjetiva e o alcance de metas pessoais independentes da organização, da sociedade
e da cultura. experiências do indivíduo ligadas ao percurso profissional, definindo o sucesso como a
realização de desejáveis resultados relativos ao trabalho em algum ponto, seja financeiramente
ou emocionalmente ao longo das suas experiências profissionais. Esta definição enfoca a
carreira subjetiva e o alcance de metas pessoais independentes da organização, da sociedade
e da cultura. Ao realizar a comparação entre as médias de percepção de sucesso na carreira, verificou-
se que não existem diferenças significativas considerando-se os grupos demográficos. No
entanto, na comparação entre as médias de satisfação com a carreira, observou-se diferenças
significativas considerando o estado civil (com média mais alta para os casados), a idade
(com média mais alta para os participantes de 33 a 34 anos), a renda (com média mais alta
para os participantes com renda acima de R$ 4.000,00) e se consegue cobrir as despesas
(com média mais alta para os participantes que conseguem cobrir). Após levantamento e análise dos dados mensurados, pode-se visualizar neste estudo um
perfil profissional dos psicólogos, demonstrando que os profissionais com maior satisfação
com a carreira são casados, estão com idade média entre 33 e 34 anos, dispõe de renda
superior a R$ 4.000,00 e são capazes de cobrir as despesas. Ou seja, são participantes com
maior autonomia na vida e conseguem se sustentar financeiramente. Observamos que essas
variáveis para este estudo tornaram-se relevantes para uma sensação de satisfação com a
carreira profissional, demostrando que a responsabilidade com a condução das próprias vidas
torna-se indispensável no senso de autonomia e realização. Esses resultados corroboram com
a pesquisa de Bedin (2010) e de Godim (2008), os quais em seus estudos com psicólogos
apontam a definição dos participantes de que o trabalho deve prover principalmente crescimento,
independência, reconhecimento econômico, bem como, igualitarismo, acolhimento, dignidade
e humanização. Outro fator relevante nos resultados e que vem ao encontro com o estágio de
estabelecimento (dos 25 aos 44 anos), é que os participantes com maior grau de satisfação
com a carreira enquadraram-se com a idade nesta faixa etária característica da teoria de Super
e também em estudo de (BEDIN, et al, 2013). Discussão dos Resultados Esta nova
percepção da carreira vem ao encontro de romper com a carreira tradicional e com as teorias
clássicas de carreira como as de Super (1980), que tem estágios estabelecidos para o ciclo
cronológico do ser humano e se adequa à situação atual de condição de emprego e mudanças
no mercado de trabalho. Ao correlacionar-se a escala de percepção de sucesso com a satisfação na carreira,
observa-se que as 11 dimensões de sucesso se correlacionam significativamente e
positivamente com a satisfação dos profissionais. A dimensão remuneração mostra que quanto
maior a percepção de recompensas financeiras, maior a satisfação do profissional com a
carreira, sendo esta a variável com maior correlação. Dessa forma, salienta-se a importância
da remuneração para a satisfação dos entrevistados; esta é a dimensão de sucesso que mais
impacta na satisfação Como as médias de satisfação com a remuneração são as mais baixas,
significa que os salários não são bons, demonstrando uma insatisfação da classe. Bastos,
Gondim e Borges-Andrade (2010) apontam que apesar de os psicólogos estarem ampliando
o seu campo de atuação profissional e se inserindo no mercado de múltiplas formas, poucos
profissionais conseguem obter rendimentos mais elevados. Ademais, em seus estudos com
psicólogos, Bedin, et al (2013) apontou que eles alcançaram a estabilização na carreira,
embora muitos reconheçam as dificuldades de prover seu sustento a partir de suas atividades
profissionais. E a dimensão valores foi a que apresentou menor correlação com a satisfação, mas
mesmo assim se correlaciona significativamente. Seibert et al (2001) conclui que o sucesso
na carreira é o acúmulo de resultados positivos psicológicos e profissionais provenientes de
experiências de trabalho. Salientando, os resultados psicológicos e a sensação de satisfação
são subjetivos, já os resultados profissionais são percebidos externamente e reconhecidos
socialmente. Arthur et al (2005) afirmam que o sucesso na carreira é o resultado das Página 47 aPe Revista de Carreiras e Pessoas São Paulo. Volume VI - Número 01 - Jan/Fev/Mar/Abr 201 O estágio de estabelecimento caracteriza-se
pelo início da vida profissional, em que o profissional usufrui da formação e das capacidades
adquiridas e desenvolvendo um estilo de vida próprio. Observam-se comportamentos de
estabilização da vida pessoal e profissional, desenvolvimento de um estilo próprio de vida e
consequente autonomia financeira. Evidenciam-se comportamentos de procura de segurança
no trabalho e procura de autonomia e aumento do nível financeiro, com consequente aumento
da responsabilidade para avançar ainda na carreira, é necessário que o indivíduo continue a
planejar e explorar oportunidades dentro e fora da organização, possibilitando a progressão
na carreira (SAVICKAS, 2001). Outra questão ligada ao sucesso na carreira é a empregabilidade. Zaccarelli, et al (2008)
definem a empregabilidade como a capacidade de obter trabalho e renda, o que corrobora com
dados desta pesquisa. Clarke (2009) enfatiza a empregabilidade como um fator importante
nas carreiras da nova economia e considera que o sucesso e a satisfação envolvem: as
recompensas da carreira tradicional como, por exemplo, aumento de status, responsabilidade
e recompensas financeiras; o sucesso psicológico no nível individual como, por exemplo, a
realização pessoal e o sentimento de orgulho; e também a percepção de empregabilidade
futura. Pá i
48
O papel do trabalho seria um dentre vários papéis, como estudo, comunidade, casa,
família e tempo livre, papéis considerados principais neste modelo (SUPER, 1980). Os papéis
centrais constituem o cerne do que o indivíduo é, e são essências para sua identidade e
para a satisfação de vida (SAVICKAS, 2005). A saliência de um papel pode ser atribuída ao
valor que a pessoa deposita em cada papel, e este valor é uma crença que pode ser estável Página 48 Zardo, Bedin,
Páginas 36-52 dentro de um período, agindo como padrões que orientam a conduta de um indivíduo de
como se deve viver. São cognições de acordo com o ambiente ao qual a pessoa está inserida
(BROWN, 2002). Savickas et al. (2009) apontam que as teorias vocacionais contemporâneas devem enfatizar
a construção da carreira em consonância com diferentes domínios da vida ao invés de limitar-
se apenas ao trabalho. Assim, as concepções vocacionais pós-modernas exigem ênfase nos
processos reflexivos e de construção de sentidos sobre os autoconceitos e o contexto social,
buscando construir alternativas de ação que devem ser revistas constantemente e viabilizando
a adaptabilidade do indivíduo frente às inúmeras transições que caracterizam o mundo do
trabalho atual. O próprio conceito de carreira está numa fase de mudança. A carreira deixa de
ser a sequência relativamente estável de posições e papéis ocupacionais para abranger os
sentidos que os indivíduos atribuem às suas experiências de vida e de trabalho cada vez mais
transitórias (BENDASSOLI, 2009). Duarte et al. (2010) preconiza a necessidade de intervenções individualizadas e únicas,
que orientem a pessoa na construção de um projeto único de vida e de trabalho. Entretanto,
pontua a importância da pesquisa, que foi realizada através de instrumentos científicos e de
estudos de casos estruturados de forma a tornarem-se comparáveis para que se apreendam
as circunstâncias individualizadas do desenvolvimento de carreira na modernidade com
objetivos de ajustar constantemente o modelo que, como a nova ordem socioeconômica,
deve ser dinâmico e integro, tanto para as questões do sujeito como para a sociedade. Os resultados desta pesquisa mostraram-se significativos e evidenciaram as questões
do estudo a respeito de como os psicólogos pesquisados se percebem em suas carreiras
em termos de sucesso e de satisfação profissional e como planejam suas carreiras. Todavia,
alguns fatores limitam o estudo como a amostra pequena, o que impossibilita generalizações. A atual crise vivenciada no país gera incertezas e estas podem intervir nas respostas dos
participantes. Além disso, os psicólogos que não responderam ao questionário representam
um viés a ser considerado como limitador deste estudo, já que não se sabe o motivo que os
levou a não participar da pesquisa, podendo ser pelo fato de não estarem satisfeitos ou por
não perceberem sucesso em suas carreiras, além de outros fatores desconhecidos. REFERÊNCIAS BIBLIOGRÁFICAS ARAÚJO, Greicy Bones de; PARADISO, Angela Carina; LASSANCE, Maria Celia Pacheco;
& SARRIERA, Jorge Castellá. Carreira e narrativa: contribuições para a intervenção. Revista
Brasileira de Orientação Profissional. jul.-dez. 2013, Vol. 14, No. 2, 191-201. 2013. ARTHUR, Michael B. et al. Career success in a boundaryless career world. Journal of
Organizational Behavior, v. 26, p. 177-202, 2005. BASTOS, Antônio Virgílio Bittencourt; GODIN, Sonia Maria Guedes; BORGES-ANDRADE,
Jairo. O Psicólogo Brasileiro: Sua Atuação e Formação Profissional. O que mudou nas últimas
décadas, Revista. CFP,2010. BEDIN, Lívia Maria. Desenvolvimento de Carreira em Psicólogos: Tarefas evolutivas do
Estágio de Estabelecimento. Porto Alegre / Lívia Maria Bedin – Porto Alegre, 2010. 83p. BEDIN, Lívia Maria; SARRIERA, Jorge Castellá e PARADIDO, Ângela Carina. Desenvolvimento de carreira em psicólogos: tarefas evolutivas de estabelecimento. Revista
Brasileira de Orientação Profissional. jan.-jun. 2013, Vol. 14, No. 1, 87-98. BENDASSOLI, Pedro Fernando. Recomposição da relação sujeito-trabalho nos modelos
emergentes de carreira. RAE. 49,387-400, 2009. BROWN, Duane. Introduction to theories of career development and choice: Origins,
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multidimensional model. Journal of Vocational Behavior, v. 73, n. 2, p. 254-267, 2008. DUARTE, Maria Eduarda; LASSANCE, Maria Célia Pacheco; SAVICKAS, Mark L; NOTA,
Laura; ROSSIER, Jerome; DAUWALDER, Jean-Pierre; et al. (2010). A construção da vida:
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pessoas. São Paulo: Atlas, 2004. PERCEPÇÃO, In: FERREIRA, Aurélio Buarque de Hollanda. Novo dicionário da língua
portuguesa. Disponível em: www.dicionariodoaurelio.com. Acesso em 26/09/2014. FERRAMENTA, In: HOUAISS, Antônio; VILLAR, Mauro de Salles. Considerações Finais Os achados deste estudo apontam que os fatores autonomia e responsabilidade são
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a análise direciona a entender que a interrelação dos fatores internos, característicos de
pessoas de sucesso que se percebem mais satisfeitas com sua carreira, são atributos de
sensação de responsabilidade e direção de sua própria vida. A condução da própria vida
fortalece, dá poder na capacidade de agir perante as decisões pessoais, na condução das
responsabilidades, nas competências profissionais e nos valores humanos. Assim, para ampliar o entendimento e a reflexão da gestão da carreira de psicólogos,
novas pesquisas são sugeridas, buscando aprofundar os aspectos que podem ser favoráveis
na gestão de carreira. Toma-se como exemplo avaliar como a atual crise econômica
vivenciada no país pode interferir no planejamento de carreira e que consequências geram
nos profissionais da psicologia, além de analisar qual o papel da competitividade nas ações e
nas decisões da carreira de psicólogos e estudar quais são as competências que favorecem
a gestão de carreira e os fatores que geram insatisfação profissional para a classe. Página 49 ReCaPe Revista de Carreiras e Pessoas São Paulo. Volume VI - Número 01 - Jan/Fev/Mar/Abr 2016 REFERÊNCIAS BIBLIOGRÁFICAS Dicionário da língua
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GONDIM, Sonia Maria Guedes. O psicólogo brasileiro: identidade profissional. In:
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CBPOT.2008. Zardo, Bedin,
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Páginas 36-52 JANEIRO, Isabel Nunes. Escala de Atribuições em relação à Carreira (EAC): Um
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Publishers, 1996. LASSANCE, Maria Célia Pacheco. Adultos com dificuldades de ajustamento ao trabalho:
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de Orientação Profissional, 6(1), 41-51, 2005. LASSANCE, Maria Célia Pacheco; & SARRIERA, Jorge Castellá. Saliência, valores e
carreira. Revista Brasileira de Orientação Profissional jan.-jun. Vol. 13, No. 1, 49-61, 2012. LASSANCE, Maria Célia Pacheco; & SARRIERA, Jorge Castellá. Saliência do papel
de trabalhador, valores de trabalho e desenvolvimento de carreira. Universidade Federal
do Rio Grande do Sul, Porto Alegre - RS, Revista Brasileira de Orientação Profissional. prof vol.13 no.1 São Paulo jun. 2012. MAGALHÃES, Mauro Oliveira. Quinta demanda-chave para a orientação profissional:
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profissional e de carreira: Perspectivas históricas e enfoques teóricos clássicos e modernos
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Prentice Hall, 2005. SAVICKAS, Mark L. The theory and practice of career construction. In S. D. Brown & R. W. Lent (Eds.), Career development and counseling – putting theory and research to work (pp. 42-70). Hoboken, NJ: John Wiley & Sons, 2005. SAVICKAS, Mark L. Envisioning the future of vocational psychology. Journal of Vocational
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Maria Eduarda; GUICHARD, Jean, et al. Life-design: A paradigm for career construction in the
21st century. Journal of Vocational Behavior, 75, 239-250;2009. REFERÊNCIAS BIBLIOGRÁFICAS Maria Eduarda Duarte SEIBERT, Scott.E, KRAIMER, Maria.L. & LIDEN, Robert.C. A social capital theory of
career success. Academy of Management Journal, Vol. 44 No. 2, pp. 219-38. 2001. SUPER, Donald Edwin. A Life-Span, Life-Space approach to career development. Journal
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ZACCARELLI, Laura Menegon, DOMENICO, Silva Marcia Rossi De; TEIXEIRA, Maria
Luísa Mendes. O outro lado da moeda: desenvolvendo a empregabilidade e a carreira. In:
HANASHIRO, Darcy Mitiko Mori; TEIXEIRA, Maria Luísa Mendes; ZACCARELLI, Laura aPe Revista de Carreiras e Pessoas São Paulo. Volume VI - Número 01 - Jan/Fev/Mar/Abr 201 Menegon. (Org.). Gestão do fator humano: uma visão baseada em stakeholders. São Paulo:
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Springer. Página 52
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http://biomed.papers.upol.cz/doi/10.5507/bp.2012.084.pdf
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Beta 2 adrenergic receptor polymorphisms, at codons 16 and 27, and bronchodilator responses in adult Venezuelan asthmatic patients
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Biomedical Papers/Biomedical Papers of the Faculty of Medicine of Palacký University, Olomouc Czech Republic
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cc-by
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Nancy Laroccaa,b, Dolores Morenob, Jenny Valentina Garmendiaa, Olga Velasqueza, Joana Martin-Rojoa, Carlos Talamoc,
Alexis Garciaa, Juan Bautista De Sanctisa Background. One of the gene polymorphisms often studied in asthmatic patients is the β2 adrenergic receptor
(ADRβ2). Even though in the Venezuelan Mestizo population there is a high incidence of asthma, there are no direct
reports of ADRβ2 gene polymorphism, and treatment response. The aim of this study was to assess, in this popula-
tion, the gene frequency of ADRβ2 polymorphisms at codons 16 Arg/Gly and 27 Gln/Glu, allergen sensitization, and
its relationship to bronchodilator response. Methods. Purified genomic DNA was obtained form 105 Mestizo asthmatic and 100 Mestizo healthy individuals from
Venezuela. The two polymorphisms were assessed by PCR-RFLP. Patient sensitization to aeroallergens and their response
to bronchodilatation were correlated. Results. Significant differences between patients and controls were recorded in: 1) the prevalence of Arg/Arg at codon
16 (28.6% in patients vs. 47% in controls, P<0.01), 2) the frequency of heterozygotes Arg/Gly (55% in patients vs. 35%
in controls, P<0.01). Conversely, no differences in polymorphism frequencies were found at codon 27. The haplotypes
Arg/Gly-Gln/Gln were more common in patients than controls (P <0.01), whereas the Arg/Arg-Gln/Glu combination
prevailed in the control group (P<0.01). The Arg/Gly and Gln/Glu genotypes were associated with better responses
after salbutamol. The asthmatic homozygotes Arg/Arg have higher sensitivity to aeroallergens. Conclusion. The difference in Arg/Arg frequency between groups suggests that this could be a protective genotype
although the asthmatic group had a higher sensitivity to aeroallergens. The asthmatic heterozygotes had better bron-
chodilator responses than the homozygotes. Key words: aeroallergen, asthma, β2 adrenergic receptors, bronchodilator response, gene polymorphism Received: January 4, 2012; Accepted with revision: July 27, 2012; Available online: November 2, 2012
http://dx.doi.org/10.5507/bp.2012.084 Received: January 4, 2012; Accepted with revision: July 27, 2012; Available online: November 2, 2012
http://dx.doi.org/10.5507/bp.2012.084 aInstitute of Immunology, Faculty of Medicine, Universidad Central de Venezuela, Apartado 50109, Sabana Grande, Caracas, Venezuela
bInstitute of Experimental Medicine, Faculty of Medicine, Universidad Central de Venezuela, Apartado 50109, Sabana Grande, Caracas,
cDepartment of Pneumology, Luis Razetti School of Medicine, University Hospital, Faculty of Medicine, Universidad Central de Venezuela,
Caracas Corresponding author: Juan Bautista De Sanctis, e-mail: sanctisj@gmail.com Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Dec; 157(4):374-378. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Dec; 157(4):374-378. INTRODUCTION patients10. These findings correspond to Caucasians, how-
ever, in Venezuela there is a high prevalence of asthma
and a large Mestizo population which seem to have a high
genetic segregation11-13. The relevance of these polymor-
phisms in various populations and its clinical relevance
so far are unknown2. In the present study, the polymor-
phisms at amino acids 16 and 27 of ADRβ2 were assessed
along with clinical, paraclinical parameters and lung func-
tion in Venezuelan Mestizo asthmatic patients. Asthma is a highly prevalent and complex disease in
which multiple interacting genes and environmental fac-
tors are involved1. Evidence shows that alterations of ad-
renergic β-2 receptors (ADRβ2) can modulate the severity
of asthma and the response to treatment, which can be
highly variable and difficult to predict2-7. Nevertheless, it
has been shown that: 1) In the LARGE multicenter study,
asthmatics with Arg/Arg homozygous genotype, receiving
inhaled corticosteroids, experience no change in hyper-
responsiveness status even when long acting β2 agonists
were used8; 2) a combination of salmeterol plus inhaled
corticosteroid in the patients Gly/Gly, at amino acid 16
increased 2.4 times the PC20, resulted in decreased bron-
chial hyperresponsiveness5,8; 3) the Gly16/Gln27 haplo-
type was inversely associated with hyperresponsiveness to
methacholine, while the Arg16/Gln27 haplotype was asso-
ciated with a greater degree of hyperresponsiveness9, and
4) children homozygous and heterozygous for Arg16 had
higher bronchodilator responses than Gly16 homozygotes METHODS All participants (205) gave written consent to enter
the study according to instructions approved by the Ethics
and Bioethics Committee of the Institute of Immunology,
and of the Venezuelan Foundation of Science and
Technology (FONACIT). Genomic DNA was isolated
from blood using the QIAmp® DNA Mini kit (Qiagen®),
of 105 individuals diagnosed with asthma according to
GINA (ref.14) guidelines and 100 control subjects with 374 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Dec; 157(4):374-378. no history of asthma or COPD. The groups were gen-
der, and aged matched (between 18 and 60 years old)
and third-generation Venezuelan. A medical history and
spirometry (forced vital capacity-FVC-forced expiratory
volume in one second-FEV1, and FEV1/FVC ratio) was
performed for all patients and controls. For the diagnosis
of asthma the ATS criteria was used. A positive response
to bronchodilator was considered (salbutamol), when a
change in FEV1 was greater than or equal to 12% and at
least 200 cc. corresponding to the codon 27. The restriction products
were run on agarose gels 4% to 100 V for 45 min with
subsequent ethidium bromide staining) and visualization
on a UV light transilluminator (Bio-Rad®). We confirmed
the specificity of amplified and presence of two polymor-
phisms by sequencing performed by a four capillary se-
quencer from Applied Biosystems. The rate of success/
failure compared RFLP to sequencing was 98%. Statistical analysis Total serum IgE was assessed by a commercial kit
(Calbiotech, USA). Skin Prick tests for different aero-
allergens (Dermatophagoides, Blomia, Blattella, dog, cat,
latex, wool, mixed pollens, mixed feathers, mixed fungi
spores) were performed in all patients by a commercial
kit Alk-ABello® (Spain). The gene frequencies of polymorphisms in each group
were calculated by standard method15. A confidence in-
terval of 95%, according to the method of Wilson with
correction for continuity, was used15. The PHASE pro-
gram16 was used to calculate the frequency of haplotypes
(Bayesian method using the Markov chain-Monte Carlo
algorithm). The 95% confidence interval of the frequency
of individual haplotypes was estimated by the Chi squared
test. Gene amplification of ADRβ2 polymorphic regions
encoding positions for the amino acids 16 and 27 was per-
formed according to the protocol of Martinez et al.10. The
two polymorphisms were identified using PCR-RFLP test. The amplification of the genetic region of ADRβ2 using
the PCR technique for each of the samples was performed
in a final volume of 50 µL with: 300-500 ng of genomic
DNA, 1.5 mM magnesium chloride, 0.2 mM of each
dNTP, 10 mM Tris.HCl, 50 mM KCl and 0.2 uM of each
primer, sense: 5'-GCCTTCTTGCTGGCACCCCAT-3'
and antisense: 5’- CAGACGCTCGAACTTGGCCATG-3'. The PCR was performed on Peltier Thermal Cycler
PTC200 thermocycler (MJ Research) under the follow-
ing conditions: an initial denaturation at 94 °C for 2 min
followed by 40 cycles of 94 °C for 40 s, 64 °C for 40 s and
72 °C for 50 s followed by a final extension phase at 72 °C
for 5 min. The PCR product of 168 bp was digested with
2 U of NcoI restriction enzyme (New England Biolabs)
for the identification of polymorphism corresponding to
the codon 16 and 0.5 U of BbvI restriction enzyme (New
England Biolabs) for identification of the polymorphism are recorded in several parameters, * P<0.0001. RESULTS The characteristics of patients and controls are shown
in Table 1. Gender and age did not differ significantly
between the groups. As expected, in the asthmatic group,
the values of FEV1 and FEV1/FVC were significantly
lower than controls (P<0.0001). Most patients had moder-
ate persistent asthma (42.9%). Allergic asthma was more
prevalent (86.7%) than intrinsic (5.7%), mixed (5.7%) or
occupational (0.9%); allergic rhinitis (50.5%) and atopic
dermatitis (10.5%) were the most frequent symptoms re-
corded. Moreover, 95% of asthmatics were positive to at
least one aeroallergen in skin tests. The most common
sensitizations in allergic asthmatics were: house dust
mites (56% to Dermatophagoides pteronyssinus (Dp), 23%
to Dermatophagoides pharinae (Df)), 38.5% to cockroach Table 1. Characteristics of patients (according gina guidelines) and controls. Patients
(n = 105)
Controls
(n = 100)
N
105
100
Female Gender
76 (72.4%)
77 (77.0%)
Age X ± SD
44.4 ± 15.2
42.6 ± 13.9
FEV1 (L) X ± SD
1.9 ± 0.6 *
3.06 ± 0.8
FEV1/FVC (%)X ± SD
65.9 ± 10.0 *
81.2 ± 5.5
Total IgE IU/ml X ± SD
473.0 ± 598.6*
123.0 ± 174.0
Smokers
37 (35.2%) *
0
Rhinitis
53 (50.5%) *
0
Atopic Dermatitis
11 (10.5%) *
0
Mild Intermittent Asthma. 13 (12.4%)
-
Mild Persistent Asthma. 18 (17.1%)
-
Moderate Persistent Asthma
45 (42.9%)
-
Severe Asthma
29 (27.6%)
-
Significant differences are recorded in several parameters, * P<0.0001. Table 1. Characteristics of patients (according gina guidelines) and controls. 375 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Dec; 157(4):374-378. Table 2. Polymorphism at codons 16 and 27 of β2 adrenergic receptor in patients with asthma and controls. Polymorphism
Patients
n (%)
Controls
n (%)
16
Arg/Arg
30 (28.6)
47 (47) *
Gly/Gly
17 (16.2)
18 (18)
Arg/Gly
58 (55.2)
35 (35) *
27
Glu/Glu
11 (10.5)
10 (10.0)
Gln/Gln
37 (35.2)
30 (30.0)
Gln/Glu
57 (54.3)
60 (60.0)
Significant differences were observed at codon 16 between controls and patients * P <0.01. Table 2. Polymorphism at codons 16 and 27 of β2 adrenergic receptor in patients with asthma and controls. Table 3. Haplotypes at codons 16 and 27 of β2 adrenergic receptor in patients with asthma and controls. Part A. Association between the percentage of predicted value
(PV) in FEV1 after bronchodilator response (BD) and β2AR
polymorphism at codon 16. Significant differences are observed
in the predicted value between the homozygotes Gly/Gly and
the heterozygotes Arg/Gly, (P<0.009). Part B. Association between FEV1 after bronchodilator re-
sponse (BD) and β2AR polymorphism at codon 27. Significant
differences are observed in the VEF1 post test between the ho-
mozygotes Glu/Glu and the heterozygotes Gln/Glu (P<0.0001). Part A. Association between the percentage of predicted value
(PV) in FEV1 after bronchodilator response (BD) and β2AR
polymorphism at codon 16. Significant differences are observed
in the predicted value between the homozygotes Gly/Gly and
the heterozygotes Arg/Gly, (P<0.009). Part A. Association between the percentage of predicted value
(PV) in FEV1 after bronchodilator response (BD) and β2AR
polymorphism at codon 16. Significant differences are observed
in the predicted value between the homozygotes Gly/Gly and
the heterozygotes Arg/Gly, (P<0.009).
Part B. Association between FEV1 after bronchodilator re-
sponse (BD) and β2AR polymorphism at codon 27. Significant
differences are observed in the VEF1 post test between the ho-
mozygotes Glu/Glu and the heterozygotes Gln/Glu (P<0.0001). Part A. Association between the percentage of predicted value
(PV) in FEV1 after bronchodilator response (BD) and β2AR
polymorphism at codon 16. Significant differences are observed
in the predicted value between the homozygotes Gly/Gly and
the heterozygotes Arg/Gly, (P<0.009).
Part B. Association between FEV1 after bronchodilator re-
sponse (BD) and β2AR polymorphism at codon 27. Significant
differences are observed in the VEF1 post test between the ho-
mozygotes Glu/Glu and the heterozygotes Gln/Glu (P<0.0001). RESULTS Polymorphism
Patients
n (%)
Controls
n (%)
OR
CI
P
Arg/Arg-Gln/Gln
10 (9.5)
18 (18)
0.54
0.27-1.08
NS
Gly/Gly-Glu/Glu
3 (2.9)
4 (4)
1.42
0.39-5.08
NS
Arg/Arg-Glu/Glu
3 (2.9)
2 (4)
0.69
0.15-3.17
NS
Gly/Gly- Gln/Gln
3 (2.9)
3 (3)
1.05
0.27-4,11
NS
Arg/Gly-Gln/Glu
29 (27.6)
22 (22)
0.74
0.43-1.26
NS
Arg/Gly-Glu/Glu
5 (4.8)
4 (4)
0.83
0.27-2.57
NS
Arg/Gly-Gln/Gln
24 (22.9)
9 (9)
0.33
0.17-0.67
<0.01
Arg/Arg-Gln/Glu
17 (16.2)
27 (27)
1.92
1.08-3.39
<0.01
Gly/Gly-Gln/Glu
11 (10.5)
11 (11)
1.06
0.50-2.22
NS
OR: Odds Ratio, CI: Confidence Interval. NS stands for not significant. Table 3. Haplotypes at codons 16 and 27 of β2 adrenergic receptor in patients with asthma and controls. OR: Odds Ratio, CI: Confidence Interval. NS stands for not significant. OR: Odds Ratio, CI: Confidence Interval. NS stands for not significant. Gly/Gly
Arg/Arg
Arg/Gly
0
10
20
30
40
50
60
70
80
90
% VEF1 PREDICTED
P < 0.009
P < 0.0001
Glu/Glu
Gln/Gln
Gln/Glu
0
1
2
3
VEF1 post BD (L)
A
B
Codon 16
Codon 27
Fig. 1. Association between bronchodilator response and gene
polymorphism. (B. germanica), 23% to Blomia tropicalis, 28% to Mold I
panel, 33% to cat and latex, 25.6% Pollen V panel, and
20.5% to Dog. The rest of the allergens were on or below
10%. (B. germanica), 23% to Blomia tropicalis, 28% to Mold I
panel, 33% to cat and latex, 25.6% Pollen V panel, and
20.5% to Dog. The rest of the allergens were on or below
10%. Gly/Gly
Arg/Arg
Arg/Gly
0
10
20
30
40
50
60
70
80
90
% VEF1 PREDICTED
P < 0.009
A
Codon 16 Table 2 shows the frequency for both polymorphisms
at codons 16 and 27. The Arg/Arg homozygous was more
frequent in the control group as compared to the patient
group, 47.0% vs 28.6%, P<0.01, and the opposite was for
the Arg/Gly heterozygous, 35.0% vs. 55.2% (P<0.01). No
differences were found in the polymorphism at amino
acid 27. No relationship was found between the different
polymorphisms (16 and 27) and asthma severity; however,
nocturnal symptoms predominated among patients with
16 Gly/Gly. In this group, 70.6% had nocturnal symptoms
(P=0.05). P < 0.0001
Glu/Glu
Gln/Gln
Gln/Glu
0
1
2
3
VEF1 post BD (L)
B
Codon 27 B Fig. 1. Association between bronchodilator response and gene
polymorphism. 376 Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2013 Dec; 157(4):374-378. ACKNOWLEDGMENT Financed by a grant from the Venezuelan Foundation
for Research (FONACIT) G-2005-000389 for a project
on Pharmacogenetics of chronic respiratory diseases. The
authors received the prize of the Venezuelan Foundation
of Medications (CAVEME) on 2010. CONFLICT OF INTEREST STATEMENT Author’s conflict of interest disclosure: The authors
stated that there are no conflicts of interest regarding the
publication of this article. The results of our study showed that the frequency of
Arg/Arg at amino acid 16 was higher in the Venezuelan
Mestizo normal population and the Arg/Gly heterozy-
gous was more prevalent in our asthmatic population
contrasting with reports on Caucasian or Asian popula-
tions8-10,17. In similar fashion, the haplotype Arg/Arg-Gln/
Glu haplotype appears to protect the Venezuelan Mestizo
population from asthma and the haplotype Arg/Gly-Gln/
Gln haplotype appears to increase susceptibility. Probably,
these differences are due to the mixture of our population,
as demonstrated by the Fortes et al study18 in which the
Venezuelan Mestizo population with autoimmune hepa-
titis showed some disease-protective HLA alleles which
differ from those reported for Caucasians and Japanese
populations18. The results of our study showed that the frequency of
Arg/Arg at amino acid 16 was higher in the Venezuelan
Mestizo normal population and the Arg/Gly heterozy-
gous was more prevalent in our asthmatic population
contrasting with reports on Caucasian or Asian popula-
tions8-10,17. In similar fashion, the haplotype Arg/Arg-Gln/
Glu haplotype appears to protect the Venezuelan Mestizo
population from asthma and the haplotype Arg/Gly-Gln/
Gln haplotype appears to increase susceptibility. Probably,
these differences are due to the mixture of our population, DISCUSSION The most common polymorphisms of the ADRβ2 are
at codons 16 and 27 and several studies have suggested
that these polymorphisms have a modulatory effect on
asthma severity and response to therapy. An association
between the Gly at codon 16 and severe asthma, requiring
oral corticosteroids, immunotherapy, or both treatments
was suggested5. RESULTS We conclude that genetic typing of ADRβ2 polymor-
phism may help identify individuals with asthma resistant
to β2-adrenergic agonists or with severe or difficult to
treat asthma. Even though, the Arg/Arg polymorphism
at amino acid 16 is common in the healthy Venezuelan
Mestizo population, as well as the Arg/Arg-Gln /Glu hap-
lotype, the relevant issue is that in Venezuelan asthmatic
Mestizo Arg/Arg are polysensitized to different aeroal-
lergens prevalent in the American tropics and that the
patients with Arg/Gly-Gln/Glu haplotype showed the
greatest changes in post-bronchodilator FEV1 increasing
therapy effectiveness. These results indicate a marked dif-
ference in prevalence and therapeutic response compared
to other ethnic groups. Further studies are required to
ascertain the importance of polymorphism at amino acid
16 as the key element for immune response and treatment
outcome in Venezuelan Mestizo population. In the haplotype analysis, (Table 3), as expected from
the previous results, Arg/Gly-Gln/Gln was more prevalent
in patients (22.9%) than controls (9%), (P<0.01), and the
haplotype Arg/Arg-Gln/Glu was more frequent in the con-
trol group (27% vs. 16.2%, P<0.01). The relationship between predicted FEV1 and FEV1
post bronchodilator response and gene polymorphisms
are represented in Fig. 1A and 1B. In part A of the figure,
even though the parameters of pre-bronchodilator lung
function were similar among patients, the group with Gly/
Gly showed a lower percentage of predicted FEV1 post
bronchodilator test than the Arg/Gly group (P<0.009). In similar fashion as shown in part B of the figure, the
Glu/Glu group showed post bronchodilator FEV1 val-
ues significantly less than Gln/Glu heterozygous patients
(P<0.0001). Interestingly, patients Arg/Arg homozygotes were
more sensitized to aeroallergens (78%) than the hertero-
zygotes (40%) and the Gly/Gly (28%) counterparts
(P<0.05). This effect was not recorded at codon 27. The
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English
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IDENTIFYING MENTAL HEALTH FACTORS OF CANCER PATIENTS IN HOSPITAL X
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Jurnal Berkala Epidemiologi/Jurnal berkala epidemiologi
| 2,023
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cc-by-sa
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entifikasi Faktor-Faktor Kesehatan Mental pada Pasien Kanker di Rumah Sakit X Solikhah Solikhah1, Rochana Ruliyandari2
, Wulan Rahmadhani 3, Fatma Nuraisyah4
1Faculty of Public Health, Universitas Ahmad Dahlan, Yogyakarta 55166, Indonesia, solikhah@ikm.uad.ac.id
2Faculty of Public Health, Universitas Ahmad Dahlan, Yogyakarta 55166, Indonesia,
ruliyandari27@gmail.com 3Department of Midwifery, Universitas Muhammadiyah Gombong, 54411, Gombong, Central Java,
Indonesia, wulannnnn02@gmail.com 4Faculty of Public Health, Universitas Ahmad Dahlan, Yogyakarta 55166, Indonesia,
fatma.nuraisyah@ikm.uad.ac.id 4Faculty of Public Health, Universitas Ahmad Dahlan, Yogyakarta 55166, Indonesia,
fatma.nuraisyah@ikm.uad.ac.id Corresponding Author: Rochana Ruliyandari, ruliyandari27@gmail.com, Faculty of Public Health,
Universitas Ahmad Dahlan, Yogyakarta 55166, Indonesia Corresponding Author: Rochana Ruliyandari, ruliyandari27@gmail.com, Faculty of Public Health,
Universitas Ahmad Dahlan, Yogyakarta 55166, Indonesia IDENTIFYING MENTAL HEALTH FACTORS OF CANCER PATIENTS IN
HOSPITAL X IDENTIFYING MENTAL HEALTH FACTORS OF CANCER PATIENTS IN
HOSPITAL X Identifikasi Faktor-Faktor Kesehatan Mental pada Pasien Kanker di Rumah Sakit X Jurnal Berkala Epidemiologi Jurnal Berkala Epidemiologi DOI: 10.20473/jbe.v11i12023.92–100
https://e-journal.unair.ac.id/JBE/
Email: jbe@fkm.unair.ac.id / jbepid@gmail.com DOI: 10.20473/jbe.v11i12023.92–100
https://e-journal.unair.ac.id/JBE/
Email: jbe@fkm.unair.ac.id / jbepid@gmail.com DOI: 10.20473/jbe.v11i12023.92–100
https://e-journal.unair.ac.id/JBE/
Email: jbe@fkm.unair.ac.id / jbepid@gmail.com ARTICLE INFO Background: Physical health of people living with cancer causes mental
health disorders and unknowingly affects the overall quality of patients’ life. As many as 34.40% of cancer patients in Indonesia experience depression
due to anxiety and fear. Objective: Therefore, this study was conducted to
determine the mental health of cancer patients in the hospital so that the
relationship between the mental health of cancer patients and other factors,
such as the characteristics of the respondents, family support, and
spirituality, is known. Methods: The type of research for this study is
quantitative with a cross-sectional design using the Pearson Chi-Square
analysis. There were 96 respondents in this study who were cancer patients
from Hospital X. The independent variables used were the respondent’s
demographic characteristics (age, gender, occupation, and education), family
support, and spirituality, with the dependent variable being mental health
(stress and depression). Results: The results of the study showed that cancer
patients had the latest elementary school education (40.60%), status not
working (61.50%), female (66.70%), early elderly (35.40%), 70.80%
received moderate family support, 76% had moderate spirituality, 51%
experienced moderate stress, and 57.30% had depression. The characteristics
of respondents in this study, namely gender, education, occupation, and
spiritual factors, were not associated with mental health in cancer patients. Conclusion: The results showed that age and family support were factors
associated with the mental health of cancer patients, while other
demographic characteristics (gender, occupation, and education) also
spiritual factors were not associated with the mental health of the cancer
patients. Article History:
Received January, 10th, 2022
Revised form August, 1st, 2022
Accepted November, 29th, 2022
Published online January, 29th, 2023 Article History:
Received January, 10th, 2022
Revised form August, 1st, 2022
Accepted November, 29th, 2022
Published online January, 29th, 2023 ©2023 Jurnal Berkala Epidemiologi. Published by Universitas Airlangga. This is an open access article under CC-BY-SA license 93 of 100 Solikhah Solikhah, et al / Jurnal Berkala Epidemiologi, 11 (1) 2023, 92-100 ABSTRAK How
to
Cite:
Solikhah,
S.,
Ruliyandari, R., Ramdani, W., &
Nuraisyah, F. (2023). Identifying
mental health factors of cancer
patients
in hospital
X. Jurnal
Berkala Epidemiologi, 11(1), 92-
100. How
to
Cite:
Solikhah,
S.,
Ruliyandari, R., Ramdani, W., &
Nuraisyah, F. (2023). Identifying
mental health factors of cancer
patients
in hospital
X. Jurnal
Berkala Epidemiologi, 11(1), 92-
100. Latar Belakang: Kesehatan fisik penderita kanker menyebabkan gangguan
pada kesehatan mental dan secara tidak sadar akan memengaruhi kualitas
hidup penderita secara keseluruhan. Sebanyak 34,40% penderita kanker di
Indonesia mengalami depresi akibat kecemasan dan ketakutan. Tujuan:
Oleh karena itu, penelitian ini dilakukan untuk mengetahui kesehatan jiwa
pasien kanker di rumah sakit, sehingga diketahui hubungan antara kesehatan
jiwa pasien kanker dengan faktor lainnya, seperti karakteristik responden,
dukungan keluarga, dan spiritualitas. Metode: Jenis penelitian ini adalah
kuantitatif dengan desain cross sectional. Analisis yang digunakan dalam
penelitian ini adalah analisis Pearson Chi-Square. Terdapat 96 responden
pada penelitian ini yang merupakan pasien kanker dari Rumah Sakit X. Variabel bebas yang digunakan adalah karakteristik responden (umur, jenis
kelamin, pekerjaan, dan pendidikan), dukungan keluarga, dan spiritualitas,
dengan variabel terikatnya adalah kesehatan jiwa (stress dan depresi). Hasil:
Hasil penelitian menunjukkan pasien kanker berpendidikan terakhir SD
(40,60%), status tidak bekerja (61,50%), berjenis kelamin perempuan
(66,70%), berumur lanjut usia dini (35,40%), 70,80% mendapat dukungan
keluarga sedang, 76% memiliki spiritualitas sedang, 51% mengalami stres
sedang, dan 57,30% mengalami depresi. Karakteristik responden dalam
penelitian ini yaitu jenis kelamin, pendidikan, pekerjaan, dan faktor spiritual
tidak berhubungan dengan kesehatan jiwa pada pasien kanker. Kesimpulan:
Hasil penelitian menunjukkan bahwa usia dan dukungan keluarga
berhubungan
dengan
kesehatan
mental
pasien
kanker,
sedangkan
karakteristik lain dan spiritualitas tidak berhubungan dengan kesehatan
mental. ©2023 Jurnal Berkala Epidemiologi. Penerbit Universitas Airlangga. Jurnal ini dapat diakses secara terbuka dan memiliki lisensi CC-BY-SA https://dx.doi.org/10.20473/jbe.11i1
2023. 92-100 How
to
Cite:
Solikhah,
S.,
Ruliyandari, R., Ramdani, W., &
Nuraisyah, F. (2023). Identifying
mental health factors of cancer
patients
in hospital
X. Jurnal
Berkala Epidemiologi, 11(1), 92-
100.
https://dx.doi.org/10.20473/jbe.11i1
2023. 92-100 Characteristics of Respondents Patient characteristics are important as an
essential reference for a study. The characteristic
describes the subjects to be studied. The following
are the characteristics of the respondents in this
study described in the frequency distribution based
on the factors of cancer patients in Hospital X, as
shown in Table 1. Table 1 shows the frequency distribution of
the characteristics of cancer patients in Hospital X. It can be seen from the age of cancer patients; 35.4
percent are the early elderly aged 46-55. For the
gender, it mainly occurred in women, as many as
64 patients (66.70%). Most respondents were not
working (61.50%), but as many as 39 patients had
a final elementary school education, which is
40.60%. Family support and spirituality have a very
close relationship. With family support, other
family members can give each additional strength
and help solve problems. In family support,
spiritual support is one way to overcome
difficulties. This study aims to determine the
factors associated with the mental health of cancer
patients at Hospital X, such as family support and
spirituality. Through this research, doctors and
family members can maintain cancer patients’
mental health. 94 of 100 Solikhah Solikhah, et al / Jurnal Berkala Epidemiologi, 11 (1) 2023, 92-100 94 of 100 Solikhah Solikhah, et al / Jurnal Berkala Epidemiologi, 11 (1) 2023, 92-100 Univariate Analysis The frequency distribution of family support
in cancer patients in hospitals can be seen in Table
2. Most cancer patients received moderate family
support (70.80%). It is shown that most cancer
patients had an intermediate spirituality level
(76%). The frequency distribution of spirituality
regarding proximity to God in cancer patients at X
Hospital is known that most cancer patients felt
somewhat close to God, as many as 49
respondents, equal to 51%. Table 2 shows that
most cancer patients experienced moderate stress,
with as many as 49 respondents (51%), and
experiencing
depression,
as
many
as
55
respondents (57.30%). METHODS This
research
is
quantitative
with
an
observational analytic method conducted with a
cross-sectional
approach. The
independent
variables in this study were the characteristics of
the respondents (age, gender, occupation, and
education of the cancer patients), family support
(the relationship between the cancer patients with
the family), and the condition of cancer patients,
especially about their spirituality. The dependent
variable in this study is the mental health of cancer
patients. The sample in this study used a purposive
sampling technique. RESULTS cancer is Indonesia's most essential and reasonable
support. The family is the leading party that
provides support when someone experiences
problems or is sick (9). Family support will affect
the psychology of cancer patients because cancer
patients will feel reassured, consoled, and loved
(10). Spiritual support can also have a positive
impact. For example, praying can provide peace
because it can improve the individual's relationship
with God. Besides that, prayer enhances the
patient's quality of life, including physical and
mental (11). INTRODUCTION patients usually begins when a person is diagnosed
with cancer, and then the stage status of cancer
continues. This condition will make people with
cancer feel depressed and stressed (5). This cancer
does not only cause physical problems but also
impacts psychology and causes psychological
pressure resulting in the patients becoming
stressed (7). One of the leading causes of death in the world
today is cancer. In 2018, 9.80% of deaths occurred
due to cancer. There are various types of cancer,
but the cancers that contribute to morbidity and
mortality are lung, breast, colorectal, prostate,
skin, and liver. Approximately 70% of cancer
deaths occur in low and middle-income countries
(1). Based on previously done research, pediatric
cancer patients, primarily males aged 6-10 years,
and suffering from leukemia (2), and based on
2013 Basic Health Research, the prevalence of
people living with cancer in Indonesia's population
of all ages is 1.40%. Indonesia is one of the lower-
middle economic category countries. Central Java
province has the highest estimate of people living
with cancer, 68,638 (3), and a relatively high
cancer incidence. Cancer is still the leading cause
of morbidity and mortality, with 1.50% of cervical
cancer cases and 0.60% of breast cancer (4). Cases of mental health disorders such as
discouragement, stress, and depression in cancer
patients are influenced by several factors,
including age, education level, occupation, and
place of residence. The advanced age of cancer
patients can increase their anxiety because the
elderly are physically old, and uncertainty can also
arise because of the low cure rate and other
congenital diseases. Lack of knowledge and
education can also cause stress which develops
into a mental disorder while dealing with the
disease. The limitations of doing activities for the
elderly or with chronic diseases result in
limitations in socializing, making people living
with cancer feel more lonely and isolated (5). Mental illness affects 34% of female cancer
patients (5). Another study also found that 25.71%
of cancer patients had low depression, 45.71% had
moderate depression, and 28.58% had severe
depression (6). Depression experienced by cancer Family support also affects severe depression
in cancer patients indirectly (8). Support from
family or people closest to people living with Table 2 Table 2
Frequency
Distribution
of
Family
Support,
Spirituality, and Mental Health of Cancer Patients Bivariate Analysis Age has a significant relationship with mental
health (stress) in cancer patients, as shown in
Table 3, and family support has a substantial
connection with mental health (depression), as
shown in Table 4. It is known that from a total of
96 cancer patients, eight people are categorized
with high-stress levels, ranging from late adults
(36-45 years old) to late elderly (56-65 years old). The sample size was calculated based on the
sample formula to calculate the cross-sectional
survey. The research instruments and tools used
are the Questionnaire Centre for Epidemiological
Studies Depression Scale (CES-D), Perceived
Stress Scale (PSS), Development of The Family
Support Scale (FSS) for Elderly People, and Daily
Spiritual Experience Scale (DSES). This study
already has an ethical permit from Ahmad Dahlan
University and has been approved under number
012103019. 95 of 100 Solikhah Solikhah, et al / Jurnal Berkala Epidemiologi, 11 (1) 2023, 92-100 95 of 100 Solikhah Solikhah, et al / Jurnal Berkala Epidemiologi, 11 (1) 2023, 92-100
Table 1
Frequency Distribution of Characteristics of
Cancer Patients in Hospital X
Characteristic
n
%
Age
Late Adolescence (17-25
Years Old)
5
5.2
Early Adults (26-35 Years
Old)
7
7.3
Late Adults (36-45 Years
Old)
20
20.8
Early Elderly (46-55
Years Old)
34
35.4
Late Elderly (56-65 Years
Old)
19
19.8
Seniors (>65 Years Old)
11
11.5
Gender
Male
32
33.3
Female
64
66.7
Occupation
Employment
37
38.5
Unemployment
59
61.5
Education
Not going to school
13
13.5
Elementary School
39
40.6
Junior High School
26
27.1
Senior High School
18
18.8
Associate degree
0
0
Bachelor/Master/PhD
0
0
Source: Primary Data, 2021
DISCUSSION
The Relationship between Cancer Patient
Characteristics and Mental Health in Hospital
X
The relationship between the characteristics of
cancer patients, namely age and mental health, and
stress, has a significant relationship—the result
aligns research regarding women's emotional and
mental health with cancer in Indonesia. The study
In contrast to the characteristics of can
patients,
such
as
gender,
occupation,
education, both stress and depression at Hospita
did not have a significant relationship. Th
results follow the research conducted by Utam
Mustikasari
(13),
which
examined
psychosocial aspects of breast cancer patients,
the research undertaken by Suwistianisa et
(2015) regarding the factors that influe
depression in cancer patients treated at RS
Arifin Achmad Riau Province. Source: Primary Data, 2021 Bivariate Analysis Table 2
Frequency
Distribution
of
Family
Supp
Spirituality, and Mental Health of Cancer Patie
Variable
n
%
Family Support
Low
16
16.70
Moderate
68
70.80
High
12
12.50
Total
96
100
Spirituality
Low
0
0
Moderate
73
76
High
23
24
Total
96
100
Spirituality
Absolutely not
0
0
Somewhat close
49
51
Close
43
45
Very close
4
4
Total
96
100
Stress
Low
39
40.60
Moderate
49
51
High
8
8.30
Total
96
100
Depression
No
41
42.70
Yes
55
57.30
Total
96
100 Table 1
Frequency Distribution of Characteristics of
Cancer Patients in Hospital X
Characteristic
n
%
Age
Late Adolescence (17-25
Years Old)
5
5.2
Early Adults (26-35 Years
Old)
7
7.3
Late Adults (36-45 Years
Old)
20
20.8
Early Elderly (46-55
Years Old)
34
35.4
Late Elderly (56-65 Years
Old)
19
19.8
Seniors (>65 Years Old)
11
11.5
Gender
Male
32
33.3
Female
64
66.7
Occupation
Employment
37
38.5
Unemployment
59
61.5
Education
Not going to school
13
13.5
Elementary School
39
40.6
Junior High School
26
27.1
Senior High School
18
18.8
Associate degree
0
0
Bachelor/Master/PhD
0
0
Source: Primary Data, 2021 In contrast to the characteristics of cancer
patients,
such
as
gender,
occupation,
and
education, both stress and depression at Hospital X
did not have a significant relationship. These
results follow the research conducted by Utami %
Mustikasari
(13),
which
examined
the
psychosocial aspects of breast cancer patients, and
the research undertaken by Suwistianisa et al. (2015) regarding the factors that influence
depression in cancer patients treated at RSUD
Arifin Achmad Riau Province. Table 2
Frequency
Distribution
of
Family
Support,
Spirituality, and Mental Health of Cancer Patients
Variable
n
%
Family Support
Low
16
16.70
Moderate
68
70.80
High
12
12.50
Total
96
100
Spirituality
Low
0
0
Moderate
73
76
High
23
24
Total
96
100
Spirituality
Absolutely not
0
0
Somewhat close
49
51
Close
43
45
Very close
4
4
Total
96
100
Stress
Low
39
40.60
Moderate
49
51
High
8
8.30
Total
96
100
Depression
No
41
42.70
Yes
55
57.30
Total
96
100
Source: Primary Data, 2021 Table 3 Table 3
Relationship between Characteristics of Cancer Patients and Mental Health (Stress) at Hospital X
Characteristics
Stress
n
P-value
Low
Moderate
High
n
%
n
%
n
%
n
%
Age (Years Old)
Late Adolescence: 17-25
4
4.2
1
1 0
0
5
5.20
0.00
Early Adults: 26-35
1
0.0
7
7.30 0
0
7
7.30
Late Adults: 36-45
12 12.50
7
7.30 1
1 20 20.80
Early Elderly: 46-55
16 16.70 13 13.50 5 5.20 34 35.40
Late Elderly: 56-65
5
5.2 12 12.50 2 2.10 19 19.80
Seniors: >65
2
2.1
9
9.40 0
0 11 11.50
Gender
Male
11 11.50 18 18.80 3 3.10 32 33.30
0.68
Female
28 29.20 31 32.30 5 5.20 64 66.70
Occupation
Employment
27 28.10 25
26 7 7.30 59 61.50
0.05
Unemployment
12 12.50 24
25 1
1 37 38.50
Education
Not going to school
4
4.20
6
6.30 3 3.10 13 13.50
0.54
Elementary School
16 16.70 21 21.90 2 2.10 39 40.60
Junior High School
13 13.50 11 11.50 2 2.10 26 27.10
Senior High School
6
6.30 11 11.50 1
1 18 18.80
Associate’s Degree
0
0
0
0 0
0
0
0
Bachelor/Master/PhD
0
0
0
0 0
0
0
0
Family Support
Low
2
2.10 12 12.50 2 2.10 16 16.70
0.19
Moderate
32 33.30 33 34.40 3 3.10 68 70.80
High
5
5.20
4
420 3 3.10 12 12.50
Spirituality
Low
0
0
0
0 0
0
0
0
0.15
Moderate
32 33.30 37 38.50 4 4.20 73
76
High
7
7.30 12 12.50 4 4.20 23
24 Table 3
Relationship between Characteristics of Cancer Patients and Mental Health (Stress) at Hospi ble 3
lationship between Characteristics of Cancer Patients and Mental Health (Stress) at Hospital X (14). In addition, not everyone who does not work
will be mentally disturbed because some people
think that not working can be a time for them to be
calmer and reduce external stressors. There is no relationship between education
and works with stress or depression (13). A study
by Khesht-Masjedi et al (15) also states no
significant relationship between gender depression
levels. Women generally can be more expressive
of their feelings and emotions. For example,
psychologically, women are easier to cry as a form
of emotional outburst and easier to persuade or
change their beliefs (16). Table 3 This psychological
change can make it easier for women to finally
express concerns that are one of the beginnings of
stress or depression and easier to return to positive
thinking about what they are experiencing. In this
study, education and work had no relationship to
mental health, either stress or depression. It can
happen because not everyone with low education
will have insufficient knowledge (13). A person's
knowledge can be obtained from family, society,
or electronic media as a source of information DISCUSSION The Relationship between Cancer Patient
Characteristics and Mental Health in Hospital
X The relationship between the characteristics of
cancer patients, namely age and mental health, and
stress, has a significant relationship—the result
aligns research regarding women's emotional and
mental health with cancer in Indonesia. The study
explains that the older a person is, the quicker their
psychological condition will be disturbed. It can be
because a higher age will cause different anxiety
levels, such as feelings of anxiety because there is
no certainty about the disease he is suffering or
because of his physical independence (5). It is also
said that older cancer patients score higher in
depression than young patients (12). 96 of 100 Solikhah Solikhah, et al / Jurnal Berkala Epidemiologi, 11 (1) 2023, 92-100 96 of 100 Solikhah Solikhah, et al / Jurnal Berkala Epidemiologi, 11 (1) 2023, 92-100 Relationship between Family Support for
Cancer Patients and Mental Health at Hospital
X Relationship between Family Support for
Cancer Patients and Mental Health at Hospital
X This study found that family support is related
to mental health, such as depression. The result
aligns with the research conducted by Suwistiana
et al (14) regarding the factors that influence the
level of depression in cancer patients treated at the
Arifin Achmad Hospital, Riau Province, and
research conducted by Yulianti et al (17) regarding
the relationship between family support and
depression in breast cancer patients. Good family 97 of 100 Solikhah Solikhah, et al / Jurnal Berkala Epidemiologi, 11 (1) 2023, 92-100 97 of 100 Solikhah Solikhah, et al / Jurnal Berkala Epidemiologi, 11 (1) 2023, 92-100 Cancer patients with low family support feel
they do not get enough support from their families. It might happen due to the lack of family
interaction and the lack of information that cancer
patients can obtain. Research also explains that
low family support triggers depression. It can
cause patients to experience severe pain (22). The
source of human strength that leads to happiness in
life can increase a person's immunity to overcome
the pain they suffer (23). support was associated with lower anxiety levels
(18). Family support can influence cancer patients'
psychology and emotional responses and will, of
course, aid their healing process (19). The family
will act as mediators to help patients manage their
emotions healthily, leading to self-acceptance and
the capacity for a happy, optimistic life (20). Through motivation and spirituality, families
provide meaningful support for cancer patients. Family members and cancer patients are also
recommended to socialize (21). Table 4
Relationship between Characteristics of Cancer Patients and Mental Health (Depression) at Hospital X
Depression Table 4
Relationship between Characteristics of Cancer Patients and Mental Health (Depressio CONCLUSION The
analysis
results
show
that
the
characteristics of cancer patients with mental
health are influenced by age. Meanwhile, the
respondent's gender, education, and occupation
characteristics are unrelated to their mental health. Family support affects cancer patients' mental
health, while spirituality has no relationship with
their mental health. The previous research explains that spiritual
well-being positively correlates with mental health
(26). t is because high spirituality will change the
patients' mindset to accept their condition better
and face their illness with gratitude to God. In a
previous study, Komariah et al (27) used an
Islamic-based practice approach for breast cancer
patients. As a result, there was a change in positive
responses, which made them closer to God and
have a peaceful mind in their daily life. Besides,
depression and anxiety in Muslim cancer patients
have a low tendency if they are more religious,
which is understanding several definitions of Al-
Islam, Al-Iman, and Al-Ihsan (28). Cancer patients
will assume that their disease is the will of God. This situation will make cancer patients more
intensive in carrying out worship to drive them
calmer and more hopeful for recovery (11). Based on the research, the researcher provides
several suggestions. For Hospital X, it is hoped
that it will continue to provide services or facilities
to provide mental support for cancer patients and
assist them in preventing mental health disorders
due to their conditions. Meanwhile, for cancer
patients in Hospital X, it is expected that they will
continue to increase their confidence and ability to
undergo treatment to prevent mental disorders that
can affect their physical condition. Lastly, for
other
researchers,
the
researchers
suggest
conducting further research on the factors that
affect the mental health of cancer patients with
more in-depth or qualitative methods. CONFLICT OF INTEREST No potential conflict of interest is relevant to
this article. However, other studies have shown that
spiritual coping can give excellent or bad side
effects on mental health. If a person can associate
his illness with a constructive attitude based on
faith, it can encourage adaptation to health and
psychological adjustment to stress. If a person sees
their illness as a punishment for an existential
crisis, spiritual coping becomes a negative side
effect (29). The
Relationship
between
Spirituality
of
Cancer Patients and Mental Health in Hospital
X The spirituality of cancer patients with mental
health did not have a significant relationship in
Hospital X. The result is different from the
Wiksuarini et al (11) research about cancer
patients' spirituality and quality of life. According
to Chaar et al (25), regarding evaluating the impact
of spirituality on the quality of life, anxiety, and
depression, a transverse observational study
explains that spirituality relates to cancer patient's
mental health. 98 of 100 Solikhah Solikhah, et al / Jurnal Berkala Epidemiologi, 11 (1) 2023, 92-100 meanings. Spirituality can be related to one's
religion. However, it can also be related to other
things, such as the experience of seeking meaning,
purpose, and well-being between oneself and
others, with oneself, or with the ultimate reality
(30). The findings of the distribution of spirituality
frequencies regarding the closeness of cancer
patients to God support the hypothesis from earlier
research. Of the majority of Muslim cancer
patients, not all of them have faith or attachment to
their God; Most of them have moderate spirituality
(73 patients), and only 23 patients have high
spirituality. Therefore, it can be stated that even
though a patient has religion, it does not mean they
are spiritual enough toward their God. Therefore, special attention is needed for the
treatment of cancer patients. One of them is family
support, especially from the closest family. Family
caregivers will have more time to interact and be
close to the patients (24). With family support,
cancer patients will feel more excited, valued, and
motivated
to
undergo
treatment
for better
conditions (17). Support from family or close
people with cancer gives a feeling of being
protected so that patients feel more comfortable
with the disease they are suffering. Table 4 ble 4
lationship between Characteristics of Cancer Patients and Mental Health (Depression) at Hospital X
Characteristics
Depression
n
P-value
No
Yes
n
%
n
%
n
%
Age (Years Old)
Late Adolescence: 17-25
3
3.10
2
2.10
5
5.20
0.30
Early Adults: 26-35
2
2.10
5
5.20
7
7.30
Late Adults: 36-45
8
8.30
12
12.50
20
20.80
Early Elderly: 46-55
19
19.80
15
15.60
34
35.40
Late Elderly: 56-65
5
5.20
14
14.60
19
19.80
Seniors: >65
4
4.20
7
7.30
11
11.50
Gender
Male
14
14.60
18
18.8
32
33.30
0.88
Female
27
28.10
37
38.50
64
66.70
Occupation
41
42.70
55
57.30
96
100
Employment
28
29.20
31
32.30
59
61.50
0.23
Unemployment
13
13.50
24
25
37
38.50
Education
Not going to school
9
9.40
4
4.20
13
13.50
0.10
Elementary School
18
18.80
21
21.90
39
40.60
Junior High School
9
9.40
17
17.70
26
27.10
Senior High School
5
5.20
13
13.50
18
18.80
Associate’s Degree
0
0
0
0
0
0
Bachelor/Master/PhD
0
0
0
0
0
0
Family Support
Low
2
2.10
14
14.60
16
16.70
0.01
Moderate
31
32.30
37
38.50
68
70.80
High
8
8.30
4
4.20
12
12.50
Spirituality
Low
0
0
0
0
0
0
Moderate
31
32.30
42
43.80
73
76
0.93
High
10
10.40
13
13.50
23
24 Relationship between Characteristics of Cancer Patients and Mental Health (Depression) etween Characteristics of Cancer Patients and Mental Health (Depression) at Hospital X 98 of 100 Solikhah Solikhah, et al / Jurnal Berkala Epidemiologi, 11 (1) 2023, 92-100 AUTHOR CONTRIBUTIONS SS handled the research's concept and idea,
RR was responsible for the final version of the
manuscript, and WR and FN experimented. All
authors shaped the research. Although the concepts of religiosity and
spirituality are related, they have different 99 of 100 Solikhah Solikhah, et al / Jurnal Berkala Epidemiologi, 11 (1) 2023, 92-100 REFERENCES 12. Estapé T. Cancer in the Elderly: Challenges
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A Physiologically-Based Pharmacokinetic Modeling Approach Using Biomonitoring Data in Order to Assess the Contribution of Drinking Water for the Achievement of an Optimal Fluoride Dose for Dental Health in Children
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International journal of environmental research and public health/International journal of environmental research and public health
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cc-by
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International Journal of
Environmental Research
and Public Health
Article
A Physiologically-Based Pharmacokinetic Modeling
Approach Using Biomonitoring Data in Order to
Assess the Contribution of Drinking Water for the
Achievement of an Optimal Fluoride Dose for Dental
Health in Children
Keven J. Jean 1,2 , Nancy Wassef 1 , Fabien Gagnon 1,3 and Mathieu Valcke 1,2, *
1
2
3
*
Institut National de Santé Publique du Québec (INSPQ), Montréal, QC H2P 1E2, Canada;
keven.biomed@outlook.com (K.J.J.); nancy.wassef@inspq.qc.ca (N.W.); fabien.gagnon@inspq.qc.ca (F.G.)
Département de Santé Environnementale et Santé au Travail, École de Santé Publique de l’Université de
Montréal (ESPUM), Montréal, QC H3C 3J7, Canada
Centre de Recherche du Centre Hospitalier Universitaire de Sherbrooke (CRCHUS), Sherbrooke,
QC J1H 5N4, Canada
Correspondence: mathieu.valcke@inspq.qc.ca; Tel.: +1-514-864-1600 (ext. 3226)
Received: 27 April 2018; Accepted: 21 June 2018; Published: 28 June 2018
Abstract: Due to an optimal fluoride concentration in drinking water advised for caries prevention
purposes, the population is now exposed to multiple sources of fluoride. The availability of
population biomonitoring data currently allow us to evaluate the magnitude of this exposure.
The objective of this work was, therefore, to use such data in order to estimate whether
community water fluoridation still represents a significant contribution toward achieving a suggested
daily optimal fluoride (external) intake of 0.05 mg/kg/day. Therefore, a physiologically-based
pharmacokinetic model for fluoride published in the literature was used and adapted in Excel
for a typical 4-year-old and 8-year-old child. Biomonitoring data from the Canadian Health
Measures Survey among people living in provinces with very different drinking water fluoridation
coverage (Quebec, 2.5%; Ontario, 70% of the population) were analyzed using this adapted model.
Absorbed doses for the 4-year-old and 8-year-old children were, respectively, 0.03 mg/kg/day and
0.02 mg/kg/day in Quebec and of 0.06 mg/kg/day and 0.05 mg/kg/day in Ontario. These results
show that community water fluoridation contributes to increased fluoride intake among children,
which leads to reaching, and in some cases even exceeding, the suggested optimal absorbed dose
of 0.04 mg/kg/day, which corresponds to the suggested optimal fluoride intake mentioned above.
In conclusion, this study constitutes an incentive to further explore the multiple sources of fluoride
intake and suggests that a new balance between them including drinking water should be examined
in accordance with the age-related physiological differences that influence fluoride metabolism.
Keywords: biomonitoring; dental health; drinking water; fluoride; pharmacokinetic modeling
1. Introduction
Intentional fluoridation of drinking water has been in use as early as the 1950s in order to prevent
tooth decay both in children and adults. Based on data extracted from McClure’s [1] work, for the past
several decades, an intake (that is, an external dose) of 0.05 mg/kg/day of fluoride has been considered
to correspond to an “optimal fluoride intake” for caries prevention while minimizing fluorosis risk.
It is the underlying population intake target behind the artificial fluoridation of drinking water at a
concentration varying between 0.5 mg/L and 1 mg/L. This is now generally considered by many dental
Int. J. Environ. Res. Public Health 2018, 15, 1358; doi:10.3390/ijerph15071358
www.mdpi.com/journal/ijerph
Int. J. Environ. Res. Public Health 2018, 15, 1358
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health professionals as a key public dental health measure [2]. Such fluoridation is recommended
by the World Health Organization and supported by several organizations such as Health Canada,
the Canadian Dental Association, the Centers for Disease Control and Prevention (CDC), and the
United States Environmental Protection Agency (US EPA) [3]. Fluoride’s mechanism of action to
prevent caries relies on its effect on tooth enamel, which is mainly composed of hydroxyapatite crystals.
Exposure to fluoride results in the substitution of the hydroxy group by the fluoride anion to produce
fluoroapatite. At the same time, hydroxyapatite is soluble at a pH of 5.5 and fluoroapatite is soluble at
pH 4.5. Lactic acid produced by cariogenic bacteria can cause the pH at the surface of enamel to drop
to between 5.5 and 4.5, which causes demineralization of the enamel. Therefore, the caries preventive
effect of fluoride stems from its ability to be incorporated into dental enamel and form fluoroapatite,
which is resistant to demineralization at a lower pH [4].
In Canada, the proportion of the population with access to intentionally fluoridated water
is currently about 37% [3], but interprovincial disparities on this proportion, which do not vary
throughout the year as a function of the season (contrary to some other places in the world),
is important. Furthermore, approximately 70% of Ontario’s population now benefit from this public
health measure. The public health concern has decreased from 12% in the early 2000s to less than
2.5% today in the neighboring Province of Quebec [5]. In this context, it is interesting to note that,
according to the Ontario Association of Public Health Dentistry (OAPHD), for the 2012–2013 school
year, Grade 2 students in Ontario had a mean DMFT index (decayed, missing, or filled teeth due to
caries) in primary and permanent teeth (dmft+DMFT) of 2.22 with 50% of students being caries free.
This result is based on screening data voluntarily reported by participating public health units to the
OAPHD [6]. In Quebec, the Clinical Study on the Oral Health of Quebec Elementary School Students
in 2012–2013 revealed, for the same age group, a mean dmft+DMFT of 2.67 with 44% of students being
caries-free [7]. It should be recalled that dental caries have a multifactorial etiology and that the study
groups were neither sampled nor weighted, according to fluoride exposure.
Proponents of community water fluoridation (CWF) highlight its contribution to address social
health disparities [8]. In addition, nearly one in three Canadians does not have dental insurance and
cannot receive the treatments needed due to their cost [9]. In Canada, dental caries treatments are
second only to mental health problems with regard to societal costs ever since the mid-1970s [10].
Since tooth decay can affect children and adults as well as the elderly, the protective effect of
fluoridated water consumption benefits everyone regardless of age. However, overexposure to fluoride
during the tooth development can cause dental fluorosis. Mild to severe dental fluorosis is observed
more frequently when the concentrations of fluoride in drinking water increase. Chronic exposure
to high levels of fluoride may also cause more severe effects such as skeletal fluorosis and bone
fractures [3,11–14], but the relevant studies have many limitations inherent to their ecological design.
In addition, Health Canada’s fluoride expert panel concluded a decade ago that the scientific studies
available at the time did not support a link between intentional fluoridation of drinking water and
cancer or any other adverse effect than fluorosis [15]. Since that time, further studies have raised some
concerns regarding the neurodevelopmental toxicity of fluoride. However, at exposure levels that are
generally well above the exposure levels correspond to intentional fluoridation of drinking water [16].
Additional studies have further supported the presumption that drinking water fluoridation is not
associated with an increased risk of osteosarcoma [17–21] nor with altered thyroid function [22].
In contrast with the era when the optimal fluoride concentration in drinking water was
determined, the population is now exposed to multiple sources of fluoride that contribute to
the total daily fluoride intake (TDFI) such as through the use of fluoride toothpaste but also
by ingesting a greater variety of foods and beverages as well as existing exposure routes such
as soil ingestion and inhalation of residual air concentrations. Therefore, it appears relevant
to re-examine the contribution of drinking water fluoride for caries prevention purposes today.
The availability of both a physiologically-based pharmacokinetic (PBPK) model for fluoride [23]
as well as population biomonitoring data for this element within the Canadian Health Measures
Int. J. Environ. Res. Public Health 2018, 15, 1358
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Survey (CHMS) [24], which is combined with the acknowledged disparity between drinking water
fluoridation coverage between two provinces mentioned above, represent a unique opportunity in
this regard. The considerable statistical weight of the participants from the provinces of Quebec
and Ontario within the whole CHMS allows the extraction of data specific to these provinces and
considered as reasonably representative of their population’s exposure to the measured chemicals
including fluoride [25].
The objective of this work was, therefore, to apply a PBPK modeling approach based on fluoride
biomonitoring data from two CHMS provinces with very different drinking water fluoridation
coverage in order to examine the resulting contribution of drinking water to achieving an optimal dose
of fluoride in children from a caries prevention perspective.
2. Materials and Methods
The approach followed here relies on the use of a PBPK model in order to estimate internal
exposure in the provinces of Quebec and Ontario using corresponding fluoride biomonitoring data
from the second and third cycles (2009–2013) of the CHMS. The obtained doses are then compared to
the suggested optimal intake for dental health of 0.05 mg/kg/day. Lastly, the internal dose metrics
(IDM) in blood and urine corresponding to the chronic consumption of 0.7 mg/L fluoridated water
were compared to the same IDM associated with the suggested optimal intake of 0.05 mg/kg/day.
2.1. PBPK Model Development and Validation
2.1.1. Model Structure
The PBPK modeling approach used in this work is based on the model developed by Rao et al. [23]
to simulate the bone kinetics of fluorides for life-long chronic exposure in rats and humans (Figure 1).
Figure 1. Conceptual diagram of (A), the original PBPK model of Rao et al. [23] and (B) the PBPK
model simplified for the purposes of this work. Each box represents a compartment (an organ or a set
of organs). The arrows symbolize the distribution of fluoride through the bloodstream.
The initial model of Rao et al. [23] considers the subdivision of the bone compartment into the
surface bone and the inner bulk bone in order to simulate the bone kinetics of fluoride in detail.
The original objective of these authors was to model the variation of the bone fluoride concentration
for a period of several years. For the purposes of the current work, however, solely the blood and
urinary concentrations of fluoride were of interest. In addition, since the model aimed to simulate
fluoride’s kinetics in children, the targeted population in the current study, in whom there is virtually
no bone resorption because of growth, the inclusion of a compartment to consider the mobilization
of bone fluoride and its release into the blood circulation was not required. Therefore, the original
Rao et al. [23] model was simplified by considering a single bone compartment, which was reproduced
Int. J. Environ. Res. Public Health 2018, 15, 1358
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in Microsoft Excel™ by Haddad et al. [26] and modified to specifically reflect the kinetics of fluoride
in children.
2.1.2. Model Parameters
In order to adapt the model of Rao et al. [23] in children, the fixed values of the physiological
parameters used in the original model were replaced by formulas described in the literature [27–30].
These equations allow for accounting the age-specific and body weight-specific variations of blood
flow and volume in the child. In doing so, average human equations for both sexes were used because
provincial biomonitoring data for the current work were not distinguished by gender. The tissue
partition coefficients of the Rao et al. [23] model were applied unchanged. The model considers that
fluorides are solely eliminated by renal clearance. Therefore, the plasma clearance formula used
by Rao et al. [23] was normalized to body weight to the 0.75th power. In a longitudinal dog study,
bone clearance and renal clearance accounted respectively for 90% and 10% of plasma clearance in
the pup and 50% each in the mature dog [23]. Assuming this relation is linear, this reasoning was
used to draw two lines (0, 90, 18, 50) and (0, 10, 18, 50) whose slopes make it possible to obtain the
percentages of the plasma clearance attributable to bone clearance or renal clearance as a function
of age. By multiplying these percentages by plasma clearance, values for bone clearance and renal
clearance were obtained.
Since the model aims to consider various sources of fluoride exposure such as air, tap water,
soil (dust), fluoride toothpaste, food, and beverages (bottled water included), source-specific
bioavailability factors were assigned in the model according to available literature [11]. Bioavailability
factors of 83%, 100%, 100%, and 40% were used for water [31–33], air [34], fluoride toothpaste [35],
and diet [36], respectively. For soil, the same bioavailability factor used for diet was attributed given
that the factors influencing its bioavailability are similar. Due to the lack of available data on oral
absorption constants from the GI tract in human PBPK models for fluoride, direct infusion in the
liver was assumed, which triggers the withdrawal of the GI track compartment from the initial model
(Figure 1B). Therefore, any daily dose ingested would be transformed into a minute-based infused
dose in the liver over 24 h.
2.1.3. Model Validation
For the purpose of model validation, data described in several published studies [37–41] in which
fluoride intake was estimated and its urinary excretion measured were used in order to compare model
simulations with observed data for given exposure conditions of specific child subjects. Additionally,
among the available studies with measured fluoride intake and excretion in 24-h urine samples,
the need for study participants to be aged eight years and under, in accordance with the targeted
population for the modeling, was retained as a selection criterion. Since no such studies were found for
children in Canada, studies from various countries (Venezuela, Chile, Germany, and United Kingdom)
were selected to validate the model. Table 1 shows the five studies selected for fluoride intake and the
amount of excreted urinary fluoride they measured.
Table 1. Fluoride intake and urinary fluoride measured in the studies were selected to validate the model.
Intake (mg/day)
Age (years)
Children (n)
4
4
5
5
7
7
31
20
61
11
21
12
1
Diet
Toothpaste
Water
Supplement
0.560
0.533
0.151
0.092
0.187
0.229
0.706
0.254
0.608
0.274
0.606
1.130
0.042
0.231
0.407
0.111
0.154
0.349
0.455 2
-
AuF-24 1 (mg/day)
Country
0.3682
0.358
0.3705
0.476
0.297
0.393
Venezuela [37]
Chile [38]
UK [39]
Germany [40]
UK [41]
UK [41]
Amount of urinary fluoride excreted in 24 h. 2 Fully absorbed fluoride tablets.
Int. J. Environ. Res. Public Health 2018, 15, 1358
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2.2. Exposure Scenarios
2.2.1. Subjects of Interest
Since this study aimed to interpret and use Canadian biomonitoring measures of fluorides,
specific PBPK models for children of the corresponding age were built. In doing so, the median age of
the two CHMS child age groups, was entered in the generic model described in Section 2.1. Specifically,
distinct models for 4-year-old and 8-year-old children were built. Additionally, the 3-year to 5-year
age group (median age: 4 years) was selected to evaluate the risk of dental fluorosis. In this age
group, permanent teeth are still in development. Although the esthetic effects of dental fluorosis on
permanent anterior teeth are generally considered to have taken place from birth to age 3, there was a
lack of studies permitting a suitable validation of the model for this group. Complimentarily, the 6-year
to 11-year age group (median age: 8 years) was selected in order to bring out insights on the possible
age-related physiological differences that influence fluoride metabolism. The 50th percentile values
of the World Health Organization (WHO) growth charts adapted for Canada in 2014 [42] were used
to determine body weights and heights of the targeted median children. Then, data reflecting the
Canadian exposure context were sought. The intake considered for fluoride toothpaste originated
from an internal consultant report discovered by Health Canada based on data collected in the early
2000s concerning toothpaste containing between 996 and 1455 ppm of fluoride [11] while air and soil
intakes were taken from the INSPQ [43]. Data on food and beverage intake from a survey of fluoride
intake in the Canadian diet were also used [11]. With respect to the daily rate of water consumption,
direct water consumption from a UK study [44] was selected. This was due to available Canadian data
that did not concern the specific ages targeted herein or include indirect water consumption already
taken into account by the food and beverage intake. Age-specific parameter values are presented
in Table 2.
Table 2. Age-specific fixed parameter values needed to model fluoride exposure.
Sources of Fluorides
Age (year)
Weight (kg)
Height (cm)
4
8
16
25
103
127
Toothpaste
Diet
Soil
Air
(µg/kg/day) (µg/kg/day) (µg/kg/day) (µg/kg/day)
40
30
21
17.5
1.19
0.21
0.01
0.01
Water
(L/day)
0.442
0.56
2.2.2. Model Simulations of Internal Dose Metrics of Interest
Continuous fluoride exposure until the steady-state is reached, after approximately 150 days for
both arterial blood and urinary fluoride (see example in Figure 2), was simulated with the models.
Given that CHMS biomonitoring data, which will be used further, result from spot urine samples,
an assumption that the measured urinary fluoride is a consequence of steady-state exposure is made.
This presumption is a state-of-the art hypothesis for the interpretation. For instance, Biomonitoring
Equivalents (BE) [45] of data was collected in large biomonitoring surveys such as the National
Health and Nutrition Examination Survey (NHANES) [46], the German chemical Exposure Study
(GERes) [47,48], and CHMS [49] with model-derived or model-simulated exposures.
By entering the available fluoride’s external exposure data for Quebec and Ontario as input in
the PBPK model, the resulting urinary levels can be compared to the corresponding CHMS values,
which exhibit significantly greater population mean urinary fluoride concentration in Ontario as
compared to Quebec [25]. Specifically, a 24-h steady state amount of excreted urinary fluoride was
obtained by subtracting the simulated amount of urinary fluoride excreted after 149 days of continuous
exposure from the same amount excreted after 150 days.
Int. J. Environ. Res. Public Health 2018, 15, 1358
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Figure 2. (A) Arterial and (B) urinary fluoride concentration in a 4-year-old child continuously exposed
to 0.7 mg/L of fluoridated water.
2.2.3. Exposure Scenarios Simulated
Three exposure scenarios were considered. In the first scenario, exposure for a 4-year old and
an 8-year old child from Ontario is modeled based on intakes listed in Table 2 from air, soil, diet,
fluoride toothpaste, and fluoridated water (Figure 3). Since 70% of Ontario’s population including
children has access to fluoridated water at 0.7 mg/L. An equivalent average fluoride concentration of
0.5 mg/L was calculated assuming that the remaining 30% non-fluoridated water had a concentration
of 0.05 mg/L (detection limit). In the second scenario, in the deemed representative of a 4-year old or
an 8-year old child in Quebec, it is assumed that the only difference in exposure sources lies in the much
lower access to fluoridated water at 0.7 mg/L. Therefore, this second scenario was modeled with 2.5%
of the Quebec child population consuming fluoridated water at 0.7 mg/L for an equivalent average of
0.06 mg/L (if 97.5% of inhabitants drink non-fluoridated water at a concentration of 0.05 mg/L).
Figure 3. Flow chart describing the methodological rationale underlying the fluoride exposure scenarios
simulated by PBPK modeling in order to examine the original research question.
Int. J. Environ. Res. Public Health 2018, 15, 1358
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As shown later (see Results), the simulation of the second scenario tends to overestimate the
urinary concentration of fluoride. In order to evaluate the contribution of drinking water to TDFI in
Quebec, the consistency between input parameters used in the model and reality, presumably reflected
accurately by CHMS data, is required. This contribution was evaluated differentially by modeling the
daily amounts of urinary fluoride excreted at steady-state levels assuming the exposure conditions
present in Ontario vs. Quebec and comparing them to those modeled for the TDFI using available
fluoride intake input parameters. Therefore, for determining the contribution of drinking water to the
overall fluoride exposure, it was necessary to account for the fact that fluoride concentrations in water
may affect those found in foods via cooking and preparation of processed food. Therefore, the third
scenario was modeled without fluoride intake from the diet to better reflect the exposure of a 4-year
old or an 8-year old child in Quebec.
2.3. Determination of Mean Absorbed Fluoride Dose in Average Children from Quebec and Ontario Using
Biomonitoring Data
Since the present work relies in part on biomonitoring data, an absorbed dose is deemed more
relevant to work for the purpose of the present study than an intake. Therefore, the calculated total
ingested fluoride intake from water and diet were converted into corresponding absorbed doses by
applying the relevant bioavailability factors of 83% and 40% presented above (see Appendix A
Table A1). A mean resulting absorbed dose across all ages investigated (1–12 years old) of
0.04 mg/kg/day was obtained and was, henceforth, used for the purpose of this work.
Based on the mean and 95th percentile values of urinary concentrations of fluoride measured in
the two CHMS provinces, corresponding absorbed doses in Quebec and Ontario could be modeled
using the solver complement in Microsoft Excel™. The solver provides a value for a given parameter
by modifying one or more model parameters while including constraints as needed. Therefore,
CHMS urinary concentrations were converted to excreted amounts of urinary fluoride using daily
urinary fluoride excretion rates [50]. The converted CHMS fluoride amount was entered into the
solver as a 24-h steady state parameter and the model was only allowed to change the absorbed TDFI
parameter while retaining the values and equations for all other parameters. The only constraint in
this case was that the TDFI had to be a positive number (greater than zero). The solver then made it
possible to determine the absorbed dose associated with the amount of urinary fluoride. The absorbed
doses from Quebec and Ontario obtained by using the solver with CHMS urinary fluoride amounts
at the mean and 95th percentile values could then be analyzed to determine if (1) the absorbed dose
of 0.04 mg/kg/day detailed above was reached in those provinces and (2) the resulting intakes
correspond to the total intakes computed in Section 2.2.2 considering different exposure levels via
drinking water fluoridation.
2.4. PBPK Model Sensitivity Analyses
In order to know the impact of the variation of each parameter on the predicted internal
concentrations of fluoride, a sensitivity analysis of the model parameters was carried out, according to
the following formula (Valcke & Krishnan [29]).
SIP =
AexcF2 − AexcFi
Pi
×
P2 − Pi
AexcF_i
(1)
where SI_P is the sensitivity index of the parameter “P”, AexcF_2 is the quantity of urinary fluoride
excreted over 24 h at a steady-state after reduction of the “P” parameter by 2% of its initial value,
AexcF_i is the initial value of the quantity of urinary fluoride excreted over 24 h at a steady-state, Pi is
the initial value of a parameter “P”, and P2 is its value after being reduced by 2% of its initial value.
Int. J. Environ. Res. Public Health 2018, 15, 1358
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3. Results
3.1. Model Validation
For intakes ranging from 0.04 mg/kg/day to 0.08 mg/kg/day in the studies used as validation,
the amounts of urinary fluoride predicted by the PBPK model were always greater than the amounts
measured (Figure 4). In order to overcome this systematic overestimation by the model, the average
ratios “urinary fluoride measured/urinary fluoride modeled” were calculated to obtain the average
empirical adjustment factor of 0.43. This factor was incorporated into the final model prior to running
the simulation of exposure scenarios and model-based interpretation of biomonitoring data for which
the results are presented hereafter in Sections 3.2 and 3.3.
Figure 4. Results of model simulation of exposure data in the literature as a function of experimental
biological measures in children aged 4 to 7 years. Each point represents a study while the line represents
a perfect agreement. The values at the top of the points represent the ratios between the modeled
values and the experimental values. Panels (A,B) illustrate, respectively, the over-estimation and
underestimation of the modeled values. Panels (C,D) show the predicted values around the perfect
agreement line when adjusted by multiplying by the adjustment factor of 0.43 in (C) and 1/0.43 in (D).
3.2. Modeled Exposure Scenarios
Table 3 shows that the modeled urinary concentrations of fluoride for the first scenario are slightly
higher than the concentrations measured in Ontario’s CHMS participants with ratios of 1.02 and
1.09. For the second scenario, the modeled concentrations were much higher than those measured in
Quebec’s CHMS participants with ratios of 1.81 and 1.78. This suggests that the modeled exposure
for this scenario does not correspond to the actual exposure likely due to one or more sources of
fluoride are overestimated for Quebec. The concentrations modeled for exposure in the third scenario
approximate the measured concentrations while remaining higher with ratios of 1.47 and 1.41.
Int. J. Environ. Res. Public Health 2018, 15, 1358
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Table 3. Urinary fluoride concentrations (mg/L) modeled in a 4-year-old and an 8-year-old child for
different exposure scenarios compared with Province-specific CHMS biomonitoring data.
Age-Specific Results
Province
4 year-old Child
Scenario
Model CHMS Ratio
8 year-old Child
1
Model CHMS Ratio 1
Ontario
1. (Fluoridation of drinking
water at 0.7 mg/L)
0.846
0.83
1.02
0.73
0.67
1.09
Quebec
2. (Fluoridation at 0.06 mg/L
with dietary intake)
0.708
0.39
1.81
0.605
0.34
1.78
Quebec–modified
3. (Fluoridation at 0.06 mg/L
without dietary intake)
0.574
1.47
0.48
1
1.41
Ratio of the modeled urinary concentration over the CHMS bio-monitored urinary concentration.
Table 4 shows the average absorbed fluoride doses, which were modeled based on CHMS urine
concentrations using Microsoft Excel™’s solver complement. In Quebec, it is lower than the suggested
optimal absorbed dose of 0.04 mg/kg/day. In Ontario, this dose is reached and even exceeded.
However, when looking at the 95th percentile, the suggested optimal absorbed dose is exceeded in
both provinces.
Table 4. Absorbed fluoride doses (mg/kg/d) modeled with CHMS biomonitoring data from Quebec
and Ontario.
Age
Quebec, Geometric Mean (95th Percentile)
Ontario, Geometric Mean (95th Percentile)
4
8
0.03 (0.13)
0.02 (0.05)
0.06 (0.17)
0.05 (0.12)
3.3. Contribution of Drinking Water to the Total Intake of Fluoride
Figure 5 shows the contribution, based on PBPK model simulations, of different fluoride sources
(see Table 2) to total intake as well as amounts or urinary fluoride for Quebec and Ontario converted
from CHMS biomonitoring data. The contribution of air and soil is minimal. The most important
source is toothpaste followed by fluoridated water at 0.7 mg/L and diet. The TDFI when the water
is fluoridated at 0.7 mg/L is above the suggested optimal intake, which is slightly higher than what
was measured in Ontario. In Quebec, where the majority of fluoride exposure is through ingestion of
fluoride toothpaste, the intake measured by the CHMS is even lower than the modeled intake from
fluoride toothpaste.
Figure 5. Modelled excreted fluoride over 24 h at a steady-state, according to the different intakes
considered in the 4-year-old (A) and 8-year-old (B) child. The data from Table 2 were used for fluoride
sources. These modeled data are compared to CHMS biomonitoring geometric mean data for cycles
2 and 3 combined for Quebec (QC) and Ontario (ONT) for children aged 3–5 years-old (A) and
6–11 years-old (B). Symbols: OAI: suggested Optimal absorbed intake of 0.04 mg/kg/day. TI: Total
(absorbed) intake. FT: Fluoride toothpaste. FW: 0.07 mg/L fluoridated water. FB: Food and beverages.
Int. J. Environ. Res. Public Health 2018, 15, 1358
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3.4. Sensitivity Analyses
Figure 6 shows the parameters with the highest sensitivity indexes (SI) in the model. These include
body weight, oral absorption fraction, renal clearance, bone volume, and fluoride intake (by using
water, food, and toothpaste ingestion). For concision reasons, Figure 6 only includes parameters
exhibiting an SI equal to or greater than 0.05.
Figure 6. Sensitivity indices of the model parameters above the arbitrarily determined threshold of 0.05.
Symbols: BW: Body weight. OAF: Oral absorption fraction. RC: Renal clearance. BV: Bone volume.
WI: water intake. FBI: Food and beverages intake. TI: Toothpaste intake. AUC vc 4: Area under the
curve of venous concentrations of fluorides over 24 h for a 4-year-old child. AUC vc 8: Area under the
curve of venous concentrations of fluorides over 24 h for an 8-year-old child. Aexc 4: Amount of urinary
fluorides excreted over 24 h for a 4-year-old child. Aexc 8: Amount of urinary fluorides excreted over
24 h for an 8-year-old child.
4. Discussion
To the best of the author’s knowledge, this study is the first of its kind considering a coupled
PBPK/biomonitoring data modeling approach to the comparison of fluoride intake in children living
in regions with significantly differences in their access to intentionally fluoridated drinking water.
PBPK modeling is an extensively used approach in order to interpret population biomonitoring data
on chemical exposures [51–58], but it has never been applied to fluoride in this perspective nor has it
been applied to fluoride exposure through drinking water from a dental health perspective. Therefore,
the comparison made regarding the fluoride intake in children from two Canadian provinces differing
in their access to optimally fluoridated water proposes new methodological insights with regard to
future fluoride intake assessments.
This study suggests that drinking water fluoride still represents an important contribution for
children under 8 years of age to attain the suggested daily optimal fluoride intake to prevent caries,
but that a reduction in the intake of fluoride could also be desirable from the perspective of the
most exposed children in order to prevent them from exhibiting a total exposure that may exceed
toxicological reference values. Such a reduction could still be made while continuing to benefit from
the protective effects of fluoride against tooth decay.
Although fluoride is classified as a non-essential mineral, it does have a protective effect against
tooth decay. As any other preventive measure, its relevance is directly related to the risk of developing
caries. It may not be relevant to use fluorides in populations who are not at risk of developing dental
caries. In addition, fluoride’s effect being predominantly topical, it is important not to confuse the
systemic absorption of fluoride with its topical effect since “topical fluorides” will always have some
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degree of ingestion and “systemic fluorides” will exert topical effects through saliva and crevicular
fluid [59].
The use of an “optimal fluoride dose” based on McClure’s work, though controversial, remains the
most appropriate population-level estimate for the purposes of this study because there is no other
estimate currently available to predict how much fluoride is likely to confer a caries preventive effect
while minimizing the risk of developing fluorosis. Although the relevancy of the notion of “optimal
fluoride dose” to prevent dental caries exceeds the scope of this paper, it is worth mentioning that it
has recently been suggested that the term optimal or “optimum” be dropped in favor of defining a
value that provides “appropriate outcomes” for both caries and fluorosis [60]. However, considering
the varying levels of caries risk between population subgroups as well as physiological differences
affecting fluoride metabolism and absorption, a range of values may be more appropriate for future
population level modeling studies [61].
By using the daily urinary fluoride presented in Figure 5, the contribution of drinking water
fluoridated at 0.7 mg/L was calculated as 25% of the TDFI. At first glance, it may seem different
from what other studies have found. In a US EPA relative source contribution analysis based on a
mean exposure scenario in the United States, a relative contribution of 42% was found for children
between 4 and 6 years old [62]. However, the fluoride concentration used in their scenario was
0.87 mg/L and the 90th percentile of water consumption (0.943 L/day) was used rather than the mean
(0.442 L/day), which can partly explain the difference with the present analysis. Health Canada’s
assessment [11] based on exposure scenarios using daily Canadian intakes suggests a contribution of
water to the TDFI of 48%. Both direct and indirect water consumption were included in that assessment.
If only direct water consumption was considered, as is the case in the present study, drinking water
contribution would drop to 17%. In another study [63], using a three-day food diary and samples
collected from various fluoride sources (tap water, drinks, foods, toothpastes, and tooth-brushing
expectorate) consumed by 3–4 year old children residing in the Gaza Strip, water contribution to the
TDFI was found to be 12%. The authors of that study mention that very little tap water was used
as a drink. Therefore, the lower contribution of water to the TDFI. Estimates of fluoride intake by
analysis of various sources with a fluoride ion selective electrode were made in South India in order to
assess drinking water contribution to the TDFI, which was evaluated at 39% in 3 year old to 10 year
old children [64]. On the other hand, fluoride toothpaste was not included in that assessment nor
mentioned in the article. If the fluoride toothpaste intake considered in the current study was added in
their contribution assessment, a relative water contribution to the TDFI value of 25% can be obtained.
Therefore, the contribution of drinking water to the TDFI of 25%, as found in the present study using
model simulations, is rather coherent with the values obtained in the aforementioned studies.
The results obtained here suggest that the difference in exposure levels between children from
Quebec and Ontario, brought out by the biomonitoring data, cannot be solely explained by the
fluoridated drinking water at 0.7 mg/L. Furthermore, it can be deduced from studying Figure 5 that
the difference between the two provinces also appears to result from dietary exposure. Since water
is used in the preparation of many foods and beverages, it is to be expected that the concentration
of fluoride in drinking water will affect the concentration found in the diet, which can explain the
difference between both provinces. Taking into account the halo effect, it would be expected that in
Quebec, where only 2.5% of the population has access to fluoridated water, fluoride would still be
found in foods imported from Ontario or the United States. For example, it would be found where
fluoridation is more prevalent. This effect indicates that foods produced in a fluoridated city will
contain a higher fluoride concentration than those produced in a non-fluoridated city where it could be
distributed and consumed, which increases the fluoride exposure of non-fluoridated areas’ residents
and vice-versa [65]. Conversely, modeled dietary intakes do not seem to contribute significantly
to the total intake estimated from biomonitoring data in Quebec, according to Figure 5. However,
this assertion appears rather uncertain given that modeled data on fluoride levels in foods were taken
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from a 1969 Nutrition Canada study [11] and may have changed since then based on the population’s
dietary habits.
The results also suggest that, at age 4, intake from fluoride toothpaste could be sufficient to
reach the suggested optimal intake in the specific population of the present study while, at 8 years
of age, the contribution from other sources would also be necessary (Figure 5). However, Ontario’s
modeled intake exceeds the suggested optimal intake and, therefore, it is possible that only a fraction
of the contribution from the other sources than fluoride toothpaste (drinking water and food) may be
sufficient. These fractions are to be determined. This observation is important given that, although
fluoride exposure prevents tooth decay, it may trigger adverse health effects particularly fluorosis if the
exposure is too high. Setting a drinking water fluoride concentration limit, therefore, requires an ideal
compromise between risk and benefits from a dental health perspective [66]. Still, by applying
the bioavailability factors previously mentioned to tolerable daily intakes (TDI) recommended
by Health Canada (0.1 mg/kg/day) [3,11,15] and, more recently, Australia and New Zealand
(0.20 mg/kg/day) [67], it is possible to compute absorbed TDI values determined to be 0.08 mg/kg/day
by Canadian public health authorities and 0.16 mg/kg/day by those from Australia/New Zealand.
The CHMS biomonitoring data-derived exposure dose for the mean and 95th percentile of Quebec
and Ontario can, therefore, be compared to these absorbed TDIs. This allowed us to evaluate the risk
of exceeding TDIs when drinking water contains fluoride at concentrations targeted with the aim of
reaching a suggested optimal dose against tooth decay.
When comparing those absorbed TDIs with absorbed doses calculated from CHMS data
(see Table 4), it can be noted that, on average, the fluoride intake in Quebec and Ontario is lower than
the Canadian TDI. However, at age 4, the 95th percentile in Quebec has an intake greater than the
Canadian TDI but lower than the Australia/New Zealand TDI. In Ontario, the 95th percentile exceeds
the Canadian TDI at ages 4 and 8 and exceeds the Australia/New Zealand TDI at age 4. Conversely,
in a context where water is generally not fluoridated as in Quebec, the TDI of Australia/New Zealand
is not exceeded. Likewise in Ontario, where most of the population has access to fluoridated water
at a level of 0.7 mg/L, children in the 95th percentile may have an intake greater than the TDI
of Australia/New Zealand at age 4 and, therefore, presents a potentially increased risk of severe,
otherwise rare, dental fluorosis. Additionally, this TDI is twice as permissible as the Canadian one.
Yet, while both TDIs are determined in order to be protective of any adverse health effect including
fluorosis, it is noteworthy to recall that any TDI does not necessarily correspond to an exposure over
which the prevalence of adverse health effects automatically increases, but rather an exposure below
which any effect is likely to occur based on the available weight of evidence. Therefore, we consider
whether a fraction of the population exceeding a given TDI should be thought of as a safeguard
rather than an absolute threshold. Since the dose is expressed in mg/kg, the fluoride intake would
have to increase in proportion to body weight in the long-term to remain at 0.2 mg/kg/day and,
therefore, increase risks of adverse effects. Increased monitoring using tools such as the urinary
fluoride/creatinine ratio (UF/Cr) of 1.69 mg/g suggested by Zohoori and Maguire [68] in order
to detect an excessively high fluoride intake well before the onset of dental fluorosis, could be an
alternative for amending the water fluoridation guideline.
This study has some limitations that must be acknowledged. In the first place, the PBPK models
used are built based on an original one that has seldom, if ever, been used nor validated since its
publication. Therefore, this limits the assessment of its validity. However, the validation process
realized herein shows that, when corrected appropriately, it can reproduce experimental data rather
well (Figure 4). The models are deterministic and represent theoretical average exposure levels
in the two ages considered. They, therefore, do not consider the physiological variations in the
individuals of the same age group nor of different ages. Neither did they account for the variations in
fluoride concentrations in the different exposure sources contrary to the population biomonitoring
data used. This may have contributed in some cases to the lack of agreement between modeled
and measured urinary fluoride values. In this line, a systematic overestimation by a factor of about
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2.5 of the modeled urinary concentration and, therefore, an underestimation of the associated fluoride
intake was observed. The model parameters attributable to such overestimations are revealed by the
sensitivity analysis (Figure 6).
First, the bone clearance of the model, related to the bone volume in the model, which exhibits
a negative AUC-based sensitivity index, may be too low. As a result, the plasma concentration is
overestimated with correspondingly overestimated modeled urine concentration. Since bone clearance
was calculated with a two-point line assuming that the relationship was linear (see Methods), it is
also possible that the values used are not accurate enough. In addition, the model’s overestimation
of urinary fluoride could be explained by an excessive modeled renal clearance, which also exhibits
a negative AUC-based sensitivity index and a positive Aexc-based one. However, according to
the values found in the literature, this does not appear to be the case [8]. Lastly, the absorption
fraction appears to be the main contributor to the overestimation. Direct infusion into the liver was
considered herein rather than via an intermediate gastrointestinal compartment from which part of
the ingested fluoride is absorbed following a rate dictated by an oral absorption constant. The highly
model-sensitive correspondent absorption fraction modeled herein (Figure 6) is probably too high as
a result. Excessive modeled bioavailability for toothpaste is also a valid explanation. Bioavailability
studies are often performed on an empty stomach while tooth brushing is recommended after eating
a meal. The ingestion of toothpaste is more likely to happen in the presence of food, which could
decrease its true bioavailability. This is supported by a lower modeled Aexc for toothpaste exposure
alone than what CHMS data suggests (Figure 5). Unfortunately, the lack of availability of a relevant
oral absorption constant and toothpaste bioavailability for different states of stomach emptiness have
precluded correcting the model in any other way than applying an empirical modification of the model
by a factor of 0.43, which did, however, correct the initial systematic error (Figure 4).
Another limitation related to the fluoride intake may come from dental care or dental hygiene
measures other than the use of fluoride toothpaste. Furthermore, this intake was not taken into
account in the model due to a lack of data precise enough to be used in the model. However,
because professional dental care is provided only a few times each year, it does not appear to
be a significant source of long-term exposure, which, in principle, is what is reflected in CHMS
biomonitoring data. For other personal fluoride products such as mouthwash, these are not
recommended for children under 12 and, therefore, should not affect the estimates in this work.
The lack of information on water fluoride levels associated with CHMS biomonitoring data is a source
of uncertainty as well.
Two final sources of uncertainties in this study that preclude drawing firm conclusions include
the low statistical power of the provincial biomonitoring data for the age groups corresponding to
the typical individuals whose exposure was modeled as well as the fact that the child data used to
validate the model did not come from Canadian studies despite Canadian biomonitoring data being
used in this work. Therefore, since this is the first study of its kind, it needs to be replicated in future
studies to validate the findings. In this regard, obtaining more robust data on fluoride levels in foods
and their bioavailability in foods for cities with variable levels of fluoride content in the drinking water
would improve our assessment particularly with respect to the halo effect of drinking water on food.
Probabilistic modeling should be considered, e.g., via Monte Carlo simulations, in order to address
the limitation issue related to the inter-individual variability of the various fluoride intakes and of the
physiological determinants of its kinetics in children.
5. Conclusions
According to the present model, the currently recommended absorbed fluoride dose of
0.04 mg/kg/day to prevent tooth decay is not attained in Quebec children where drinking water
fluoridation is sparse while it is surpassed by children in Ontario where such fluoridation is extended.
Since this study is the first of its kind regarding the use of biomonitoring data under a PBPK modeling
approach in order to compare fluoride intake in children from two regions differing in their access
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to intentionally fluoridated water, further research needs to be undertaken in order to confirm these
results. In addition, these results are an incentive to further explore the multiple sources of fluoride
intake since they suggest that a new balance between them are sought, which is in accordance with
the physiological differences that influence fluoride metabolism in each age group. This is important
from a public health perspective since the aim is to maximize the number of individuals capable of
achieving a daily fluoride intake that provides appropriate outcomes in terms of caries prevention and
minimizing the risk of fluorosis.
Author Contributions: M.V. and F.G. conceived and designed the study. K.J.J. performed the study. K.J., F.G.,
and M.V. analyzed the data. N.W. contributed to the dental health considerations. K.J.J. wrote the first draft of the
manuscript. M.V. commented and reviewed the first few drafts of the paper. All authors commented and edited
on the following version of the paper until the approval of the final version. M.V. coordinated the overall work
and submitted the paper.
Funding: This research received no external funding.
Acknowledgments: In-kind contributions of INSPQ’s staff are acknowledged. The authors thank André Lavallière
from the Public Health Directorate of Estrie (Sherbrooke, QC, Canada) for his very relevant comments especially
regarding the notion of suggested optimal fluoride intake.
Conflicts of Interest: The authors declare no conflict of interest.
Appendix A
Table A1. Absorbed doses calculated from the McClure [1] data on intakes from water and diet 1 .
Age
(year)
Weight
(kg)
Water
(mL/day)
Diet
(mg/day)
Total
(mg/day)
Intake (mg/kg/day)
Absorbed Dose
(mg/kg/day) 1
1 to 3
8 à 16
0.39–0.56
0.027–0.265
0.417–0.825
0.026–0.103 (0.065)
0.036–0.058 (0.047)
4 to 6
13 à 24
0.52–0.745
0.036–0.360
0.556–1.105
0.023–0.085 (0.054)
0.030–0.049 (0.040)
7 to 9
16 à 35
0.65–0.93
0.045–0.450
0.695–1.380
0.020–0.086 (0.053)
0.029–0.048 (0.039)
10 to 12
25 à 54
0.81–1.66
0.056–0.560
0.866–1.725
0.016–0.069 (0.043)
0.023–0.037 (0.031)
1
Absorbed optimal doses were calculated by multiplying mean water and diet intake by their respective
bioavailability factors of 83% and 40% and dividing that total absorbed dose by the mean weight for that age group.
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article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Point prevalence of SARS-CoV-2 infection in Sweden at six time points during 2020
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Background To estimate the prevalence and understand the dissemination of SARS-CoV-2 in Sweden, the Public Health Agency of Sweden,
with support from the Swedish Armed Forces, conducted a series of point prevalence surveys between March and December
2020. Findings The first survey in the Stockholm region in March, including 707 participants, showed a SARS-CoV-2 prevalence of 2.5%. The
following five surveys, performed on a national level, with between 2,461 and 2,983 participants, showed SARS-CoV-2
prevalences of 0.9% (April), 0.3% (May), 0.0% (August), 0.0% (September), and 0.7% (December). All positive cases responding to the questionnaires regarding experienced symptoms had experienced symptoms two weeks
before sampling up until the day of sampling. Interpretation None of the individuals shown to be PCR-positive were asymptomatic at the time of sampling, including 14 days prior to
sampling. This is in contrast to many other surveys, where a substantial proportion of positive cases are reported to be
asymptomatic. In line with an increasing testing capacity and consecutive inclusion of all symptomatic individuals in the case definition for
testing, our surveys demonstrate a decreasing ratio between notified cases and shown prevalence throughout the year. Point prevalence of SARS-CoV-2 infection in Sweden at six
time points during 2020 time points during
Ramona Groenheit
(
ramona.groe
Public Health Agency of Sweden
Jessica Beser
Public Health Agency of Sweden
Sharon Kühlmann Berenzon
Public Health Agency of Sweden
Ilias Galanis
Public Health Agency of Sweden
Edward Straten
Public Health Agency of Sweden
Jan Duracz
Public Health Agency of Sweden
Marie Rapp
Public Health Agency of Sweden
Disa Hansson
Public Health Agency of Sweden
Mikael Mansjö
Public Health Agency of Sweden
Sandra Söderholm
Public Health Agency of Sweden
Shaman Muradrasoli
Public Health Agency of Sweden
Anna Risberg
Public Health Agency of Sweden
Richard Ölund
Swedish Armed Forces
Andreas Wiklund
Swedish Armed Forces
Kristoffer Metzkes
Swedish Armed Forces
Matilda Lundberg
Swedish Armed Forces
Philip Bacchus
Swedish Armed Forces
Karin Tegmark Wisell
Public Health Agency of Sweden
Andreas Bråve
Public Health Agency of Sweden Edward Straten
Public Health Agency of Sweden Edward Straten
Public Health Agency of Sweden Jan Duracz
Public Health Agency of Sweden Jan Duracz
Public Health Agency of Sweden Marie Rapp
Public Health Agency of Sweden Marie Rapp
Public Health Agency of Sweden Disa Hansson
Public Health Agency of Sweden Disa Hansson
Public Health Agency of Sweden Mikael Mansjö
Public Health Agency of Sweden Sandra Söderholm
Public Health Agency of Sweden Sandra Söderholm
Public Health Agency of Sweden Shaman Muradrasoli
Public Health Agency of Sweden Anna Risberg
Public Health Agency of Sweden Anna Risberg
Public Health Agency of Sweden Research Article Page 1/14 Page 1/14 Keywords: SARS-CoV-2, COVID-19, point prevalence, large-scale prevalence surveys, symptoms
Posted Date: April 12th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-1365777/v1 License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License nse:
This work is licensed under a Creative Commons Attribution 4.0 International
Lice censed under a Creative Commons Attribution 4.0 International
License. Read Full License Page 2/14 Methods Sampling material and instructions on how to perform self-sampling of the upper respiratory tract were delivered to the homes
of the participants. Samples were analysed by real-time PCR, and the participants completed questionnaires regarding
symptoms. Background From July 11, 2020 and onwards, all regions had gained sufficient testing capacity to include also
the general public in the testing. An overview of the testing strategy is available at ECDC COVID-19 country overviews (4). Due to initial limited testing capacity and the subsequent sharp increase in capacity throughout the spring and summer, the
ratio of detected and notified cases versus the true number of cases was assumed to increase throughout the year. In order to
estimate the true number of cases for mathematical transmission models of the spread of SARS-CoV-2 in the population, which
were important tools for evaluating and planning control measures of the spread of SARS-CoV-2 and predicting future
scenarios, point prevalence and seroprevalence estimates are needed to more accurately estimate the population prevalence at
different time points and to calibrate the number of infected individuals in the mathematical models. Self-sampling of the upper respiratory tract at home or at drive-in test centres is an efficient alternative to sampling by trained
health care professionals in health care facilities. With large-scale global demand and shortage of personal protective
equipment (PPE) during the early phase of the pandemic the concept of self-sampling was developed to allow for large-scale
testing in the society of individuals not in need of medical care. Additionally, this sampling methodology reduces the risk of
potentially infected individuals leaving their homes and exposing others to the disease. Moreover, self-sampling at home
requires less effort for individuals to participate in surveys, which may increase the level of participation. With these
considerations in mind, the Public Health Agency of Sweden together with the Swedish Armed Forces performed a pilot study
evaluating the self-sampling methodology (5) and furthermore developed concepts for the distribution of material for self-
sampling and the collection of samples, enabling nationwide surveys. Our main aim was to estimate the prevalence of COVID-19 in the population in Sweden and the Stockholm Region at different
time points in order to provide estimates of the true number of cases. Results that could be used in mathematical models of the
spread of SARS-CoV-2 in the population. A secondary aim was to estimate the proportion of asymptomatic cases in the
population and describe the most common symptoms among those positive for SARS-CoV-2. Survey design and participants Six population-based cross-sectional surveys (surveys 1–6) were conducted in Sweden between March and December 2020. Survey 1 was carried out in the Stockholm Region from 26 March–3 April. Surveys 2 to 6 were conducted at a national level
during the following time periods: 21–24 April, 25–28 May, 24–28 August, 21–25 September, and 30 November–4 December. Six population-based cross-sectional surveys (surveys 1–6) were conducted in Sweden between March and December 2020. Survey 1 was carried out in the Stockholm Region from 26 March–3 April. Surveys 2 to 6 were conducted at a national level Six population-based cross-sectional surveys (surveys 1–6) were conducted in Sweden between March and December 2020. Six population-based cross-sectional surveys (surveys 1–6) were conducted in Sweden be Survey 1 was carried out in the Stockholm Region from 26 March–3 April. Surveys 2 to 6 were conducted at a national level
during the following time periods: 21–24 April, 25–28 May, 24–28 August, 21–25 September, and 30 November–4 December. Participants in surveys 1 to 5 were recruited from a pre-existing web panel regularly used for health-related questionnaires at the
Public Health Agency of Sweden. The web panel was built in 2015 from a simple random sample of 35,000 individuals from
the general Swedish population; in 2016 (n=15,000) and in 2018 (n=25,000), top-up samples were drawn by age, sex and
education (6). At the time of the first survey, the web panel included 4,500 individuals. Survey 6 was designed as an age-stratified random sample from the general population 16 years and older. The calculated
sample sizes for the age groups 16–29 years, 30–59 years, and 60 years and older were 2,486, 1,257, and 1,700, respectively,
assuming point prevalence of 2.0%, 1.0%, and 0.4% and a precision of 0.6%, 0.6%, and 0.3%, respectively, at the 5% significance
level (7). From previous experience, we assumed response rates of 30%, 40%, and 40%, yielding a total of 15,701 individuals to
be invited. Statistics Sweden sent invitations to individuals in survey 6 and provided population figures for 2020 (8). Background The first confirmed case of coronavirus disease 2019 (COVID-19) in Sweden was reported on 31 January 2020 (1). Since then,
multiple independent introductions have resulted in the disease spreading throughout the country, with 457,379 reported cases
(4,429 cases per 100,000 inhabitants) and 9,818 deaths by the end of 2020. In accordance with a request from the Public Health Agency of Sweden, COVID-19 was by the Swedish Government 2 February
2020, included in the Communicable Diseases Ordinance as a disease dangerous to public health and to society (2). This in
turn tasks all individuals and all physicians with an obligation to investigate all suspected cases of COVID-19, including
laboratory sampling when necessary to determine the diagnose. The nationally recommended prioritizing indications for testing
of COVID-19 were throughout 2020 revised several times according to the increasing testing capacity (3). Up until March 13,
2020 all individuals with a travel history to any countries with community transmission according to WHO and any of the
symptoms cough, elevated body temperature or dyspnoea were sampled. March 13, 2020 community transmission was
declared in Sweden. The need for testing for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) then exceeded the
national capacity why prioritizing of testing to the health care and elderly care sector was recommended at a national level. Simultaneously, all individuals having any kind of symptoms of respiratory disease or other symptoms consistent with COVID-
19 were recommended to stay at home and avoid contact with other individuals. Consequently, individuals in the community
without medical indication and not belonging to any of the prioritized groups were in general not prioritized for testing. As
testing capacity was building up additional groups in society could be included in the prioritized groups for testing according to
a national testing strategy (3). On June 11, 2020 the Swedish government and The Swedish Association of Local Authorities Page 3/14 Page 3/14 and Regions agreed to the Regions committing to have a testing capacity available that would provide testing to all individuals
with symptoms of COVID-19. From July 11, 2020 and onwards, all regions had gained sufficient testing capacity to include also
the general public in the testing. An overview of the testing strategy is available at ECDC COVID-19 country overviews (4). and Regions agreed to the Regions committing to have a testing capacity available that would provide testing to all individuals
with symptoms of COVID-19. Procedures Invitations to potential participants were sent three to five weeks before the start of each survey and included instructions on
how to perform self-sampling at home, information on how the sample would be collected and when to fill in the questionnaire. Participants were provided with access to a 13-hour/day telephone helpline and signed up for the survey by filling out an online
form and providing consent to the survey; for those under 16 years of age, the legal guardian signed up. Participation was
voluntary and could be withdrawn at any time. In survey 1, material for self-sampling was delivered to the participant’s home by the Swedish Armed Forces, while material for
the other surveys was sent by regular post. For all surveys, the Swedish Armed Forces coordinated and collected the samples at
the homes of the participants. The samples were collected the same day as the self-sampling or the day after. The participants
were asked to store the samples in a refrigerator until collection. For surveys 1 to 5, self-sampling material consisted of sterile cotton swabs and one test tube containing 1 ml phosphate-
buffered saline. Participants were asked to use one cotton swab to perform a throat swab by scraping the posterior pharyngeal
wall for 10–20 seconds and then to swirl the cotton swab in the buffer. A second cotton swab was used to sample the distal
nasal cavity through both nostrils, followed by swirling the cotton swab in the buffer. In surveys 1 and 2, saliva was separately
collected in a collection tube wherein the participants had spat 3–4 times, while the participants in the following surveys had
swirled a third cotton swab in their saliva before the swab was swirled in the buffer. For survey 6, participants received one swab and a test tube containing 0.5 ml sample storage reagent consisting of 0.9%
normal saline. A swab was used to sample the posterior pharyngeal wall, the distal nasal cavity, and saliva. In all surveys, participants younger than 16 years, had their sampling performed by a caregiver. The samples were analysed using real-time RT–PCR assays routinely utilized to diagnose COVID-19. Altogether, three different
laboratories performed analyses using different targets for real-time RT–PCR for SARS-CoV-2 (9-11). The presence of hBeta-
actin or hRNAse P was analysed to confirm that a sample was taken correctly in terms of swabbing against mucosal surfaces
in the nose or throat. Procedures Samples lacking human cell material were excluded from analysis. Data on the age, sex, and region of the participant were gathered when the sample was collected. In survey 6, the educational
level of the participants was retrieved from administrative registers. On the day of self-sampling, the participants answered an
online questionnaire about specific symptoms experienced in the past 24 hours and two weeks prior to self-sampling. An
additional symptom questionnaire was distributed seven days after self-sampling to those who tested positive for SARS-CoV-2. The number of reported COVID-19 cases and number of individuals tested for COVID-19 were retrieved from the Public Health
Agency of Sweden. Ethics approval and consent to participate The evaluation was performed as part of the Public Health Agency of Sweden’s assignment to monitor communicable diseases
and evaluate infection control measures, in accordance with §§ 18 of the ordinance (2021:248) from the Swedish Parliament. For this reason, specific ethical clearance for the surveys described in this manuscript is not required. All methods were carried
out in accordance with relevant guidelines and informed consent was obtained by all participants and/or their legal guardians. Page 4/14 Page 4/14 Statistical analysis The point prevalence was calculated as the weighted proportion of individuals testing positive for SARS-CoV-2 among those
with valid test results. In surveys 1 to 5, weighting included the sample weights from the web panel cohort, nonresponse in the
survey, and population size by age, sex, and region. In survey 6, the weighting was based on the age, sex, region, and
educational level of the participants to consider the sampling error, nonresponse, and population size. Confidence intervals (CIs)
were calculated using the Clopper-Pearson exact method. Estimates were reported by sex and age group (0–15 years, 16–29
years, 30–59 years, 60 years and older) for Sweden and for the Stockholm Region. We calculated the proportion of each
symptom among those who tested positive for SARS-CoV-2; the proportion and its 95% CI were not weighted. Differences in
prevalence between sexes were tested with univariate weighted logistic regression. Analyses were carried out in R v.3.6.2 and
survey package v.4.0. Point prevalence Overall, 74 of 14,197 samples tested positive for SARS-CoV-2 (Table 2). The weekly prevalence at the national level was highest
in April, with 0.9% (95% CI 0.6–1.5) thereafter decreasing in May. In August and September, no samples positive for SARS-CoV-2
were found, while the weekly prevalence increased to 0.7% (95% CI 0.4–1.2) in December. The same pattern was seen
in the Stockholm region, although the weekly prevalence in April was almost three times higher than the prevalence at the
national level during the same week (Table 2). Figure 1 shows the weighted population prevalence estimates as well as the number of newly reported cases and tested
individuals per week during 2020. The Public Health Agency of Sweden utilized a number of sources in order to determine the
burden of disease, this was of particular importance during the initial phase of the outbreak as the testing capacity was not
sufficient to meet the demands. If looking only at the notified number of cases, the first wave took place between the middle of
March and the end of June. The number of notified cases peaked around middle of June and coincided with an increase in
testing. The second wave, with a much higher reported incidence as many more cases were confirmed as compared to during
the initial wave, started around the middle of September and peaked at the end of the year; the number of tested individuals
increased weekly during that period. While point weekly prevalence estimates in April and December were similar, the reported
weekly incidence and number of tested individuals at the end of the year were approximately ten times higher than those in
April. At the national level, the highest weekly prevalence among 16–29-year-olds and 30–59-year-olds was seen in April, while
among those 60 years and older, it was observed in December (Figure 2, Supplementary Table 2). All age groups had the
highest weekly prevalence in the March survey conducted in the Stockholm Region, with results varying slightly between age
groups. We could not find any significant differences (p > 0.05) between sexes in terms of the prevalence of SARS-CoV-2 in any of the
surveys (Table 3). Description of participants In survey 1 in the Stockholm Region, 738 individuals participated, while in the following five national surveys, between 2,471
and 3,038 individuals participated each time (Supplementary Table 1). Participation rates for the first five surveys from the
web panel were between 55–67% and 19% for survey 6 from the general population. In general, individuals between 16 and 29
years old tended to participate to a lower degree than other age groups, and males to a lower degree than females (Table 1). The distribution of participants by region followed a similar pattern as the general population. In total, 14,329 samples were collected from 6,608 individuals. Among the individuals participating, 3,545 participated in one
survey, 605 in two, 739 in three, 1,374 in four, 344 in five and one person participated in all six surveys. We excluded 132
samples that had invalid test results; thus, 14,197 were included in the estimations (Table 2). In addition to the lack of hBeta-
actin or hRNAse P, invalid test results were also due to the participant failing to properly close the test tube, resulting in leakage,
or that parts from the swab remained in the test tube. Results Page 5/14 Page 5/14 Discussion We conducted six cross-sectional surveys in the Swedish population to estimate the weekly prevalence of SARS-CoV-2 infection
between April and December 2020. The point prevalence estimates varied between 0.0% and 0.9%, with high levels during the
first wave in the spring and again in the second wave in the autumn. The Stockholm region generally had higher point
estimates than the country as a whole. While the estimated weekly prevalence was similar in early spring (April) and at the end of the year (December), the reported
incidence according to notified cases was several times higher in the latter month compared to the former. These observed
differences between the estimated weekly prevalence in our surveys and the weekly reported incidence are in line with the
increasing testing capacity throughout the year. The proportion of unreported cases can thus be assumed to have decreased
during the year, with a higher proportion of unreported cases during the first half of 2020. The proportion of infections that are asymptomatic has not yet been fully elucidated. A meta-analysis of 13 studies with follow-
up of symptoms found the proportion of asymptomatic cases to be 17% (12), but various figures ranging from 4% to 100%
have been reported (12-20). Studies aimed at establishing SARS-CoV-2 transmission potential from the proportion of
asymptomatic infection, generally face two challenges. Firstly, studies relying solely on reported symptoms at one specific time
point do not allow for distinguishing between asymptomatic, pre- or post-symptomatic SARS-CoV-2 infection (19). Secondly, the
requisite for asymptomatic categorization varies greatly, where some studies include all symptoms whatsoever, and others
fewer than two specified symptoms (20). Combined, these challenges illustrate the uncertainty that exists in many studies. In
contrast to the surveys conducted in England (21), where a substantial proportion (45–68%) of the infected individuals reported
no symptoms around their sampling date, our surveys show that all positive cases that answered the symptom questionnaire
had experienced symptoms within two weeks before sampling. Our timeframe for identifying symptoms, i.e. within two weeks
prior to sampling and one week follow-up for those testing positive, could explain the difference for other studies using either
no follow-up period and/or a period prior to the testing (12). Our implicit definition of asymptomatic cases, no symptoms
whatsoever, in combination with our timeframe significantly narrows the set for identifying asymptomatic cases. Positive cases and reported symptoms Of the 74 individuals positive for SARS-CoV-2, 44 participated in at least one sampling after having tested positive; none were
positive more than once. In total, 72 of the 74 positive individuals answered the first questionnaire about symptoms in the past
24 hours and past two weeks (Supplementary Table 3). In the past two weeks prior to self-sampling, 70 of the 74 positive
individuals reported having had symptoms, while 69 of the 74 did so when asked about the 24 hours before self-sampling. All
74 individuals positive for the virus answered the follow-up questionnaire one week after self-sampling, and 68 reported having
experienced symptoms within those seven days. All individuals positive for SARS-CoV-2, responding to the questionnaires,
reported at least one symptom during at least one of the recall periods. Page 6/14 Page 6/14 The most common symptoms two weeks before self-sampling were runny nose, cough, headache, extreme fatigue, and loss of
taste, while 24 hours before self-sampling, the most common symptoms were headache, runny nose, extreme fatigue, cough
and fever, and one week later, headache, extreme fatigue, runny nose, cough, and loss of smell (Table 4). Conclusion None of the survey participants positive for SARS-CoV-2 were asymptomatic, an important finding that at large can be
attributed to how and when information on symptoms was collected. The estimated prevalence of SARS-CoV-2 in the population during 2020 show that the fraction of unreported cases decreased
during the second wave compared to the first wave. While the estimated weekly prevalence was similar in early spring (April)
and at the end of the year (December), the reported incidence according to notified cases was approximately ten times higher at
the end of the year. This is in line with an increasing testing capacity and consecutive broadened indication for sampling
throughout the year. The results were instrumental to optimize and find values to parameters in the mathematical models
developed to understand the spread of COVID-19 in Sweden and the Stockholm region (25, 26). Consent for publication Not applicable. Discussion symptoms before an individual knows the test result, as the knowledge of a positive test can lead to over-interpretation of
symptoms (22). Asking for symptoms up to two weeks (23) before sampling, not only on the day of sampling, also reduces the
risk of misclassifying post asymptomatic cases into asymptomatic cases. Issues with recall bias, however, need to be
considered when interpreting the results. For the first five surveys, participants were invited from a pre-existing web panel (6). This web panel was readily available, and
individuals could swiftly be invited to our surveys. Web panel participation rates in the prevalence surveys were just above 50%,
which is lower compared to studies conducted with the same panel during 2020 (80–90%) (24). A weakness of survey 6 was
the low participation rate of 19%. A requirement of the prevalence surveys was for participants to self-sample using swabs, as
well as to be available at their home address during a specific day and time, albeit chosen by them, for sample collection. This
additional effort may have discouraged individuals from participating. Although we applied survey weights to account for
nonresponse, bias due to other unknown factors may remain. Additionally, those who previously had been positive for SARS-
CoV-2 may have been less willing to participate, even though the invitation encouraged individuals to participate regardless of
previous infection or the presence of antibodies. Ethics approval and consent to participate The evaluation was performed as part of the Public Health Agency of Sweden’s assignment to monitor communicable diseases
and evaluate infection control measures, in accordance with §§ 18 of the ordinance (2021:248) from the Swedish Parliament. For this reason, specific ethical clearance for the surveys described in this manuscript is not required. All methods were carried
out in accordance with relevant guidelines and informed consent was obtained by all participants and/or their legal guardians. Availability of data and materials The datasets generated and analysed during the surveys are not publicly available due to existing general data protection rules
and the official secrecy but are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Discussion This
discussion accentuates the risk of categorizing infected cases as asymptomatic, instead of pre-symptomatic, which in turn can
lead to wrongly formulated counter-pandemic strategies. The low number of individuals positive for SARS-CoV-2 in our surveys,
hampered our ability to conduct several planned comparisons with respect to reported symptoms, including changes over time,
differences by age and sex, and negative vs. positive test results. The surveys were conducted using self-sampling at home, a concept developed and evaluated by the Public Health Agency of
Sweden with support from the Swedish Armed Forces (5). The Public Health Agency of Sweden was granted support from the
Swedish Armed Forces in accordance with the regulation regarding Swedish Armed Forces support to civilian authorities. Overall, participants seemed positive to the use of this approach, as it allowed them to participate without leaving their home. This was an important aspect since the national recommendations in Sweden included that symptomatic individuals should
avoid leaving their homes and, of importance for the results, we did not want to exclude any individuals with symptoms. Sampling at home prevented potentially presymptomatic individuals from exposing other individuals to the virus. In contrast to
surveys performed in England (21), where study workers monitored self-swabbing in the homes of participants, our participants
performed the swabbing on their own, following instructions included in the sampling kit. The few samples that had to be
excluded due to lack of hBeta-actin or hRNAse P indicate that both adults and caregivers to children were successful in
performing the sampling. In the surveys, we used a combination of samples from the throat, nose, and saliva, since this showed
the highest sensitivity in our pilot study (5). A strength of our surveys is that we collected information on symptoms experienced within two weeks and 24 hours before
sampling and, importantly, before the participants knew if they were positive for SARS-CoV-2. It is favourable to ask about Page 7/14 Page 7/14 symptoms before an individual knows the test result, as the knowledge of a positive test can lead to over-interpretation of
symptoms (22). Asking for symptoms up to two weeks (23) before sampling, not only on the day of sampling, also reduces the
risk of misclassifying post asymptomatic cases into asymptomatic cases. Issues with recall bias, however, need to be
considered when interpreting the results. Authors' contributions RG, KTW and AB designed and planned the study. EvS, JD, MR, MM, SS, SM and AR contributed to the laboratory analysis. IG,
SKB, and DH contributed to the statistical analysis. RÖ, AW, KM, ML and PB developed concepts for the distribution of sampling
material and sample collection. RG, JB and SKB drafted the report. All authors contributed to the data interpretation and report
revisions. All authors approved the final version of the report and agree to be accountable for all aspects of the work. Acknowledgements This work was funded by governmental grants provided to the Public Health Agency of Sweden for assignments
S2020/0281/FS and S2020/08532 FS. We would like to thank all the participants in the prevalence surveys for volunteering to
perform self-sampling and completing symptom questionnaires. We thank the Swedish Armed Forces, whose personnel from
regular units, national guard units, and conscripts collected all the samples. We thank members from the Swedish voluntary
defence organizations for assistance with packing the material for self-sampling that was sent to participants in the prevalence
surveys. We also thank Leah Martin and Nina Lagerqvist at the Public Health Agency of Sweden for careful review of earlier
versions of the manuscript. Funding Page 8/14
This work was funded by governmental grants provided to the Public Health Agency of Sweden for
assignments S2020/02681/FS and S2020/08532 FS. The funders of this work had no role in study design, data collection, Page 8/14
This work was funded by governmental grants provided to the Public Health Agency of Sweden for
assignments S2020/02681/FS and S2020/08532 FS. The funders of this work had no role in study design, data collection, Page 8/14 Page 8/14 data analysis, data interpretation, writing of the report or in the decision to submit the paper for publication. References 1. Public Health Agency of Sweden. Bekräftat fall i Jönköping av nya coronaviruset (2019-nCoV) [Available from:
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in a non-hospitalized population: results from the large Italian web-based EPICOVID19 cross-sectional survey. JMIR Public
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outbreak in the Italian municipality of Vo'. Nature. 2020;584(7821):425–9. 16. Menachemi N YC, Dixon BE et al. Population Point Prevalence of SARS-CoV-2 Infection Based on a Statewide Random
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coronavirus infections (COVID-19). Int J Infect Dis. 2020;94:154–5. 19. Oran DP, Topol EJ. The Proportion of SARS-CoV-2 Infections That Are Asymptomatic. Annals of Internal Medicine. 2021;174:655–62. 20. Yanes-Lane M, Winters N, Fregonese F, Bastos M, Perlman-Arrow S, Campbell JR, et al. Proportion of asymptomatic
infection among COVID-19 positive persons and their transmission potential: A systematic review and meta-analysis. PLoS
One. 2020;15(11):e0241536. 21. Pouwels KB, House T, Pritchard E, Robotham JV, Birrell PJ, Gelman A, et al. Community prevalence of SARS-CoV-2 in
England from April to November, 2020: results from the ONS Coronavirus Infection Survey. Lancet Public Health. 2021;6(1):e30-e8. 22. Robertson CG, Kesselheim A. Blinding as a Solution to Bias: Strengthening Biomedical Science, Forensic Science, and Law. 1st edition.: Academic Press; 30th January 2016. 22. Robertson CG, Kesselheim A. Blinding as a Solution to Bias: Strengthening Biomedical Science, Forensic Science, and Law. 1st edition.: Academic Press; 30th January 2016. 23. Broeck JVd, Brestoff JR. Epidemiology Principles and Pracical Guidelines: Springer Dordrecht Heidelberg New York London
2013. 24. Public Health Agency of Sweden. Hälsorapport [2021-06-14]. Available from: https://www.halsorapport.se/sv/. 25. Public Health Agency of Sweden. Skattning av peakdag och antal infekterade i covid-19-utbrottet i Stockholms län februari-
april 2020 [Available from: https://www.folkhalsomyndigheten.se/publicerat-material/publikationsarkiv/s/skattning-av-
peakdag-och-antal-infekterade-i-covid-19-utbrottet-i-stockholms-lan-februari-april-2020/. 26. Public Health Agency of Sweden. Scenarier för fortsatt spridning - Interrimrapport inom regeringsuppdraget att löpande
uppdatera scenarier för hur smittspridningen av det virus som orsakar sjukdomen covid-19 kan komma att utvecklas
framöver: Public Health Agency of Sweden; 2021 [Available from:
https://www.folkhalsomyndigheten.se/contentassets/fa397252cd614d1d91663d7a844426b6/scenarier-fortsatt-
spridning-21018.pdf. 26. Public Health Agency of Sweden. Scenarier för fortsatt spridning - Interrimrapport inom regeringsuppdraget att löpande
uppdatera scenarier för hur smittspridningen av det virus som orsakar sjukdomen covid-19 kan komma att utvecklas
framöver: Public Health Agency of Sweden; 2021 [Available from:
https://www.folkhalsomyndigheten.se/contentassets/fa397252cd614d1d91663d7a844426b6/scenarier-fortsatt-
spridning-21018.pdf. Table 2. Number of samples with valid test results, number of samples positive for SARS-CoV-2, and weighed population
prevalence with 95% confidence intervals at the national level and for the Stockholm region in Sweden in 2020. Tables Distribution of participants by age group, sex and region by survey in Sweden in 2020. Page 10/14 Page 10/14 Survey
26
March–
3 April
21–24
April
25–28
May
24–28
August
21–25
September
30
November–
4
December
Swedish
population
(n=738)
(n=2,586)
(n=2,969)
(n=2,527)
(n=2,471)
(n=3,038)
(n=10,379,295)
Age
group
0–15 years
20.8%
18.9%
17.2%
15.1%
15.1%
-
18.8%
16–29 years
7.2%
6.9%
8.2%
6.9%
6.9%
37.8%
16.9%
30–59 years
42.3%
39.5%
38.0%
38.0%
38.0%
26.6%
38.6%
60+ years
29.7%
34.7%
36.6%
40.0%
40.0%
35.6%
25.6%
Sex
Females
51.6%
54.5%
53.9%
53.3%
53.4%
55.5%
49.7%
Males
48.4%
45.5%
46.1%
46.7%
46.6%
44.5%
50.3%
Region
Stockholm
100%
26.4%
25.7%
24.7%
25.7%
26.5%
23.0%
Västra
Götaland
-
16.1%
16.2%
16.1%
16.0%
17.9%
16.7%
Skåne
-
13.7%
13.2%
13.7%
13.8%
11.7%
13.4%
Östergötland
-
4.6%
4.4%
4.0%
4.2%
5.5%
4.5%
Uppsala
-
4.3%
4.2%
3.9%
4.1%
4.3%
3.7%
Jönköping
-
3.3%
3.1%
3.6%
3.4%
3.1%
3.5%
Halland
-
3.3%
3.7%
3.6%
3.4%
2.8%
3.2%
Södermanland
-
2.8%
2.9%
3.0%
3.0%
3.1%
2.9%
Örebro
-
2.6%
2.6%
2.5%
2.4%
3.2%
2.9%
Dalarna
-
2.6%
2.6%
2.9%
2..9%
2.6%
2.8%
Gävleborg
-
2.6%
2.5%
2.8%
2.6%
2.3%
2.8%
Västmanland
-
2.4%
2.6%
2.9%
2.8%
2.1%
2.7%
Värmland
-
1.3%
1.9%
1.9%
1.7%
2.3%
2.7%
Västerbotten
-
3.0%
2.8%
3.1%
2.9%
2.6%
2.6%
Västernorrland
-
2.1%
2.3%
2.2%
2.1%
1.7%
2.4%
Kalmar
-
1.9%
1.8%
1.7%
1.7%
1.7%
2.4%
Norrbotten
-
1.8%
2.0%
2.0%
1.9%
2.3%
2.4%
Kronoberg
-
2.4%
2.3%
2.4%
2.4%
1.7%
1.9%
Blekinge
-
1.2%
1.2%
1.3%
1.2%
0.9%
1.5%
Jämtland
-
1.0%
1.1%
1.1%
1.0%
1.0%
1.3%
Gotland
-
0.7%
0.8%
0.8%
0.9%
0.7%
0.6%
Table 2. Number of samples with valid test results, number of samples positive for SARS-CoV-2, and weighed population
prevalence with 95% confidence intervals at the national level and for the Stockholm region in Sweden in 2020. Table 4. Proportion of participants positive for SARS-CoV-2 (N=74) with a 95% confidence interval (95% CI) in six cross-
sectional population surveys by reported symptom and recall period in Sweden in 2020. Tables Page 11/14 Page 11/14 Sweden
Stockholm Region
Survey
Dates of
survey
Samples with
valid test
results
Positive
samples
Weighted
population
prevalence (95%
CI)
Samples with
valid test
results
Positive
samples
Weighted
population
prevalence (95%
CI)
(n=13,490)
(n=56)
(n=4,192)
(n=45)
1
26 March–
3 April
707
18
2.5% (1.4–4.1)
2
21–24 April
2,571
23
0.9% (0.6–1.5)
679
12
2.3% (1.1–4.2)
3
25–28 May
2,957
9
0.3% (0.1–0.5)
761
5
0.7% (0.2–1.6)
4
24–28
August
2,518
0
0.0% (0.0–0.2)
623
0
0.0% (0.0–0.6)
5
21–25
September
2,461
0
0.0% (0.0–0.2)
632
0
0.0% (0.0–0.6)
6
30
November–
4 December
2,983
24
0.7% (0.4–1.2)
790
10
1.0% (0.4–2.1)
T bl 3 W i h
d
l
i
l
i h 95%
fid
i
l b
d
i
S
d
i
2020 Stockholm Region n prevalence with 95% confidence interval by sex and survey in Sweden in 2020. Table 3. Weighted population prevalence with 95% confidence interval by sex and survey in Sweden in 2020. Table 3. Weighted population prevalence with 95% confidence interval by sex and surve Table 3. Weighted population prevalence with 95% confidence interval by sex and survey in Sweden in 2020. Survey
Dates of survey
Weighted population prevalence (95% CI)
Female
Male
P value
1*
26 March–3 April
3.7% (0.8–7.0)
1.4% (0.1–12.7)
0.07
2
21–24 April
0.7% (0.1–1.7)
1.2% (0.6–2.2)
0.21
3
25–28 May
0.3% (0.1–0.7)
0.2% (0.1–0.6)
0.70
4
24–28 August
0.0% (0.0–0.3)
0.0% (0.0–0.3)
-
5
21–25 September
0.0% (0.0–0.3)
0.0% (0.0–0.3)
-
6
30 November–4 December
1.0% (0.5–1.7)
0.5% (0.2–1.3)
0.26 *Stockholm region Table 4. Proportion of participants positive for SARS-CoV-2 (N=74) with a 95% confidence interval (95% CI) in six cross-
sectional population surveys by reported symptom and recall period in Sweden in 2020. Tables Page 12/14 Page 12/14 Recall period
2 weeks before self-
sampling
24 hours before self-
sampling
1 week after self-
sampling
(n=72)
(n=72)
(n=74)
Symptom
%
(95% CI)
%
(95% CI)
%
(95% CI)
Chills
13.9
(6.9–24.1)
43.1
(31.4–55.3)
18.1
(10.0–28.9)
Cough
50.0
(38.0–62.0)
62.5
(50.3–73.6)
45.8
(34.2–58.0)
Diarrhoea
18.1
(10.0–28.9)
25.0
(15.5–36.6)
16.7
(8.2–27.0)
Ear pain
6.9
(2.3–15.5)
12.5
(5.9–22.4)
8.3
(3.1–17.3)
Extreme fatigue, exhaustion
41.7
(30.2–53.9)
63.9
(51.7–74..9)
52.8
(40.7–64.7)
Eye discharge
15.3
(7.9–25.7)
16.7
(8.9–27.3)
16.7
(8.9–27.0)
Fever
19.4
(11.1–30.5)
54.2
(42.0–66.0)
20.8
(12.2–32.0)
Headache
48.6
(36.7–60.7)
77.8
(66.4–86.7)
52.8
(40.7–64.7)
Joint pain
20.8
(12.2–32.0)
37.5
(26.4–49.7)
25.0
(15.5–36.6)
Loss of smell
27.8
(17.9–39.6)
37.5
(26.4–49.7)
45.8
(34.2–58.0)
Loss of taste
34.7
(23.9–46.9)
37.5
(26.4–49.7)
43.1
(31.4–55.3)
Myalgia
25.0
(15.5–36.6)
40.3
(28.9–52.5)
30.6
(20.2–42.5)
Nausea
18.1
(10.0–28.9)
31.9
(21.4–44.0)
18.1
(10.0–28.9)
Nosebleeds
5.6
(1.5–13.6)
13.9
(6.9–24.1)
8.3
(3.2–17.3)
Runny nose
58.3
(46.1–69.9)
65.3
(53.1–76.1)
50.0
(38.0–62.0)
Shortness of breath/difficulty breathing
11.1
(4.9–20.7)
20.8
(12.2–32.0)
31.9
(21.4–44.0)
Skin rashes such as hives, dots, pustules
or blisters
6.9
(2.3–15.5)
6.9
(2.39–15.5)
5.6
(1.5–13.6)
Sore throat
23.6
(14.4–35.1)
48.6
(36.7–60.7)
31.9
(21.4–44.0)
Stomach ache
18.1
(10.0–28.9)
31.9
(21.4–44.0)
19.4
(11.1–30.5)
Vomiting
2.8
(0.3–9.7)
4.2
(0.9–11.7)
4.2
(0.9–11.7)
No symptoms
4.2
(0.9–11.7)
2.8
(0.3–9.7)
5.6
(1.5–13.6) Recall period Figure 2 Weighted population weekly prevalence with 95% confidence interval by age group in Sweden in 2020. Weighted population weekly prevalence with 95% confidence interval by age group in Sweden in 2020. Figure 1 A. Estimated weekly prevalence of SARS-CoV-2 infection with 95% CI from the five national population surveys in Sweden in
2020. . Number of notified cases of COVID-19 and number of tested individuals per week in Swede Page 13/14 Figure 2
Weighted population weekly prevalence with 95% confidence interval by age group in Sweden in 2020. Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. SupplementaryTable1.docx SupplementaryTable2.docx SupplementaryTable3.docx Page 14/14 Page 14/14 Page 14/14
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Who should operate patients presenting with emergent colon cancer? A comparison of short- and long-term outcome depending on surgical sub-specialization
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World journal of emergency surgery
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Abstract Background Colorectal cancer presents as emergencies in 20% of the cases. Emergency resection is associated
with high postoperative morbidity and mortality. The specialization of the operating team in the emergency settings
differs from the elective setting, which may have an impact on outcome. The aim of this study was to evaluate short-
and long-term outcomes following emergent colon cancer surgery depending on sub-specialization of the operating
team. Methods This is a retrospective population study based on data from the Swedish Colorectal Cancer Registry
(SCRCR). In total, 656 patients undergoing emergent surgery for colon cancer between 2011 and 2016 were included. The cohort was divided in groups according to specialization of the operating team: (1) colorectal team (CRT); (2)
emergency surgical team (EST); (3) general surgical team (GST). The impact of specialization on short- and long-term
outcomes was analyzed. Results No statistically significant difference in 5-year overall survival (CRT 48.3%; EST 45.7%; GST 42.5%; p = 0.60)
or 3-year recurrence-free survival (CRT 80.7%; EST 84.1%; GST 77.7%21.1%; p = 0.44) was noted between the groups. Neither was any significant difference in 30-day mortality (4.4%; 8.1%; 5.5%, p = 0.20), 90-day mortality (8.8; 11.9; 7.9%,
p = 0.37) or postoperative complication rate (35.5%, 35.9 30.7, p = 0.52) noted between the groups. Multivariate analy-
sis adjusted for case-mix showed no difference in hazard ratios for long-term survival or postoperative complications. The rate of permanent stoma after 3 years was higher in the EST group compared to the CRT and GST groups (34.5%
vs. 24.3% and 23.9%, respectively; p < 0.0.5). Conclusion Surgical sub-specialization did not significantly affect postoperative complication rate, nor short- or
long-term survival after emergent operation for colon cancer. Patients operated by emergency surgical teams were
more likely to have a permanent stoma after 3 years. Keywords Emergency surgery, Colorectal cancer, Sub-specialization, Large bowel obstruction World Journal of
Emergency Surgery World Journal of
Emergency Surgery Arnarson et al. World Journal of Emergency Surgery (2023) 18:3
https://doi.org/10.1186/s13017-023-00474-y Arnarson et al. World Journal of Emergency Surgery (2023) 18:3
https://doi.org/10.1186/s13017-023-00474-y Open Access Open Access © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://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. Who should operate patients presenting
with emergent colon cancer? A comparison
of short‑ and long‑term outcome depending
on surgical sub‑specialization Örvar Arnarson*, Ingvar Syk and Salma Tunå Butt Örvar Arnarson*, Ingvar Syk and Salma Tunå Butt Introduction Colorectal cancer (CRC) is among the most common
cancers globally and counting for over 7000 new cases
in Sweden annually [1]. Apart from tumor biology and
stage, survival is dependent on various factors including
the quality of the surgical resection, number of examined *Correspondence:
Örvar Arnarson
orvar.arnarson@med.lu.se
Department of Surgery, Skane University Hospital Malmo, Lund
University, Lund, Sweden *Correspondence:
Örvar Arnarson
orvar.arnarson@med.lu.se
Department of Surgery, Skane University Hospital Malmo, Lund
University, Lund, Sweden Approximately 20% of the colon cancer cases present as
emergencies, in which acute resection is associated with
increased perioperative morbidity and mortality, but also
impaired long-term survival compared to elective cases,
independent of tumor stage [7, 8]. Patients undergoing
emergency surgery tend to have more advanced cancer
with higher T-stage and N-stage compared to electively
operated patients [9]. Radical resection rate has also been
shown to be lower among emergency presented cases
[10]. lymph nodes and adequate adjuvant therapy [2–6]. Approximately 20% of the colon cancer cases present as
emergencies, in which acute resection is associated with
increased perioperative morbidity and mortality, but also
impaired long-term survival compared to elective cases,
independent of tumor stage [7, 8]. Patients undergoing
emergency surgery tend to have more advanced cancer
with higher T-stage and N-stage compared to electively
operated patients [9]. Radical resection rate has also been
shown to be lower among emergency presented cases
[10]. In Sweden, as in many western countries, surgical care
is characterized by centralization and sub-specialization. Most high-volume hospitals have both colorectal teams
with colorectal surgeons managing all elective cases of
CRC and emergency surgery teams dealing with a wide
spectrum of emergency cases, including emergent colon
cancer surgery. Smaller- or low-volume hospitals usually
do not have this division of specialized teams, and colo-
rectal surgery as well as emergency surgery is performed
by general surgeons with or without colorectal speciali-
zation. Some studies suggest that surgical specialization,
hospital volume and caseload are important prognos-
tic factors in elective colorectal cancer surgery [11–14],
whereas the impact of surgical specialization on emer-
gent colon cancer is more elusive [15–18].h Date of death was registered by linking to the Swed-
ish Population Registry, which is continuously updated. Last retrieval from both registers was performed 3rd mars
2020, giving a mean follow-up time of 4.2 (S.D. ± 1.7)
years. Data retrieved from the SCRCR included: patient
demographics, tumor characteristics, operative details,
complete pathology results, detailed information on
postoperative events, and date and localization of any
recurrence. All patients were routinely followed up with
a CT scan at 3 years, whereas the number of examina-
tions up to three years differed among centers. Further, if
the patients were operated on with a diverting ileostomy
or a permanent stoma and whether they had undergone
reversal of any diverting ileostomy within 3 years postop-
eratively were noted. © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Arnarson et al. World Journal of Emergency Surgery (2023) 18:3 Page 2 of 10 Page 2 of 10 the study. Whether the surgeon was a qualified specialist
in surgery, sub-specialist in colorectal surgery or general
surgery was noted. In the SCRCR, an emergency resec-
tion was defined as a resection performed in a patient
admitted via the emergency department due to acute
symptoms emanating from the tumor and requiring
immediate resection. No data on duration of symptoms
were available in the register. The coverage in SCRCR,
as compared to the Swedish cancer registry where regis-
tration is compulsory, was 98.5–99.6% during the study
period. The cohort was divided into three categories:
(1) operated by colorectal team (CRT); (2) operated by
emergency surgical team (EST); and (3) operated by gen-
eral surgical team (GST). Patients that did not undergo
primary resection of the tumor, but were operated with
intestinal by-pass, bowel deviation, endoluminal stent or
just open and close procedure were excluded from the
study. Cases operated in the specialized hospitals with
missing data regarding sub-specialization of operating
team were excluded, whereas missing data on specializa-
tion in the small hospitals were considered operated by
general surgical teams. lymph nodes and adequate adjuvant therapy [2–6]. Methodsh This was a retrospective study, based on the Swedish
Colorectal Cancer Registry (SCRCR), in which all pri-
mary tumors of invasive adenocarcinomas are registered. Hence, no recurrent cancer was included in this study. In
the south region of Sweden, with approximately 1.8 mil-
lion inhabitants, an addition has been made in the regis-
try, regarding whether the patient was operated on by a
colorectal team, an emergency surgical team or not appli-
cable. The latter refers to smaller hospitals lacking spe-
cialized surgical teams but handled by general surgeons,
often with a broad surgical experience. In total, 8 hospi-
tals in the region performed surgery for colorectal cancer
during the study period, of which 5 had sub-specialized
teams (colorectal and emergency teams) and 3 did not. All patients operated on with emergent resection for
colon cancer in the south region of Sweden between 2011
and 2016 were identified via SCRCR and were included in Postoperative complications were
graded by the Clavien–Dindo classification system, and
grade 3b or higher was defined as severe complications. Patients were considered radical resected if judged both
macroscopically radical by the surgeon and microscopi-
cally radical according to the pathology report. Doubtful
and undefinable resections were considered as not radi-
cal. Curative operation was defined as radical resection in
M0 patient. Whether the operation started after daytime
hours (i.e., 16:00) on any day of the week was noted. The aim of this study was to evaluate the impact of sur-
gical specialization on short- and long-term outcomes in
patients undergoing emergent colon cancer surgery. Pri-
mary endpoints were 5-year overall survival and 3-year
recurrence-free survival. Secondary endpoints were rate
of radical resections, postoperative complication rate,
30- and 90-day postoperative mortality and stoma rate at
3 years. Secondary endpoints One or more postoperative complications were registered
in 232 (35.4%) patients. Neither the total complication
rate nor the rate of severe postoperative complications
(CD ≥ 3b) differed between the groups, and there was
no difference in thirty- or ninety-day mortality. Neither
was there any difference in whether the patients received
adjuvant or palliative treatment (Table 4). Patient demography and tumor characteristics are
presented in Table 1. There was no significant differ-
ence in gender or age between the three groups. Neither
were there any significant differences in the propor-
tion of high- grade or mucinous type nor T- or M-stage,
whereas for N-stage, a borderline statistical difference
was noted (p = 0.06). Notably, the rate of preoperative
staging regarding liver and lung metastases was higher
in the CRC group (90.3%), compared to the EST and
GST groups (66.7% and 63.8%, respectively, p < 0.001),
although proper M-staging was done in all but 4 patients
during hospitalization. The proportion of patients with
ASA score 3 and 4 was higher in the EST group (53.8%)
compared with the CRT group (43.9%) and the GST
group (40.9%) (p < 0.05) (Table 1). Surgical specialization was not predictive for 30-day
mortality (OR = 1.4 [95% CI 0.6–3.2] and 1.2 [95% CI
0.4–3.2]) or 90-day mortality (OR = 1.0 [95% CI 0.3–2.0]
and 0.8 [95% CI 0.3–1.8]) in multivariate logistic regres-
sion analyses. Only high ASA score was predictive for
30-day mortality, whereas besides high ASA score, both
high age and metastasized disease were risk factors for
90-day mortality (Table 5). Selection of variables adjusted
for, according to principles given in methods, is pre-
sented in Additional file 1: Table S2. i
A total of 279 of the 656 patients (42.5%) were oper-
ated on with a stoma: 46.4% in the CRT group; 41.9% in
the EST group; and 33.9% in the GST group (p < 0.05). The use of diverting ileostomy was more often used by
colorectal teams, i.e., in 16.9% (54/319) compared to 5.7%
(12/210) in the EST group and 8.7% (11/127) in the GST
group, (p < 0.05). In turn, patients in the CRT group had
higher rate of stoma reversal and of all patients alive after
3 years, 24.3% (46/189) patients in the CRT group had a
permanent stoma compared with 34.5% (40/116) in the
EST group and 23.9% (17/71) in the GST group (p < 0.05)
(Table 4). Results A total of 699 patients were identified as having under-
gone emergent colonic resection between 2011 and 2016
due to cancer with acute symptoms. Of these, 43 patients
were excluded because of missing data. Hence, a cohort
of 656 patients were included in the study of which 319
were operated by colorectal teams (CRT), 210 by emer-
gency surgical teams (EST) and 127 by general surgical
teams (GST). Median age was 75 (range 32–101), and 319
(48.6%) were male and 337 (51.4%) females. Primary endpoints
ll Five-year overall survival rates did not differ statistically
significant depending on operating team and was 48.3%
in the CRT group, 45.7% in the EST group and 42.5% in
the GST group (p = 0.57) (Table 2 and Fig. 1). Three-year
recurrence-free survival in M0 patients did not differ
either and was 80.1% in the CRT group, 84.1% in the EST
group and 77.3% in the GST group (p = 0.44) (Table 2 and
Fig. 2). Statistical analyses were performed using SPSS (IBM
SPSS version 25, Armonk, NY, USA). Survival curves
were generated using Stata (release 17; Stata Statistical
Software, College Station, TX, USA). Multivariate COX proportional risk analysis showed
no difference in impact of surgical specialization on
5-year OS or 3-year RFS (Table 3). Selection of variables
adjusted for, according to principles given in methods, is
presented in Additional file 1: Table S1. Statistical analysis Categorical variables are reported as numbers and pro-
portion; continuous variables are reported as median
and range. Comparisons of continuous and categorical
variables were analyzed with Kruskal–Wallis and Chi2
test, respectively. p values less than 0.05 were considered Arnarson et al. World Journal of Emergency Surgery (2023) 18:3 Page 3 of 10 statistically significant, and 95% confidence intervals (CI)
are presented when appropriate. Laparoscopic approach was used in only 11 cases without
any significant difference between the groups. There was no
significant difference between the three groups whether the
patients were operated during night hours or not. Formal
qualification differed among the groups, but all resections
were performed by specialists in surgery and thus having at
least 5 years of surgical experience (Table 1). Kaplan–Meier curves were used to describe overall
(OS) and recurrence-free survival (RFS) rates, which were
analyzed with log rank test to determine statistical sig-
nificance. OS and RFS were defined as time from date of
operation to death of any cause and date of recurrence,
respectively. Patients alive and without recurrence were
censored at last follow-up. Association of well-known con-
founders on postoperative mortality and long-term sur-
vival was examined with univariate analysis. Variables with
p value of 0.1 or less were entered in a multivariate model
to determine predictors for those outcomes. Regarding
postoperative mortality, logistic regression analysis was
used, whereas for overall and recurrence-free survival Cox
proportional hazards regression analysis was used. Secondary endpoints World Journal of Emergency Surgery (2023) 18:3 Table 2 5-year overall mortality and 3-year recurrence-free
survival following emergent resection of colon cancer, stratified
on specialization of operating team Table 2 5-year overall mortality and 3-year recurrence-free
survival following emergent resection of colon cancer, stratified
on specialization of operating team stoma after 3 years compared with patients operated by
colorectal surgical teams or general surgeons. This may
reflect that colorectal surgeons are more prone to opt
for primary anastomosis and diverting ileostomy in left-
sided resections. CRT Colorectal team, EST Emergency surgical team, GST General surgical team
* M0 at diagnosis only
# Log rank
OS Overall survival, RFS Recurrence-free survival
CRT (%)
EST (%)
GST (%)
p value#
5-year OS
48.3
45.7
42.5
0.57
3-year RFS*
80.1
84.1
77.3
0.44 The rate of microscopically radical resections did not
differ between the groups. In contrast, a difference in
number of examined lymph nodes was noted, which,
however, did not reflect in any difference in risk of recur-
rence. The difference was small and could as well depend
on the pathology as the surgery. Further, all groups had
totally sufficient numbers of examined lymph nodes and
we perceive the noted difference not to be of clinical
importance. Notably, a borderline difference in N-stage,
with more N0 in the GST group, was noted. Although the
reason for this is elusive, it might reflect a stage migration
albeit the sufficient number of examined lymph nodes as
any difference in N-stage depending on geography is less
likely. Nevertheless, as the whole groups of patients were
analyzed, any stage migration should not impact the pri-
mary endpoints, survival and recurrence rate. OS Overall survival, RFS Recurrence-free survival The number of examined lymph nodes was signifi-
cantly less in the GST group compared with the other
groups. Moreover, the rate of microscopically radical
resection of the primary tumor did not differ between
groups (Table 6). Secondary endpoints Colonic obstruction was the most common indication
for surgery, 80.8%, 73.8% and 76.4% in CRT, EST and GST
groups, respectively (p = 0.08), Table 1. There was no differ-
ence in tumor location, but 11 patients in the CRC group
underwent anterior resection compared to none of the
patients in the other two groups. Otherwise, there was no
significant difference in type of resection, data not shown. Arnarson et al. World Journal of Emergency Surgery (2023) 18:3 Page 4 of 10 Table 1 Patient and tumor characteristics stratified on specialization of operating team
* CRT Colorectal team
CRT* (N = 319)
EST** (N = 210)
GST*** (N = 127)
p value#
n
(%)
n
(%)
N
(%)
Gender
Male
147
(46.1)
111
(52.9)
61
(48.0)
0.31
Female
172
(53.9)
99
(47.1)
66
(52.0)
Age
< 66
77
(24.1)
52
(24.8)
27
(21.3)
0.75
66–80
149
(46.7)
91
(43.3)
60
(47.2)
0.70
> 80
93
(29.2)
67
(31.9)
40
(31.5)
0.77
ASA score
ASA 1–2
175
(54.9)
80
(38.1)
74
(58.3)
< 0.05
ASA 3
126
(39.5)
92
(43.8)
40
(31.5)
< 0.05
ASA 4
14
(4.4)
21
(10.0)
12
(9.4)
< 0.05
Missing
4
(1.3)
17
(8.1)
1
(0.8)
–
Indication for surgery
Obstruction
261
(80.8)
155
(73.8)
97
(76.4)
0.08
Bleeding
13
(4.1)
9
(4.3)
4
(3.1)
0.87
Perforation
36
(11.3)
29
(13.8)
19
(15.0)
0.50
Other
9
(2.8)
17
(8.1)
7
(5.5)
< 0.05
Tumor location
Appendix
4
(1.3)
4
(1.9)
3
(2.4)
0.68
Right colon
115
(35.1)
84
(40.5)
55
(43.3)
0.31
Transverse colon
34
(10.7)
17
(8.1)
16
(12.6)
0.39
Left colon
166
(52.0)
104
(49.5)
53
(41.7)
0.14
pT stage
T1–T2
13
(4.1)
6
(2.9)
7
(5.5)
0.48
T3
137
(42.9)
89
(42.4)
56
(44.1)
0.95
T4
167
(52.4)
114
(54.3)
64
(50.4)
0.78
TX
2
(0.6)
1
(0.5)
0
(0.0)
–
pN stage
N0
106
(33.2)
72
(34.3)
56
(44.1)
0.06
N1–2
211
(66.1)
136
(64.8)
68
(53.5)
< 0.05
Missing
2
(0.6)
2
(1.0)
3
(2.4)
–
M-stage¤
M0
246
(77.1)
164
(78.1)
97
(76.4)
0.93
M1
73
(22.9)
45
(21.4)
27
(21.3)
0.09
Missing
0
(0.0
1
(0.5)
3
(2.4)
–
Surgical specialization
CR§
278
(87.7)
60
(29.4)
71
(56.3)
< 0.05
Non-CR
39
(12.3)
144
(70.6)
55
(43.7)
< 0.05 Table 1 Patient and tumor characteristics stratified on specialization of operating team Page 5 of 10 Arnarson et al. Discussionh This study shows that neither risk of recurrence nor
survival after emergent colon cancer resection was
influenced by the specialization of the surgical team per-
forming the operation. No difference in postoperative
morbidity or mortality rate was noted. Patients oper-
ated by emergency teams had a higher rate of permanent Several studies have shown an association between
outcome and surgical sub-specialization and surgical vol-
ume, respectively, in elective surgery for colon cancer [12,
19–22], although not all were consistent. For example,
Hall et al. performed a retrospective registry-based study Fig. 1 Kaplan–Meier survival estimates for overall survival stratified by specialization of the surgical team Arnarson et al. World Journal of Emergency Surgery (2023) 18:3 Page 6 of 10 Fig. 2 Kaplan–Meier survival estimates for recurrence-free survival stratified by specialization of the surgical team. M0 patients only Fig. 2 Kaplan–Meier survival estimates for recurrence-free survival stratified by specialization of the surgical team. M0 238 patients in each group. Operations performed
by colorectal surgeons were associated with signifi-
cantly lower rates of 30-day mortality (6.7% vs 16.4%,
p = 0.001) and postoperative morbidity (45.0% vs 56.7%,
p = 0.009). However, only 13.0% of the patients had a
malignant disease [24]. A large population-based reg-
istry study from the UK showed that emergency lapa-
rotomy performed by consultants without a special
interest in colorectal surgery had an increased adjusted
30-day mortality risk (OR 1.23, 95 CI 1.13–1.33) as well
as increased risk of re-operation (OR 1.13, CI 1.05–
1.20) compared to consultants with special colorectal
interest [25]. of 21,432 patients who had undergone elective operation
for colon cancer and 5893 operated on for rectal cancer
either by colorectal specialists or general surgeons. Colo-
rectal surgeons performed 16.3% of the colon and 27% of
the rectal resections. They found no difference in overall
5-year disease-specific survival (DSS) between the spe-
cialties except in stage II rectal cancer in a multivariate
analysis. When the analysis was limited to high-volume
surgeons only, the results remained the same [2].h The impact of specialization and caseload in the
emergency settings is much less studied and unclear. Kwan et al. studied the impact of hospital volume on
30-day postoperative mortality following emergency
colorectal surgery in 864 patients, of which 63.8% had
colon cancer, operated in 15 different hospitals. The
hospitals were grouped into low, medium and high
operative volume according to caseload. Discussionh World Journal of Emergency Surgery (2023) 18:3 Page 7 of 10 Table 3 Multivariate COX proportional hazard model
Hazard ratios (HR) for 5-year overall mortality and recurrence within 3 years following emergent resection of colon cancer
CST colorectal team, EST emergency surgical team, GST general surgical team, ASA score American Society of Anaesthesiologists classification
5-year mortality
Recurrence within 3 years
HR
95% CI
p value
HR
95% CI
p value
Age
< 65
1.0
1.0
65–74
1.2
(0.8–1.6)
0.4
1.2
(0.7–2.1)
0.52
75–84
1.8
(1.3–2.5)
0.00
1.0
(0.6–1.8)
0.95
> 85
3.6
(2.5–5.1)
0.00
0.5
(0.2–1.3)
0.15
ASA score
1–2
1.0
1.0
0.90
3
1.8
(1.4–2.3)
0.00
1.0
(0.6–1.5)
0.50
4
4.0
(2.8–5.9)
0.00
1.4
(0.5–3.7)
0.60
T-stage
1–2
1.0
1.0
3
1.4
(0.7–3.0)
0.34
1.1
(0.3–4.6)
0.90
4
2.4
(1.1–4.9)
0.2
1.4
(0.3–5.9)
0.70
N-stage
0
1.0
1.0
1–2
1.6
(1.2–2.1)
< 0.05
2.9
(1.7–4.9)
< 0.05
M-stage
0
1.0
1
2.9
(2.3–3.7)
0.00
Surgical specialization
CRT
1.0
1.0
EST
1.0
(0.7–1.3)
0.88
0.7
(0.4–1.1)
0.15
GST
1.2
(0.9–1.6)
0.24
1.3
(0.8–2.2)
0.36 Table 3 Multivariate COX proportional hazard model Hazard ratios (HR) for 5-year overall mortality and recurrence within 3 years following emergent resection of colon cancer
CST colorectal team, EST emergency surgical team, GST general surgical team, ASA score American Society of Anaesthesiologis y
y
y
CST colorectal team, EST emergency surgical team, GST general surgical team, ASA score American Society of Anaesthesiologists classification Multidisciplinary approach is needed for identification of
at-risk patients, prevention of avoidable complications,
recognition of unavoidable complications and prompt
intervention in attempt to prevent avoidable death [31,
32]. Henneman et al. evaluated the association between
structural hospital characteristics (hospital volume,
teaching status and intensive care facilities (ICU) and
FTR after colorectal cancer surgery. Only higher levels
of ICU facilities were associated with lower FTR rates
(OR 0.72; 95% CI 0.65–0.88) in multivariate analysis
[28]. Intensive care in Sweden is generally of high quality
and quite standardized between hospitals which might
explain why we did not find any difference in in-hospital
mortality between the groups in the present study.h to what degree the improved postoperative mortality rate
was dependent on the specialization of the surgeon or
the competence of the whole team, caring for the patient
postoperatively. Discussionh The colorec-
tal POSSUM scoring system was used to adjust for dif-
ference in case-mix in the study. Thirty-day mortality
was 16.3% without any statistical difference in mor-
tality between hospitals of different case volume [23],
a finding in line with our result. Kulyat et al. studied
short-term outcomes in patients undergoing emergent
colectomies by colorectal surgeons compared to gen-
eral or emergency care surgeons in 3 academic hospi-
tals. A propensity score matching was performed with A Swedish registry-based retrospective study on 13,365
patients operated on for colon cancer between 2007 and
2010 focused on formal competence of the most senior
surgeon attending the procedure irrespective of surgical
team or hospital volume, of which 21.9% were emergency
procedure. The result showed superior five-year over-
all survival in patients operated by colorectal surgeons
(36.6%) compared to general surgeons (33.4%) (p < 0.05). However, after adjusting for 30- and 90-day mortality,
no statistically significant difference was noted. Hence,
the difference in long-term survival was explained by a
lower postoperative mortality in the group of patients
operated by colorectal surgeons. However, it is unclear Arnarson et al. Discussionh Probably this result was also affected by
a case-mix, such as more frail and severely ill patients,
e.g., with peritonitis, had to be operated during on-call
and thus by younger less qualified surgeons [26]. i
Moreover, high-volume hospitals not only have more
colorectal specialist, but also better intensive care and
a lower rate of failure to rescue (FTR) [27–29] which
reflects the rate of mortality after major complications. Postoperative complications greatly affect short- and
long-term survival after surgery for colon cancer and
even more so in patients operated on acutely [30]. Pre-
operative comorbidities, such as congestive heart fail-
ure and chronic renal failure, have been associated with
higher rates of FTR in emergency general surgery [31]. These patients may neither tolerate fluid shifts nor the
resuscitation required to restore physiologic parameters
postoperatively [31]. Hospital factors also influence FTR. The Union of International Cancer Control (UICC)
recommends the evaluation of a minimum of 12 LNs
for appropriate staging of patients with pN + disease
[33]. Some previous studies report insufficient examined
lymph nodes in the emergent setting [34], perhaps due
to technical difficulties and instable patients [35], with Arnarson et al. colorectal team, EST emergency surgical team, GST general surgical team, ASA score American Society of Anaesthesiologists classification Discussionh World Journal of Emergency Surgery (2023) 18:3 Page 8 of 10 Table 4 Perioperative outcome and oncological treatment, stratified on specialization of surgical team
CRT
N = 319
EST
N = 210
GST
N = 127
p value#
n
(%)
n
(%)
N
(%)
Postoperative complications
All
113
(35.5)
75
(35.9)
39
(30.7)
0.53
Severe (≥ CD* 3b)
55
(17.2)
46
(21.9)
22
(17.3)
0.37
Postoperative mortality
30-day mortality
14
(4.4)
17
(8.1)
7
(5.5)
0.20
90-day mortality
28
(8.8)
25
(11.9)
10
(7.9)
0.37
Stoma
At primary operation
148
(46.4)
88
(41.9)
43
(33.9)
< 0.05
Diverting ileostomy
54
(16.9)
12
(5.7)
11
(8.7)
< 0.05
Sigmoidostomy
33
(10.3)
30
(14.3)
13
(10.2)
0.13
Other
61
(19.1)
46
(21.9)
19
(15.0)
0.25
Stoma after 3 years**
46/189
(24.3)
40/116
(34.5)
17/71
(23.9)
< 0.05
Chemotherapy
Adjuvant
133
(41.7)
77
(36.7)
51
(40.2)
0.51
Palliative
28
(8.8)
21
(10.0)
11
(8.7)
0.87
Operated outside office hours***
84
(26.3)
59
(28.1)
34
(26.8)
0.75 Table 4 Perioperative outcome and oncological treatment, stratified on specialization of surgical team Table 5 Risk factors for 30- and 90-day postoperative mortality following emergent resection of colon cancer
Multivariate logistic regression
CST colorectal team, EST emergency surgical team, GST general surgical team, ASA score American Society of Anaesthesiologists classification
30-day mortality
90-day mortality
OR
(95% CI)
p value
OR
(95% CI)
p value
Age
< 65
1.0
Ref
65–74
0.4
(0.1–1.9)
0.29
0.7
(0.2–2.2)
0.53
75–84
1.6
(0.5–4.7)
0.40
1.9
(0.7–4.8)
0.21
> 85
1.9
(0.6–6.0)
0.26
4.2
(1.6–11.5)
< 0.05
ASA score
1–2
1.0
Ref
3
3.9
(1.4–10.9)
< 0.05
4.0
(1.7–9.1)
< 0.05
4
18.4
(5.7–58.7)
< 0.05
18.8
(7.0–53.5)
< 0.05
M-stage
0
Ref
1
2.9
(1.4–5.8)
< 0.05
Indication for surgery
Obstruction
1.0
Bleeding
0.8
(0.2–4.0)
0.80
1.8
(0.5–5.8)
0.35
Perforation
1.2
(0.4–3.0)
0.77
1.7
(0.7–3.6)
0.16
Other
1.1
(0.3–4.5)
0.86
2.1
(0.6–5.9)
0.22
Surgical specialization
CRT
1.0
Ref
EST
1.4
(0.6–3.1)
0.41
1.0
(0.5–2.0)
0.96
GST
1.1
(0.4–2.9)
0.89
0.8
(0.3–1.8)
0.60 isk factors for 30- and 90-day postoperative mortality following emergent resection of colon cancer Table 5 Risk factors for 30- and 90-day postoperative mortality following emergent resection of colon cancer Arnarson et al. Funding Open access funding provided by Lund University. This study was supported
by Skane University Hospital grants, ALF-agreement (2018-Project 0264),
between the Swedish government and the county councils, and local Grant
(Allmanna Sjukhusets i Malmö Stiftelse för bekampande av cancer). Conclusion Postoperative morbidity and mortality as well as long-
term survival following emergent resection for colon
cancer did not significantly differ depending on surgi-
cal specialization. Long-term survival and postoperative
outcomes proved good in comparison to results reported
in the literature, despite the differences in formal train-
ing and small volumes in some of the hospitals. However,
permanent stoma rate was higher in patients operated by
emergency surgeons. Further studies comprising more
detailed data on comorbidity and management of com-
plications and the impact on survival would be of value. Acknowledgements
We acknowledge the Swedish Colorectal Cancer Registry. We acknowledge the Swedish Colorectal Cancer Registry. The weakness of the present study is the retrospective
design, implying a risk of selection bias although known
confounders, such as ASA score, age and TNM stage
were adjusted for in the multivariate analysis. Although
the five larger hospitals had dedicated teams for colorec-
tal and emergency surgery, there was some overlapping
of surgeon´s specialization, especially in the GST where
over half of the surgeons had colorectal qualification. Author contributions
Ö ÖA contributed to the acquisition of data, design of the study and analysis of
the data as well as the drafting of the manuscript. IS contributed to the design
of the study, acquisition of data and revision the manuscript for important
intellectual content. STB critically revised the statistics and manuscript. All
authors reviewed, discussed and approved the final manuscript. Availability of data and materials The ethical approval for this study prohibits us from publishing any patient
data on individual level. Discussionh World Journal of Emergency Surgery (2023) 18:3 Page 9 of 10 Table 6 Primary oncological and pathology results stratified on specialization of surgical team
CST colorectal team, EST emergency surgical team, GST general surgical team
# Chi-square test
## Kruskal–Wallis test
CRT
EST
GST
p value
N = 319
N = 210
N = 127
Radical resection (R0), n (%)
289
(90.9)
178
(85.2)
114
(89.8)
0.12#
Curative operation, n (%)
228
(71.5)
147
(70.0)
89
(70.1)
0.92#
Examined lymph nodes, Mean, (S.D.)
26.6
(13.9)
26.9
(17.7)
22.8
(11.9)
< 0.05## Table 6 Primary oncological and pathology results stratified on specialization of surgical team Supplementary Information
The online version contains supplementary material available at https://doi.
org/10.1186/s13017-023-00474-y. Additional file 1. Table S1. Association between selected confounders
and 5-year mortality and recurrence within 3 years. Univariate analysis. Table S2. Association between selected preoperative variables and post-
operative mortality. Univariate analysis. a subsequent risk of not having adjuvant chemotherapy,
as the indication for adjuvant chemotherapy is deter-
mined foremost by node positivity. However, also other
risk factors for recurrence, including emergent operation
constitute indications for adjuvant treatment [36]. In the
present study, no difference in the proportion of patients
given adjuvant chemotherapy was noted, albeit a numeri-
cal difference in N0 stage, probably due to that emergent
resection is an indication for adjuvant chemotherapy.h a subsequent risk of not having adjuvant chemotherapy,
as the indication for adjuvant chemotherapy is deter-
mined foremost by node positivity. However, also other
risk factors for recurrence, including emergent operation
constitute indications for adjuvant treatment [36]. In the
present study, no difference in the proportion of patients
given adjuvant chemotherapy was noted, albeit a numeri-
cal difference in N0 stage, probably due to that emergent
resection is an indication for adjuvant chemotherapy. Supplementary Information Additional file 1. Table S1. Association between selected confounders
and 5-year mortality and recurrence within 3 years. Univariate analysis. Table S2. Association between selected preoperative variables and post-
operative mortality. Univariate analysis. Competing interests Competing interests
The authors declare that they have no competing interests. The authors declare that they have no competing interests. Abbreviations
SCRCR
Swedish Colorectal Cancer Registry
CRT
Colorectal team
EST
Emergency surgical team
GST
General surgeons
OS
Overall survival
RFS
Recurrence-free survival
ASA
American Society of Anaesthesiologists classification
CD
Clavien–Dindo
FTR
Failure to rescue
ICU
Intensive care unit Received: 22 November 2022 Accepted: 1 January 2023 Received: 22 November 2022 Accepted: 1 January 2023 Ethics approval and consent to participate Ethical approval for this study was obtained from the Regional Ethics Commit-
tee in Lund, dnr 2018/298. Received: 22 November 2022 Accepted: 1 January 2023 References
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English
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Advanced Numerical Methods for Graphene Simulation with Equivalent Boundary Conditions: A Review
|
Photonics
| 2,023
|
cc-by
| 10,439
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Review Not peer-reviewed version Advanced Numerical Methods for
Graphene Simulation With Equivalent
Boundary Conditions: A Review Yansheng Gong and Na Liu *
Posted Date: 10 May 2023
doi: 10.20944/preprints202305.0697.v1 Article
Advanced Numerical Methods for Graphene
Simulation with Equivalent Boundary Conditions: A
Review Yansheng Gong and Na Liu *
The Institute of Electromagnetics and Acoustics, Xiamen University, Xiamen 361005, China, and also with the
Fujian Provincial Key Laboratory of Electromagnetic Wave Science and Detection Technology, Xiamen
University, Xiamen 361005, China
* Correspondence: liuna@xmu.edu.cn (Na Liu) Abstract: Since the discovery of graphene, due to its excellent optical, thermal, mechanical
and electrical properties, it has a broad application prospect in energy, materials, biomedicine,
electromagnetism and other fields. A great quantity of researches on the physical mechanism
of graphene has been applied to engineering in electromagnetism and optics. To study the
properties of graphene, different kinds of numerical methods have been developed for simulating the
electromagnetic field effects of graphene, and equivalent boundary conditions have been employed to
replace graphene in the computational domain. In this work, the numerical methods with equivalent
boundary conditions are reviewed, and some examples are provided to illustrate their applicability. Keywords: numerical algorithm; computational electromagnetics; graphene; equivalent boundary
conditions Preprints.org is a free multidiscipline platform providing preprint service that
is dedicated to making early versions of research outputs permanently
available and citable. Preprints posted at Preprints.org appear in Web of
Science, Crossref, Google Scholar, Scilit, Europe PMC.
Copyright: This is an open access article distributed under the Creative Commons
Attribution License which permits unrestricted use, distribution, and reproduction in any
medium, provided the original work is properly cited. Preprints.org is a free multidiscipline platform providing preprint service that
is dedicated to making early versions of research outputs permanently
available and citable. Preprints posted at Preprints.org appear in Web of
Science, Crossref, Google Scholar, Scilit, Europe PMC. Copyright: This is an open access article distributed under the Creative Commons
Attribution License which permits unrestricted use, distribution, and reproduction in any
medium, provided the original work is properly cited. doi:10.20944/preprints202305.0697.v1 Disclaimer/Publisher’s Note: The statements, opinions, and data contained in all publications are solely those of the individual author(s) and
contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting
from any ideas, methods, instructions, or products referred to in the content. 1. Introduction Graphene [1–3] is an allotropy of 3-D crystal graphite, a true two-dimensional material composed
of a single layer of carbon atoms. Due to its unique electrical, electromagnetic, and optical
characteristics, it has attracted widespread attention, leading to the design of many new systems
and equipment [4–6]. In 2010y, Novoselov and Heim [1] won the Nobel Prize for their research and
observation of graphene properties. Graphene exhibits semi metallic properties and has a strong
bipolar electric field effect. And graphene has been shown to possess electrical properties similar to
semiconductors (although with a zero band gap) [7–9]. Due to its excellent properties, many methods
have been developed to fabricate graphene, such as micromechanical cleavage technique [10], chemical
vapor deposition [11], solvent exfoliation [12,13], solvothermal method [14], etc. The development
of preparation technology has made it easy to separate high-quality graphene, triggering a research
boom in the 2-D material family. In fact, the optical properties generated by the unique electronic
band structure of graphene are considered attractive features in the design of nanophotons and
optoelectronic components [15,16]. Graphene has a strong interaction with electromagnetic fields,
and its response to light is nonlinear, manifested by plasmonic characteristics. Graphene-based
plasmonic can not only limit the electromagnetic field to a smaller transverse propagation range, but
also modulate it over a wide frequency range through gating and chemical doping [17–20]. In the past few years, in addition to the physical research on graphene and graphene-based
devices, another important topic has been the numerical simulation of graphene. Kubo formula puts
forward the expression of graphene conductivity, which is a function formed by physical parameters
such as wavelength, chemical potential and temperature [21–24]. A surface conductivity model is also
proposed to describe graphene as an isotropic infinitesimal sheet. Although the model can provide
problematic results for some tests, the electromagnetic simulation of specific graphene devices still
remains a challenge when it comes to practical graphene problems. Fortunately, with the development
of computational electromagnetics, many efficient and accurate numerical methods can have been
proposed to analyze the electromagnetic response of graphene related devices. Among them, the most © Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 May 2023 doi:10.20944/preprints202305. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 May 2023 doi:10.20944/preprints202305.0697.v1 2 of 19 popular methods are the finite difference time domain method (FDTD), finite element method (FEM),
and spectral element method (SEM) [25–30]. 2. Advanced Numerical Methods With the development of computer performance and new efficient and accurate algorithms,
electromagnetic numerical methods have become more applicable and efficient. In order to analyze
the electromagnetic field interaction related to graphene and the shielding effect of graphene, several
different numerical methods can be used, including mixed finite element method (Mixed FEM), the
mixed spectral element method (Mixed SEM), auxiliary source method (MAS) and interior penalty
discontinuous Galerkin time domain (IPDG), and equivalent boundary conditions such as ITBC, SCBC
and IMBC are introduced. Regardless of the boundaries used by these methods, a mathematical
model is required to describe the conductivity of graphene. The following subsection depicts the
mathematical model employed in this review. 1. Introduction The numerical approaches of processing graphene include:
(a) Taking graphene as a zero-thickness sheet [31–34]; (b) Graphene is regarded as a thin plate with
finite thickness, and its surface conductivity is converted into a volumetric dielectric constant [35]. Due
to the easy realization of (b), some published papers and commercial software will model graphene
as a thin sheet with limited thickness [36–38]. However, direct discretization of graphene thin plates
result in extremely fine grids and a large number of unknowns. Especially in time-domain simulation,
extremely small time steps are required to ensure stability, which consumes an enormous amount
of CPU time and memory costs. Therefore, equivalent boundary conditions are preferred methods
used to eliminate thin plates in the computational domain, which include impedance transmission
boundary condition (ITBC) [39,40], surface current boundary condition (SCBC) [41] impedance matrix
boundary condition (IMBC) [42]. These numerical methods and equivalent boundary conditions have
been proved that can greatly improve the design and manufacturing speed. In this review, we will focus on the electromagnetic simulation of graphene, and select several
numerical cases to show their serviceability in practical engineering applications. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 May 2023 doi:10.20944/preprints202305.0697.v1 3 of 19 The interband conductivity, which is smaller than the intraband conductivity, is in the order of
magnitude of e2/¯h. The dielectric constant of graphene is determined by bε = 1 −jσg/(ε0ωτ). The
electrons and holes close to the band edges "blocke" interband transitions at lower frequencies (i.e., in
the THz range). Graphene exhibits the characteristics of a conductive film, and a straight forward
Drude model is used to describe its conductivity (primarily caused by in-band contributions). The
electrons and holes close to the band edges "blocke" interband transitions at lower frequencies 2.2. Mixed Finite Element Method The graphene layer is assumed to have a finite thickness and a specific volumetric conductivity. 2.2. Mixed Finite Element Method With the increasing complexity of microwave and optical waveguide structures, it is difficult to
effectively conduct modal analysis of waveguide problem, which seriously delays the subsequent
adjustment of geometric and material parameters in waveguide design. Scholars have developed
different numerical methods to solve this problem, among which the FEM is a successful representative. The mathematical theory of the finite element method was completed by Feng Kang and others scholars. Compared with the FDTD, the FEM can utilize unstructured meshes to discretize the computational
domain, which is convenient for solving problems in complex calculation regions. However, the
traditional FEM based on scalar node basis functions inevitably generate spurious modes with non-zero
eigenvalues. The FEM based on vector basis functions is a reliable solution, where there are no non-zero
spurious solutions for solving Maxwell problems, but it will generate zero spurious solutions (also
known as DC spurious modes) [46]. All modes can be suppressed by a mixed FEM based on a new
weak form. The mixed FEM is a powerful technique that not only maintains the flexibility of accurately
modeling complex geometries shapes with finite elements, but also suppresses all spurious modes with
new weak forms [47–49]. It applies systematic and rigorous mathematical techniques to the solution of
equations and boundary conditions and uses meshes (for example, using triangle and quadrilateral
elements) to discrete the computational domain involving geometrically complex structures that
have potentially heterogeneous material properties. The medium in a waveguide structure is a
two-dimensional variable with a transverse anisotropic tensor, and its longitudinal component is
constant where the electromagnetic field is a three-dimensional function (assumed to be in the
z-direction). Thus, the electric field in an arbitrary cross section is described as e(x, y)e−jkzz. This
results in the expression of the vectorial Helmholtz equation and Gauss’ law can be written as: ∇t × µ−1
rz ∇t × et + jkz ˆz×µ−1
rt ˆz × ∇tez −k2
z ˆz × µ−1
rt ˆz × et −k2
0εrtet = 0
∇t × µ−1
rt (ˆz × ∇tez + jkz ˆz × et) + k2
0εrtez ˆz = 0
∇t · εrtet −jkzεrzez = 0
(4) (4) where µr = diag(µrt, µrz) and εr = diag(εrt, εrz) are the magnetic permeability and relative dielectric
permittivity tensors with the transversely anisotropic medium µrt and εrt, respectively. k0 is the
wavenumber in vacuum. 2.1. Mathematical Model The numerical methods listed above are commonly used to simulate graphene as a thin sheet with
a conductivity that is both complex and dependent on frequency. The graphene surface conductivity is
usually expressed using the Kubo formula [43–45]: σg = je2(ω −j2Γ)
π¯h2
[−
Z ∞
0
ε
fd(−ε) −fd(ε)
(ω −j2Γ)2 −4( ε
¯h)2 dε
+
1
(ω −j2Γ)2
Z ∞
0
ε(∂fd(ε)
∂ε
−∂fd(−ε)
∂ε
)dε]
(1) (1) where ε is the electron charge, ω is the radian frequent, Γ is the phenomenological scattering rate, ¯h
is the reduced Planck constant and e is the energy state. fd = (e(ε−|µc|)/kBT + 1)−1 is the Fermi-Dirac
distribution, where µc is the chemical potential or Fermi level, T is the temperature and kB is the
Boltzman constant. Equations (1) contains both intraband and interband contributions that can be
expressed as σg = σintra(ω, µc, Γ, T) + σinter(ω, µc, Γ, T), where: σintra(ω, µc, Γ, T) =
−2je2T
¯h2π(ω −jΓ)
ln
2cosh( µc
2T )
(2) (2) σinter(ω, µc, Γ, T) = e2
4¯h
1
2 + 1
π arctan( ¯hω −2µc
2T
) +
j
2π ln
(¯hω + 2µc)2
(¯hω −2µc)2 + (2T)2
(3) σinter(ω, µc, Γ, T) = e2
4¯h
1
2 + 1
π arctan( ¯hω −2µc
2T
) +
j
2π ln
(¯hω + 2µc)2
(¯hω −2µc)2 + (2T)2
(3) (3) 2.2.1. Impedance Transmission Boundary Condition In the traditional FEM, due to its simplicity and feasibility, the graphene is assumed to be a layer of
finite-thickness with a specific volumetric conductivity [50]. However, a graphene sheet having finite
thicknesses results in exceedingly small meshes and a substantial number of unknowns when they are
directly discretized. Therefore, to ensure numerical accuracy, a large amount of CPU time and memory
costs are required. The impedance boundary condition (IBC) is a useful way for eliminating thin plates
from the computational field, such as impedance network boundary condition (INBC), impedance
transmission boundary condition (ITBC) and surface impedance boundary condition (SIBC) [51,52]. By
using two-port network equations to describe the field inside the conductive sheet, INBC is effectively Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 May 2023 doi:10.20944/preprints202305.0697.v1 4 of 19 implemented in both FDTD and FEM. Meanwhile, Leontovich proposed a hypothetical SIBC for thin
plates that the thickness is less than or equal to the skin depth. However, when the frequency of
graphene reaches the terahertz band, the thickness of graphene is much lower than the skin depth. Therefore, ITBC is a better fit method to describe the interdependent tangential electromagnetic field
on each side of graphene surface [53]. In order to clearly describe the implementation of ITBC, Figure 1
uses two triangular elements that share ITBC to illustrate. L
Figure 1. The Degree distribution of triangular elements on both sides of ITBC edge L. Reproduced
with permission of Ref. [50]. Copyright of ©2016 IEEE. Figure 1. The Degree distribution of triangular elements on both sides of ITBC edge L. Reproduced
with permission of Ref. [50]. Copyright of ©2016 IEEE. The prerequisite for employing ITBC is that the wavelength and skin depth δ are larger than the
conducting sheet’s thickness τ. As shown in Figure 2, it can be observed that using line segments to
represent thin conductive sheets with finite thickness in a 2-D waveguide cross-section model [50]. The
condition for using ITBC is that the wavelength and the skin depth δ are greater than the conducting
sheet’s thickness τ, while avoiding sharp edges and angles. At this dot, the electromagnetic field is
transmitted on either side of a sheet. 2.2.1. Impedance Transmission Boundary Condition Meanwhile, considering the interdependence of electromagnetic
fields above and below the thin film, ITBC uses transmission line theory to represent the relationship
between tangential electromagnetic fields above and below the line segment L: "
n1 × h1
n2 × h2
#
=
"
Y11
Y12
Y12
Y11
# "
n1 × n1 × e1
n2 × n2 × e2
#
(5) "
n1 × h1
n2 × h2
#
=
"
Y11
Y12
Y12
Y11
# "
n1 × n1 × e1
n2 × n2 × e2
#
(5
1
2
e1 h1
e2 h2
n1
n2
1
2
e1 h1
e2 h2
n1
n2
L
Figure 2. Geometric representation of thin conductive sheets with finite thickness. Left: Treating
graphene as a single atom thick thin sheet; Right: The finite thickness graphene sheet is replaced by a
one-dimensional line . Reproduced with permission of Ref. [50]. Copyright of ©2016 IEEE. (5) Figure 2. Geometric representation of thin conductive sheets with finite thickness. Left: Treating
graphene as a single atom thick thin sheet; Right: The finite thickness graphene sheet is replaced by a
one-dimensional line . Reproduced with permission of Ref. [50]. Copyright of ©2016 IEEE. Graphene nanoribbon sandwich waveguide [54] is considered which placed in vacuum
background, with two identical graphene ribbons on both sides and a dielectric strip with a refractive
index of nd = 1.4 in the middle as shown in Figure 3 [50]. In the model, the graphene is an isotropic
medium which thickness is 0.5 nm and Γ = 0.1 meV, µc = 0.5 eV, and T = 300 K. The computing
domain of 20 µm × 20 µm yields 11 212 elements, including 81 011 unknowns. Figure 4 shows the
calculation results |kz/k0| for the first twelve modes. We can clearly see that the mixed FEM-ITBC
results and FEM solutions have good consistency. In addition to the accuracy of the mixed FEM-ITBC, Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 May 2023 doi:10.20944/preprints202305.0697.v1 5 of 19 Table 1 also shows that the mixed FEM-ITBC incurs lower memory costs and requires less CPU time
than the traditional FEM. Table 1 also shows that the mixed FEM-ITBC incurs lower memory costs and requires less CPU time
than the traditional FEM. W
d
nd
Figure 3. Structural schematic diagram of graphene nanostrip sandwich waveguide, where d = 2 nm,
W = 300 nm. Reproduced with permission of Ref. 2.2.1. Impedance Transmission Boundary Condition The nanowire with a radius of Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 May 2023 doi:10.20944/preprints202305.0697.v1 6 of 19 R = 100 nm has a relative permittivity of εr1 = 3 and the external region has a relative permittivity of
εr2 = 1. The surface conductivity s of graphene σs is calculated using Kubo’s formula with µc = 0.5 e
V, T = 300 K, and τ = 200 ns. By using PML which thickness is 0.5 µm to truncate the open GNW
structure. The length and width of the calculation area are both 5 µm. Through comparing the results
calculated by the mixed FEM-SCBC method with the analysis results [58], the field distributions of the
first three propagation modes (m0, m1, m2), propagation lengths L = 1/[2Im(kz)], and the dispersion
relations as a function of frequency is investigated. The proposed method has been proven to be
accurate, as presented in Figure 6a,b, with maximum relative errors of 0.4 % and 0.15 %, respectively. The field distributions of the modes (m0, m1, m2) in Figure 6c are also in well-alignment with the field
distribution of the analytical method. Furthermore, Table 2 demonstrates that the mixed FEM-SCBC
is also a more valuable option to studying the propagation characteristics of plasmonic waveguides
which is based on graphene, as it requires only 91 997 DoFs and avoids the demand for a dense mesh
around the graphene. In contrast, the traditional FEM requires 384 737 DoFs to discretize thin graphene
sheets with a thickness of 0.5 nm. In summary, this method offers both effectiveness and computational
efficiency for the analysis of graphene-based plasmonic waveguides. R = 100 nm has a relative permittivity of εr1 = 3 and the external region has a relative permittivity of
εr2 = 1. The surface conductivity s of graphene σs is calculated using Kubo’s formula with µc = 0.5 e
V, T = 300 K, and τ = 200 ns. By using PML which thickness is 0.5 µm to truncate the open GNW
structure. The length and width of the calculation area are both 5 µm. Through comparing the results
calculated by the mixed FEM-SCBC method with the analysis results [58], the field distributions of the
first three propagation modes (m0, m1, m2), propagation lengths L = 1/[2Im(kz)], and the dispersion
relations as a function of frequency is investigated. 2.2.1. Impedance Transmission Boundary Condition [50]. Copyright of ©2016 IEEE. Figure 3. Structural schematic diagram of graphene nanostrip sandwich waveguide, where d = 2 nm,
W = 300 nm. Reproduced with permission of Ref. [50]. Copyright of ©2016 IEEE. 0
2
4
6
8
10
12
20
40
60
80
100
120
140
160
180
Mode order
|kz/k0|
FEM with Graphene Sheet
Mixed FEM−ITBC
Figure 4. Normalized propagation constant |kz/k0| of different modes in waveguides. Reproduced
with permission of Ref. [50]. Copyright of ©2016 IEEE. 6
8
Mode order Figure 4. Normalized propagation constant |kz/k0| of different modes in waveguides. Reproduced
with permission of Ref. [50]. Copyright of ©2016 IEEE. Table 1. Calculate the DOF, CPU Time, and Memory Consumption of the First Five Eigenvalues of
Graphene Sandwich Waveguide Using Mixed FEM-ITBC and FEM, Respectively. Reproduced with
Permission of Ref. [50]. Copyright of ©2016 IEEE. Mixed FEM-ITBC
FEM with graphene sheet
DOF
82,215
408,035
CPU time
29.9 s
375.0 s
Memory
0.07 GB
0.18 GB
2.2.2. Surface Current Boundary Condition Mixed FEM-ITBC
FEM with graphene sheet 2.2.2. Surface Current Boundary Condition 2. Surface Current Boundary Condition The previous ITBC is only applicable to model isotropic media. The modeling thin layers of
anisotropic media requires the use of SCBC. In the right figure of Figure 2, one-dimensional lines are
used to replace thin conductive sheets with finite thickness. When graphene thin layers are equivalent
to zero thickness [33,55], fine mesh generation can be avoided. SCBC can be expressed as: n × (h2 −h1) = Js = σse
(6) (6) where e is the electric field, σs is the surface conductivity of the nanomaterial sheet which can be
anisotropic media. n = n1 = −n2 is the unit normal vector from Ω1 to region 2. h1, h2 are the magnetic
fields of Ω1 and Ω2, respectively. where e is the electric field, σs is the surface conductivity of the nanomaterial sheet which can be
anisotropic media. n = n1 = −n2 is the unit normal vector from Ω1 to region 2. h1, h2 are the magnetic
fields of Ω1 and Ω2, respectively. Figure 5 shows a graphene-coated dielectric nanowire waveguide (GNW), with an inner layer of
dielectric nanowires and an outer layer covered with graphene sheet. 2.2.1. Impedance Transmission Boundary Condition The proposed method has been proven to be
accurate, as presented in Figure 6a,b, with maximum relative errors of 0.4 % and 0.15 %, respectively. The field distributions of the modes (m0, m1, m2) in Figure 6c are also in well-alignment with the field
distribution of the analytical method. Furthermore, Table 2 demonstrates that the mixed FEM-SCBC
is also a more valuable option to studying the propagation characteristics of plasmonic waveguides
which is based on graphene, as it requires only 91 997 DoFs and avoids the demand for a dense mesh
around the graphene. In contrast, the traditional FEM requires 384 737 DoFs to discretize thin graphene
sheets with a thickness of 0.5 nm. In summary, this method offers both effectiveness and computational
efficiency for the analysis of graphene-based plasmonic waveguides. Figure 5. The GNW structure. Reproduced with permission of Ref. [56]. Copyright of ©2021 IEEE. Figure 5. The GNW structure. Reproduced with permission of Ref. [56]. Copyright of ©2021 IEEE. 10
20
30
40
50
Frequency [Thz]
20
40
60
Re(kz)/k0
m0, proposed method
m1, proposed method
m2, proposed method
(a)
10
20
30
40
50
Frequency [Thz]
2
4
6
8
10
12
L [ m]
m0, proposed method
m1, proposed method
m2, proposed method
(b)
Figure 6. (a) Dispersion relations. (b) Propagation length. (c) Field distributions of |E| of three plasma
modes in GNW. The analytical solution in [58] is represented by a solid lines in (a) and (b). Reproduced
with permission of Ref. [56]. Copyright of ©2021 IEEE. 10
20
30
40
50
Frequency [Thz]
2
4
6
8
10
12
L [ m]
m0, proposed method
m1, proposed method
m2, proposed method
(b) 10
20
30
40
50
Frequency [Thz]
20
40
60
Re(kz)/k0
m0, proposed method
m1, proposed method
m2, proposed method
(a) 10
20
30
40
50
Frequency [Thz]
20
40
60
Re(kz)/k0
m0, proposed method
m1, proposed method
m2, proposed method
(a)
10
20
30
40
50
Frequency [Thz]
2
4
6
8
10
12
L [ m]
m0, proposed method
m1, proposed method
m2, proposed method
(b) Figure 6. (a) Dispersion relations. (b) Propagation length. (c) Field distributions of |E| of three plasma
modes in GNW. The analytical solution in [58] is represented by a solid lines in (a) and (b). Reproduced
with permission of Ref. [56]. Copyright of ©2021 IEEE. 2.2.1. Impedance Transmission Boundary Condition Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 May 2023 doi:10.20944/preprints202305.0697.v1 7 of 19 Table 2. The Required DOF and CPU Time for the First Three Modes of GNW Calculated Using Mixed
FEM-SCBC and FEM At 30 THz, Respectively. Reproduced with Permission of Ref. [56]. Copyright of
©2021 IEEE. Mixed FEM-SCBC
FEM with graphene sheet
DOF
91 997
384 737
CPU time
31.4 s
252.2 s
2.3. Mixed Spectral Element Method With SCBC Mixed FEM-SCBC
FEM with graphene sheet
DOF
91 997
384 737
CPU time
31.4 s
252.2 s 2.3. Mixed Spectral Element Method With SCBC Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 May 2023 doi:10.20944/preprints202305.0697.v1 8 of 19 The multilayer nanoring open waveguide was truncated using a PML with 100 nm thickness,
and the entire simulation domain size was 1.2 µm × 1.2 µm. To obtain accurate simulation results for
the graphene sheets, conventional FEM requires a dense finite element mesh with 127 620 triangular
elements, yielding 893 421 unknowns. Instead, the mixed SEM-SCBC method substitutes SCBC for
graphene sheets in the computational domain. As a result, only 1449 quadrilateral elements are
produced across the entire domain, and only 17 528 of them are unknown. Figure 8a displays the
extremely fine meshes of graphene sheet, where at least one element is discretized in the graphene
sheet, while Figure 8b displays the quadrilateral elements that result from SCBC, which are relatively
coarse. It is evident that using SCBC instead of graphene sheets can significantly decrease on the
number of elements and degrees of freedom (DOF) required for numerical calculations. (a)
(b)
Figure 8. (a) Divide graphene sheets with thickness to produce extremely fine grids. (b) Replacing
graphene by using SCBC. In (a), the red line depicts graphene sheets, while in (b), it represents SCBC. Reproduced with permission of Ref. [64]. Copyright of ©2020 IEEE. (a) (b) (b) Figure 8. (a) Divide graphene sheets with thickness to produce extremely fine grids. (b) Replacing
graphene by using SCBC. In (a), the red line depicts graphene sheets, while in (b), it represents SCBC. Reproduced with permission of Ref. [64]. Copyright of ©2020 IEEE. The mixed SEM-SCBC is employed to simulate the numerical dispersion relationship of graphene
waveguides. Figure 9 shows the relationship between the real part of the effective refractive index (real
(Neff, Neff = kz/k0) and the operating frequency. When m = 0 is a fixed, the real part of the effective
refractive index varies with n, with mode (0, 1) being the lowest among modes (0, n). The propagation
lengths, represented by Lp = λ0/4π[Im(Neff)] in Figure 10, are calculated based on the operating
frequency. It can be observed that higher order modes experience more losses compared to lower order
modes, which is in line with the characteristics of propagation modes. In this figure, the numerical
results of traditional FEM are presented by –, – –, and · lines. 2.3. Mixed Spectral Element Method With SCBC The SEM which combines spectral method with FEM extended by Patera. Due to its unique basis
function construction method and node arrangement, it can achieve higher accuracy than the FEM, and
consumes less memory costs and computational time. For solving Stokes equations in fluid mechanics,
Patera took the lead in introducing spectral methods into calculations, resulting in better numerical
accuracy and convergence compared to traditional methods [59]. Since then, the SEM has been widely
used in numerical calculations simulation in the field of fluids. Later, pseudospectral method was
proposed by Chebyshev, and Liu incorporated the pseudospectral method into the FEM [60]. The SEM
has become a new and efficient tool for electromagnetic numerical computation. The mixed SEM possesses both spectral accuracy and the capability to eliminate zero spurious
eigenvalues [61]. The combination of SCBC and mixed SEM is utilized to analyze the modes of
graphene based pasmonic waveguides due to their enormous potential in eigenvalue solver. This
method employs a new variational formulation to suppress zero spurious modes by introducing
Gauss’ law to the vectorial Helmholtz equation. Additionally, an equivalent SCBC is used in the
computational domain to replace the nanoscale graphene sheets. A tunable multilayer nanoring waveguide consisting of six layers of graphene and seven layers
of dielectric is firstly considered. The graphene and dielectric layers are alternately distributed [62]. Figure 7 depicts the cross-section and three-dimensional structure of the waveguide. The innermost
ring radius is 140 nm, and the total ring radius is 210 nm. The innermost and outermost layers have a
thickness of 10 nm, while the rest layers and graphene sheets are 9.5 nm and 0.5 nm thick, respectively. The dielectric layer has a relative permittivity of 2.1. In this situation, the environment temperature
is T = 300, τ = 0.5 ps and µc = 0.5 eV. The relative dielectric constant of graphene is expressed as
εr = 2.5 −jσs/(ε0ωd) [63], where d represents the thickness of graphene. Figure 7. Structure of the tunable multilayer nanoring waveguide: cross section view (upper) and 3-D
view (lower). Reproduced with permission of Ref. [64]. Copyright of ©2020 IEEE. Figure 7. Structure of the tunable multilayer nanoring waveguide: cross section view (upper) and 3-D
view (lower). Reproduced with permission of Ref. [64]. Copyright of ©2020 IEEE. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 May 2023 The DOF, CPU Time, and Memory Costs Required for Calculations Using the Mixed SEM-SCBC
and Traditional FEM, Respectively. Reproduced with Permission of Ref. [64]. Copyright of ©2020 IEEE. Table 3. The DOF, CPU Time, and Memory Costs Required for Calculations Using the Mixed SEM-SCBC
and Traditional FEM, Respectively. Reproduced with Permission of Ref. [64]. Copyright of ©2020 IEEE. Mixed SEM-SCBC
FEM with graphene sheet
DOF
17528
893421
CPU time
13.8 s
434.5 s
Memory
0.06 GB
3.17 GB
2.4. The Method of Auxiliary Sources with IMBC Mixed SEM-SCBC
FEM with graphene sheet Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 May 2023 The proposed mixed SEM-SCBC method
has great consistency with the traditional FEM with graphene sheet, but with significantly lower CPU
time and memory costs (only 3% and 2%, respectively), as shown in Table 3. These results demonstrate
that the mixed SEM-SCBC can enhance computing speed and save computer resources. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 May 2023 doi:10.20944/preprints202305.0697.v1 9 of 19 30
32
34
36
38
40
Frequency [THz]
30
40
50
60
70
80
90
Real(Neff)
Mode (0, 3)
Mode (0, 2)
Mode (0, 1)
Figure 9. Real part of effective refractive index for different waveguide modes. The data of conventional
FEM are represented by the symbols -, – and ··. Reproduced with permission of Ref. [64]. Copyright of
©2020 IEEE. 30
32
34
36
38
40
Frequency [THz]
30
40
50
60
70
80
90
Real(Neff)
Mode (0, 3)
Mode (0, 2)
Mode (0, 1) Figure 9. Real part of effective refractive index for different waveguide modes. The data of conventional
FEM are represented by the symbols -, – and ··. Reproduced with permission of Ref. [64]. Copyright of
©2020 IEEE. 30
32
34
36
38
40
Frequency [THz]
0.8
1
1.2
1.4
1.6
1.8
2
2.2
2.4
2.6
Lp( m)
Mode (0, 3)
Mode (0, 2)
Mode (0, 1)
Figure 10. Propagation lengths of the different waveguide modes. The data of conventional FEM are
represented by the symbols -, – and ··. Reproduced with permission of Ref. [64]. Copyright of ©2020
IEEE. Table 3. The DOF, CPU Time, and Memory Costs Required for Calculations Using the Mixed SEM-SCBC 30
32
34
36
38
40
Frequency [THz]
0.8
1
1.2
1.4
1.6
1.8
2
2.2
2.4
2.6
Lp( m)
Mode (0, 3)
Mode (0, 2)
Mode (0, 1) Figure 10. Propagation lengths of the different waveguide modes. The data of conventional FEM are
represented by the symbols -, – and ··. Reproduced with permission of Ref. [64]. Copyright of ©2020
IEEE. Table 3. The DOF, CPU Time, and Memory Costs Required for Calculations Using the Mixed SEM-SCBC
and Traditional FEM, Respectively. Reproduced with Permission of Ref. [64]. Copyright of ©2020 IEEE. Table 3. The DOF, CPU Time, and Memory Costs Required for Calculations Using the Mixed SEM-SCBC
and Traditional FEM, Respectively. Reproduced with Permission of Ref. [64]. Copyright of ©2020 IEEE. Table 3. 2.4. The Method of Auxiliary Sources with IMBC The
field of the l-th source produced in region m(m = 1, 2) is expressed as: Eml(x, y) = −ˆzkmZm
4
ImlH(2)
0 (kmρml)
(7) (7) Hml(x, y) = km
4i Iml
ˆx(yml −y) + ˆy(x −xml)
ρml
× H(2)
1 (kmρml)
(8) (8) for the TMz case, and Hml(x, y) = −ˆz km
4Zm
KmlH(2)
0 (kmρml)
(9)
Eml(x, y) = km
4i Kml
ˆx(yml −y) + ˆy(x −xml)
ρml
× H(2)
1 (kmρml)
(10) Hml(x, y) = −ˆz km
4Zm
KmlH(2)
0 (kmρml)
(9)
km K
ˆx(yml −y) + ˆy(x −xml)
H(2)(k
)
(10) Hml(x, y) = −ˆz km
4Zm
KmlH(2)
0 (kmρml)
(9) (9) Eml(x, y) = km
4i Kml
ˆx(yml −y) + ˆy(x −xml)
ρml
× H(2)
1 (kmρml)
(10) (10) for TEz polarization, where (xml, yml) denotes filament coordinates in region m, ρml denotes the
separation between the observation point (x, y) and the l-th filament in region m, Iml and Kml denote
the current and magnetic current amplitudes of the l-th filament in region m, km = ω
p
µm/εm and
Zm =
p
µm/εm denote the wave number and impedance of region m, respectively. Then, the 2-D-IMBC approach is applied to M matching pairs. This is done to associate the fields
at specific points on boundary a with the fields at corresponding points on boundary b. "
ˆna × (ˆna × Ea)
ˆnb × (ˆnb × Eb)
#
=
−ZS
q
rb
ra Ztr
−
q
ra
rb Ztr
ZS
"
ˆna × Ha
ˆnb × Hb
#
(11) (11) where Ztr = −iZ3csc(k3△) and Zs = −iZ3cot(k3△) are the transfer and surface impedance, k3 and
Z3 denote the wavenumber and characteristic impedances of the conductive layer, the curvature radii
on boundaries a and bare ra and rb, respectively. The appendix of [73] shows that ˆna = ˆn = ˆnb and
ra = △+ rb for uniformly thick conductive layers. Figure 12 studies a multi-layer cylindrical medium with a circular square cross-section. Wherein
region 3 is a graphene monolayer, region 2 is a silicon dioxide (SiO2) layer, air is filled in both regions
1 and 4; thus, ε1 = ε4 = ε0 and ε2 = 3.9 ε0. The surface conductivity of graphene with EF = 0.5 eV,
τ = 6.582 ps, T = 300 K and △= 0.5 nm is utilized. 2.4. The Method of Auxiliary Sources with IMBC 2.4. The Method of Auxiliary Sources with IMBC The electromagnetic waves are incident on a conductive layer that separates the layered medium
in a normal shielding configuration. The conductive layer can block the incident field and act as an
electromagnetic shield. The planar, cylindrical, and spherical interfaces are the most common layered
media models used to demonstrate electromagnetic shielding. However, for the high conductivity of
interior materials, the conventional MAS [67,68] approach will yield divergent results. In Figure 11,
if the standard MAS described in [69,70] is used to calculate the shielding effect, a convergent field
cannot be obtained within the highly conductive layer. Therefor, a modified version of MAS was
proposed in [71] to improve by associating the field at each point of boundary a with the electric field
at the corresponding point of boundary b. This effect can be achieved by replacing the conductive
layer with the IMBC. The shielding effectiveness of a single layer of graphene covering a cylindrical
structure can be calculated using this modified method. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 May 2023 doi:10.20944/preprints202305.0697.v1 10 of 19 10 of 19 Figure 11. Using MAS and 2-D IMBC in multi-layer cylindrical media containing conductive layers. Reproduced with permission of Ref. [72]. Copyright of ©2022 IEEE. Figure 11. Using MAS and 2-D IMBC in multi-layer cylindrical media containing conductive layers. Reproduced with permission of Ref. [72]. Copyright of ©2022 IEEE. By superposing the fields emitted by N1 and N2 filaments set on the auxiliary surfaces C1
aux and
C2
aux, it is possible to replicate the internal field in region 2 and the scattering field in region 1. The N1
and N2 filaments, which radiate in the dielectric-filled unbounded space of regions 1 and 2, respectively,
and carry unknown currents I1l(K1l), l = 0, ..., N1 −1 and I2l(K2l), l = 0, ..., N2 −1, respectively. 2.4. The Method of Auxiliary Sources with IMBC The relationship between the shielding effectiveness of frequency f with EF = 0.5 eV in
Figure 12. Left: TM-polarization. Right: TE polarization. Reproduced with permission of Ref. [72]. Copyright of ©2022 IEEE. Figure 14. The relationship between the shielding effectiveness of frequency f with EF = 0.5 eV in
Figure 12. Left: TM-polarization. Right: TE polarization. Reproduced with permission of Ref. [72]. Copyright of ©2022 IEEE. Figure 15. For f = 3 GHz, the relationship between shielding effectiveness and chemical potential EF
in Figure 12. Left: TM polarization. Right: TE polarization. Reproduced with permission of Ref. [72]. Copyright of ©2022 IEEE. Figure 15. For f = 3 GHz, the relationship between shielding effectiveness and chemical potential EF
in Figure 12. Left: TM polarization. Right: TE polarization. Reproduced with permission of Ref. [72]. Copyright of ©2022 IEEE. Figure 16. Norm of the magnetic field for the TM polarization in Figure 12 is expressed in V/m with
f = 3 GHz and EF = 0.5 eV. Reproduced with permission of Ref. [72]. Copyright of ©2022 IEEE. Discontinuous Galerkin Time-Domain Method With SIBC Figure 16. Norm of the magnetic field for the TM polarization in Figure 12 is expressed in V/m with
f = 3 GHz and EF = 0.5 eV. Reproduced with permission of Ref. [72]. Copyright of ©2022 IEEE. 5. Discontinuous Galerkin Time-Domain Method With SIBC Figure 16. Norm of the magnetic field for the TM polarization in Figure 12 is expressed in V/m with
f = 3 GHz and EF = 0.5 eV. Reproduced with permission of Ref. [72]. Copyright of ©2022 IEEE. Figure 16. Norm of the magnetic field for the TM polarization in Figure 12 is expressed in V/m with
f = 3 GHz and EF = 0.5 eV. Reproduced with permission of Ref. [72]. Copyright of ©2022 IEEE. 5. Discontinuous Galerkin Time-Domain Method With SIBC 2.5. Discontinuous Galerkin Time-Domain Method With SIBC The discontinuous Galerkin time-domain method [34,74] is a versatile approach for solving
differential equations in numerous fields such as computational science, engineering, and physics. It
incorporates the advantages of the Finite Volume Method (FVM) [75,76] and the FEM, enabling mesh
discretization of the computational domain. The spatial DGTD operations, like FVM, are localized,
and the global mass matrix can be transformed and divided into a block diagonal mass matrix. The
mass matrix block’s inversion and storage [77] are performed before initiating time marching. 2.4. The Method of Auxiliary Sources with IMBC Boundary b has collocation points with the
following coordinates: xb = 15 sgn(cos θ)|cos θ|
3
2
yb = 15 sgn(sin θ)|sin θ|
3
2
(12) (12) Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 May 2023 doi:10.20944/preprints202305.0697.v1 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 May 2023 doi:10.20944/preprints202305.0697.v1 11 of 19 Figure 12. Using MAS and 2-D IMBC in an rounded squared multilayered cylindrical medium
containing a graphene monolayer. Reproduced with permission of Ref. [72]. Copyright of ©2022 IEEE. Figure 12. Using MAS and 2-D IMBC in an rounded squared multilayered cylindrical medium
containing a graphene monolayer. Reproduced with permission of Ref. [72]. Copyright of ©2022 IEEE. A TMz or TEz plane wave incident the computational domain. There are four sets of
auxiliary sources N1 = N2 = N3 = N4 = 110, each placed on coordinate sources (xa_1, ya_1) =
0.8(xb, yb), (xa_2, ya_2)
=
2(xb, yb), (xa_3, ya_3)
=
0.8(xc, yc) and (xa_4, ya_4)
=
1.2(xc, yc) of
C1
aux, C2
aux, C3
aux, and C4
aux. Applying the boundary condition Equation (10) to a and b on the
M = 110 matching pair. The norm of the fields in regions 1 and 4 with (xo_1, yo_1) = 2(xb, yb)
and (xo_2, yo_2) = 0.6(xc, yc) are described in Figure 13. It is also shown that the modified MAS in
good agreement with the finite element calculation results of COMSOL. The relationship between the
shielding effect with EF = 0.5 and f, and the relationship between the shielding effect with f = 3 GHz
and EF are presented in Figure 14 and Figure 15, respectively. The modified MAS not only matches the
finite element calculation results, but also has advantages in memory usage. The shielding effect is
shown in Figure 16. Figure 13. The norm of the fields in regions 1 and 4 in Figure 12. TM polarization is represented by (a)
and (b), and TE-polarization is represented by (c) and (d). Reproduced with permission of Ref. [72]. Copyright of ©2022 IEEE. Figure 13. The norm of the fields in regions 1 and 4 in Figure 12. TM polarization is represented by (a)
and (b), and TE-polarization is represented by (c) and (d). Reproduced with permission of Ref. [72]. Copyright of ©2022 IEEE. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 May 2023 doi:10.20944/preprints202305.0697.v1 12 of 19 Figure 14. 2.4. The Method of Auxiliary Sources with IMBC This
makes the solver of DGTD very compact, especially when using explicit integration methods. These
characteristics make DGTD an excellent method for simulating multi-scale electromagnetic fields in
two-dimensional materials. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 May 2023 doi:10.20944/preprints202305.0697 [78]. Copyright of ©2018 IEEE. Figure 20. The calculated impedance matrix element Z21. (a) Real part of Z21. (b) Imaginary part of
Z21. Reproduced with permission of Ref. [78]. Copyright of ©2018 IEEE. i
l
i
i
G l ki
i
i
h d Wi h
C Figure 19. The calculated impedance matrix element Z11. (a) Real part of Z11. (b) Imaginary part of
Z11. Reproduced with permission of Ref. [78]. Copyright of ©2018 IEEE. Figure 19. The calculated impedance matrix element Z11. (a) Real part of Z11. (b) Imaginary part of
Z11. Reproduced with permission of Ref. [78]. Copyright of ©2018 IEEE. Figure 20. The calculated impedance matrix element Z21. (a) Real part of Z21. (b) Imaginary part of
Z21. Reproduced with permission of Ref. [78]. Copyright of ©2018 IEEE. Figure 20. The calculated impedance matrix element Z21. (a) Real part of Z21. (b) Imaginary part of
Z21. Reproduced with permission of Ref. [78]. Copyright of ©2018 IEEE. Figure 20. The calculated impedance matrix element Z21. (a) Real part of Z21. (b) Imaginary part of 2.6. Interior Penalty Discontinuous Galerkin-Time Domain Method With ITBC The IPDG, a DG method, is also a good method for solving electromagnetic problems, including
modeling graphene as an infinitely thin impedance surface using ITBC [79], which minimizes memory
usage and computation time. Equation (1) expresses the surface conductivity as a frequency-domain expression, which is
difficult to solve in time domain. To overcome this, the vector-fitting technique [80] is employed to
approximate σg as the sum of partial fractions of the extreme residual pairs, either in the form of real
or complex conjugate, on the frequency band. Equation (1) is modified as: σg =
N
∑
l=1
σl =
N
∑
l=1
rl
jω −pl
(14) (14) where N is the total number of poles and pl and rl are residuals and poles, respectively. Assuming
a computational domain Grammar which has boundary Γb. The entire computational domain is
discretized into multiple non-overlapping tetrahedrons, with Γinterior representing all inner surfaces
and ΓITBC representing the inner surfaces attached to the graphene surface. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 May 2023 doi:10.20944/preprints202305.0697 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 May 2023 doi:10.20944/preprints202305.0697.v1 doi:10.20944/preprints202305.0697.v1 13 of 19 Since the jumping depth of graphene is much greater than the thickness of the graphene layer, the
SIBC can be utilized to replace the graphene layer, which can be expressed as: ˆn × (E2 −E1) = 0
σ−1
g
· [ˆn × (H2 −H1)] = σ−1
g
· J = E
(13) (13) where E1 and E2 indicate the total electric field intensity on each side of the graphene layer, respectively. H1 and H2 represent the total magnetic field intensity on each side of the graphene layer, respectively. ˆn indicates the unit normal vector above the graphene layer, pointing outwards from the plane. J is
the induced polarization current density in the graphene layer and E represents the intensity of the
electric field. A graphene nano-ribbon transmission line is simulated and the electromagnetic field interaction
on graphene nano-ribbon (GNR) located on silicon substrate is analyzed. As is shown in Figure 17,
the GNR dimension is 0.3 µm × 3 µm × 0.05 µm. Port 1 is the current source, and both non-local and
local conductivity models are simulated. Time-voltage is recorded at ports 1 and 2 of the nanobelt
transmission line in two simulation models (see Figure 18). Figures 19 and 20 show the relationship
between Z11 and Z21 with frequency. Results in the time domain differ significantly from those in the
frequency domain, especially in the high-end terahertz band. Figure 17. Schematic of the GNR transmission line placed on a substrate. Reproduced with permission
of Ref. [78]. Copyright of ©2018 IEEE. Figure 17. Schematic of the GNR transmission line placed on a substrate. Reproduced with permission
of Ref. [78]. Copyright of ©2018 IEEE. Figure 18. Using local or non-local conductive models to measure the voltage at the port of a
transmission line over time. (a) Port 1. (b) Port 2. Reproduced with permission of Ref. [78]. Copyright
of ©2018 IEEE. Figure 18. Using local or non-local conductive models to measure the voltage at the port of a
transmission line over time. (a) Port 1. (b) Port 2. Reproduced with permission of Ref. [78]. Copyright
of ©2018 IEEE. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 May 2023 doi:10.20944/preprints202305.0697.v1 14 of 19 Figure 19. The calculated impedance matrix element Z11. (a) Real part of Z11. (b) Imaginary part of
Z11. Reproduced with permission of Ref. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 May 2023 doi:10.20944/preprints202305.0697 The enhanced IPDG
method using ITBC can be reformulated as: Z
Ωε∂2 ˜E
∂t2 · Ni dr +
Z
Ωµ−1∇× ˜E · ∇× Ni dr −
Z
ΓITBC
JS ·
n
Ni
o
dS
−
Z
Γinterior
n
˜E∗−˜E
o
·
hh
µ−1∇× Ni
ii
T dS −
Z
Γinterior
n
(µ−1∇× ˜E)∗o
·
hh
Ni
ii
T dS
+
Z
Γinterior
hh
˜E∗−˜E
ii
T ·
n
µ−1∇× Ni
o
dS +
Z
Γinterior
hh
(µ−1∇× ˜E)∗ii
T ·
n
Ni
o
dS
+
Z
Γb
n × (µ−1∇× ˜E)∗· Ni dS +
Z
Γb
n × ( ˜E∗−˜E) · µ−1∇× Ni dS = 0
(15) (15) Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 10 May 2023 doi:10.20944/preprints202305.0697.v1 15 of 19 where n indicates the unit vector, which points towards the graphene sheet. ˜E = ∂−1
t
E is expanded
with vector basis function Ni. ·
T represents tangential jump,
·
represents the average value on
the face f of an element, and the superscript "*" represents numerical flux. Assuming an infinite graphene sheet, the x direction boundary is taken as PEC while PMC is
considered as the boundary in the y direction. The truncated boundary is a first-order ABC in the z
direction . The entire domain size is 10.5 µm ×2.5 µm ×2.5 µm, and a plane wave source is created
using total/scattered field boundary conditions. Unfolding unknown fields using full two vector basis
functions, with a time step size of △t is 5e−17 s. The graphene parameter µc is estimated to be 0.15 eV. Figure 21. Comparison of reflection and transmission coefficients calculated by IPDG method with
analytical solutions. Reproduced with permission of Ref. [39]. Copyright of ©2019 IEEE. Figure 21. Comparison of reflection and transmission coefficients calculated by IPDG method with
analytical solutions. Reproduced with permission of Ref. [39]. Copyright of ©2019 IEEE. 3. Conclusions In the past decade, due to its excellent optical properties, thermal, mechanical and electrical,
widespread attention has been attracted on graphene, papers on the physical mechanisms and
applications of graphene in various fields have been constantly updated. In this work, we reviewed
some of the most commonly used algorithms for simulating graphene in the CEM field. The mixed
FEM-ITBC and mixed FEM-SCBC and mixed SEM-SCBC are employed to solve the graphene-based
plasmonic waveguide problem. The improved auxiliary source method with IMBC is used to calculate
the shielding effect, which can obtain a convergence field in a high conductive layer; The DGTD
method with SIBC is also an effective method for exploring the spatial dispersion characteristics of
graphene. The IPDG-ITBC to model graphene, saving memory and usage time. All these equivalent
boundary conditions have been proposed and significantly saved computational time and memory
costs. With all of these performances and improvements, we anticipate that equivalent boundary
conditions are promising techniques to simulate electromagnetic response of graphene. 1.
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of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s)
disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or
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https://openalex.org/W1972504991
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https://thescipub.com/pdf/jcssp.2013.114.121.pdf
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AN EVALUATION OF THE INFORMATION SYSTEMS FOR TEACHING AND LEARNING ONLINE OF THE PUBLIC UNIVERSITY
|
Journal of computer sciences/Journal of computer science
| 2,013
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cc-by
| 5,930
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ABSTRACT This sign up service is only
necessary for lecturers and professors who are interested in
creating an online classroom for their subjects through the
website. As a professor, there is an infinite amount of
document creation and subject creation. Simultaneously,
they can also select the group of students who will be
eligible to visualize and access information in this online
service classroom. The FAQ is the additional information
for some frequently asked questions. On the students’
interface, active member students can access the class
activity details, courses outline, download the uploaded
documents and share their suggestions in class the same
way
Facebook
shares
the
status
online. More Journal of Computer Science, 9 (1): 114-121, 2013
ISSN 1549-3636
© 2013 Pattanasethanon and Chanthinok, This open access article is distributed under a Creative Commons Attribution
(CC-BY) 3.0 license
doi:10.3844/jcssp.2013.114.121 Published Online 9 (1) 2013 (http://www.thescipub.com/jcs.toc) Journal of Computer Science, 9 (1): 114-121, 2013
ISSN 1549-3636
© 2013 Pattanasethanon and Chanthinok, This open access article is distributed under a Creative Commons Attribution
(CC-BY) 3.0 license
doi:10.3844/jcssp.2013.114.121 Published Online 9 (1) 2013 (http://www.thescipub.com/jcs.toc) Journal of Computer Science, 9 (1): 114-121, 2013
ISSN 1549-3636
© 2013 Pattanasethanon and Chanthinok, This open access article is distributed under a Creative Commons Attribution
(CC-BY) 3.0 license
doi:10.3844/jcssp.2013.114.121 Published Online 9 (1) 2013 (http://www.thescipub.com/jcs.toc) 1. INTRODUCTION Corresponding Author: Petcharat Pattanasethanon, Department of Computer Business, Faculty of Accountancy and Management,
Mahasarakham University Mahasarakham, Thailand
1. INTRODUCTION
Information system has been engaged in major parts of
our society such as the educational system, the security
system, banking system. The rapid change in a form of
information system has influenced our daily lives in various
ways (Agre, 2002). Knowledge is not restricted only in the
classrooms anymore. It can approach us anywhere and
everywhere. The education values in the teaching and
learning activity via the Internet by students, instructors and
classmates can be consulted, shared and exchanged between
one and another, which is similar to a regular classroom but
in a different form of information delivery (Son, 2008). In
addition, by using modern communication and technology,
learning and knowledge management is the most cost-
effective and limitless form of communication. This
research introduces the use of information system
technology in public Universities, as they are government
organizations
that
literally
provide
education
and
manipulate media as a tool to educate both teachers and
learners (Oblinger and Oblinger, 2005). This research
composes of two major purposes of information system
aspects. It develops the system to become the media center
for professors and students in terms of efficiency in
information delivery as well as study how to make this
system work properly and appropriately for both professors
and students. The domain www.forstd.net is the main key to
access this online classroom. This sign up service is only
necessary for lecturers and professors who are interested in
creating an online classroom for their subjects through the
website. As a professor, there is an infinite amount of
document creation and subject creation. Simultaneously,
they can also select the group of students who will be
eligible to visualize and access information in this online
service classroom. The FAQ is the additional information
for some frequently asked questions. On the students’
interface, active member students can access the class
activity details, courses outline, download the uploaded
documents and share their suggestions in class the same
way
Facebook
shares
the
status
online. More Computer Business, Faculty of Accountancy and Management,
h il
d
learners (Oblinger and Oblinger, 2005). This research
composes of two major purposes of information system
aspects. Science Publications Petcharat Pattanasethanon and Kriangsak Chanthinok Department of Computer Business,
Faculty of Accountancy and Management, Mahasarakham University, Thailand Received 2013-01-06, Revised 2013-02-16; Accepted 2013-03-11 Received 2013-01-06, Revised 2013-02-16; Accepted 2013-03-11 ABSTRACT This research aims to evaluate the online information system for instructors and students at the
undergraduate level of public Universities in Thailand. Tools and data that are collected in this research
consist of two parts. The first part is the creation of a web application to educate teachers and students
online, while the second part is to create a questionnaire as a tool for assessing the satisfaction of using the
online surveys and a questionnaire prepared directly for the students. From collecting over 496 samples of
analysis, we found that most of the students and professors that use the online information system in this
group are highly satisfied. Overall, the level of significance at 0.5 was obtained from this study, which can
be used as a guide for evaluating other different angle of educational activities in the future. Keywords: Evaluation, Information System Online, Web Application, Acceptance Behavior Corresponding Author: Petcharat Pattanasethanon, Department of Computer Business, Faculty of Accountancy and Management,
Mahasarakham University Mahasarakham, Thailand
y
,
y
,
pp
,
p
1. INTRODUCTION
Information system has been engaged in major parts of
our society such as the educational system, the security
system, banking system. The rapid change in a form of
information system has influenced our daily lives in various
ways (Agre, 2002). Knowledge is not restricted only in the
classrooms anymore. It can approach us anywhere and
everywhere. The education values in the teaching and
learning activity via the Internet by students, instructors and
classmates can be consulted, shared and exchanged between
one and another, which is similar to a regular classroom but
in a different form of information delivery (Son, 2008). In
addition, by using modern communication and technology,
learning and knowledge management is the most cost-
effective and limitless form of communication. This
research introduces the use of information system
technology in public Universities, as they are government
organizations
that
literally
provide
education
and
manipulate media as a tool to educate both teachers and
learners (Oblinger and Oblinger, 2005). This research
composes of two major purposes of information system
aspects. It develops the system to become the media center
for professors and students in terms of efficiency in
information delivery as well as study how to make this
system work properly and appropriately for both professors
and students. The domain www.forstd.net is the main key to
access this online classroom. 1. INTRODUCTION It develops the system to become the media center
for professors and students in terms of efficiency in
information delivery as well as study how to make this
system work properly and appropriately for both professors
and students. The domain www.forstd.net is the main key to
access this online classroom. This sign up service is only
necessary for lecturers and professors who are interested in
creating an online classroom for their subjects through the
website. As a professor, there is an infinite amount of
document creation and subject creation. Simultaneously,
they can also select the group of students who will be
eligible to visualize and access information in this online
service classroom. The FAQ is the additional information
for some frequently asked questions. On the students’
interface, active member students can access the class
activity details, courses outline, download the uploaded
documents and share their suggestions in class the same
way
Facebook
shares
the
status
online. More Corresponding Author: Petcharat Pattanasethanon, Departmen
Information system has been engaged in major parts of
our society such as the educational system, the security
system, banking system. The rapid change in a form of
information system has influenced our daily lives in various
ways (Agre, 2002). Knowledge is not restricted only in the
classrooms anymore. It can approach us anywhere and
everywhere. The education values in the teaching and
learning activity via the Internet by students, instructors and
classmates can be consulted, shared and exchanged between
one and another, which is similar to a regular classroom but
in a different form of information delivery (Son, 2008). In
addition, by using modern communication and technology,
learning and knowledge management is the most cost-
effective and limitless form of communication. This
research introduces the use of information system
technology in public Universities, as they are government
organizations
that
literally
provide
education
and
manipulate media as a tool to educate both teachers and Science Publications JCS 114 Petcharat Pattanasethanon and Kriangsak Chanthinok / Journal of Computer Science 9 (1): 114-121, 2013 importantly, class’s homework submission and request
for appointment with the lecturer can be done online
(Gregg et al., 2009). The subsequences of this system are
that students tend to pay more attention in regular classes
and due to the convenient access of this information
system, most students are more independent and capable
of revising lessons on their own. 1. INTRODUCTION Meanwhile, lecturers
themselves are more deliberate and it is easier for them
to plan the syllabus (Muir-Herzig, 2004). They also can
upload what has been left out in class to make sure that
the lessons are equally delivered in every section. The
second part of this research is the questionnaire and
evaluation part. This part creates a survey that allows
both
instructors
and
student
to
complete
the
questionnaire regarding the system. The total of 496
individuals from Mahasarakham University (2012) have
manifests that this system is highly satisfied and that it is
effective and appropriate for their needs. system, conditions that will support/facilitate the
progress (user’s usage), trustworthy of the user toward
the system and the objective. These impacts are the habit
of acknowledging technology and intentions of the user
that has been develop on the Unified Theory of
Acceptance and Use of Technology (UTAUT theory). It
includes 8 other theories (Davis, 1989) before it is
developed. The suggestion in this research is the habitat
of approving the online information system of instructors
and learners in the higher level, which the opinion may
manifests profoundly based on the individual’s thought
toward this online system. Other researches included in
this study are described below. The objective of this
research is to study the problems and needs for
communicating of administrators and professors in the
royal Thai air force (Onkhao, 1999). The air education
and
training
command
sample
consist
of
52
administrators and 56 professors of the royal Thai air
force. The air command center receives complete
questionnaires of 108 sets to study with this research,
which is calculated in percentage. The research found
that the environment that the manager and professor uses
are as follows; most of the teaching methods that were
used are lectures, discussions, invited lecturer from other
agencies and teaching seminars. Most tools in the
teaching lessons are overhead projectors and video
projectors. While most materials used in this lesson are
documents about the lecture, books and picture. However, the problem occurred while using this teaching
were the appropriate materials, option of the materials
and the dysfunctional ties of the tools and graphical
managements. Sawasdiyakorn (1998), has aim to explore
the problem and demand of using the information system
in the undergraduate level of professor at Arts
department institution. Sumak et al. (2010), had studied
about an acceptance of virtual learning by using the
UTAUT model. 1. INTRODUCTION This research has used the UTAUT
theory to study about the needs and emotions of student
that had used the Moodle program, this project consists
the total of 235 undergraduates individual. The results
indicated that the indicator of capability and social power
affects
the
students’
attitude
to
used
Moodle
significantly. AlAwadhi and Morris (2008) had studied
the UTAUT theory in accepting the E-government of
Kuwait country. This research has used the UTAUT
theory to apply for studying the factor in managing the
E- government of Kuwait, by exploring through 880
people. The research discovers that the capability,
expectation and attempt affect the usage of E-
government. Khongmalai and Poungmali (2009),
studied about factors that influence the purpose of E-
learning, which affects the analysis and comparison of The aim of this research is to evaluate the online
information system of instructors and students. After
developing the online information system for 3 years, we
found out that the population of this system increases
annually, which indicates that the online information
system have taken an important role in our lives and it
includes guidelines for lecturer to gain confidence in
creating and providing the diversity of education through
certain aspects of learning behavior. This system
influences students to study with their maximum
potential and encourages them to be engaged in
individual learning process. Since, self- study is a
requirement and characteristic that the students in the
modern days should have, if that student is already eager
to learn and they were to be practiced and trained
seriously, the knowledge they obtained would last their
whole lives. Online information system is an innovative
education that is expected to be creative. The creation of
other new academic subject is designed to satisfy the
needs of student and lecturer to raise the academic
standard and the quality of education system at
Mahasarakham University and other institution in the
country to achieve their goal effectively and efficiently. Science Publications 2.1. Population Sampling Population sample is denoted through students and
instructors from Mahasarakham University year 2555,
with the total of 133 instructors and 9320 students. The
sample group in this research is 450 students and 46
lecturer of the MSU as described in Table 1 below. 2.2. Building Tools for Storing Data A research questionnaire assesses the use of
information technology in teaching and learning online. It is generated according to the methods of questionnaire
and the accuracy of the tools. Two experts will have to
consider and determine based on the content validity. The appropriateness of the language used is analyzed
through the IOC and the selection is more than or
equal to 0.5. 1.1. Relative Works The conception of information system has 2
variables; the instructors’ and students’ statuses. These
statuses have an impact on the six-part evaluation of the
online information system for instructors and students,
which consists of the expectations about the performance
of the online information system, expectation about the
users effort, attitude toward the use of online information JCS 115 Petcharat Pattanasethanon and Kriangsak Chanthinok / Journal of Computer Science 9 (1): 114-121, 2013 students from each instructor in the classroom, which
the details are as listed below. learners and developers by using UTAUT theory. As a
consequence, studying through E-learning in working
places is acceptable and holds a potential growth in
terms of population (Wagner et al., 2008). 2. MATERIALS AND METHODS In the first part, this research has developed the
information system to browse online by registering
the domain name “www.forstd.net” and has developed
into a web application for students and teachers inside
the
faculty
of
accounting
and
management
in
Mahasarakham University, Thailand. The structures are
shown in the picture below. Table 1 represent the data of population and sample
groups by dividing into 2 parts, which includes 133
instructors and 9320 students. A sample calculation for a
confidence level of 95 percent and 5 percent deviation
equal to the number of sample is 46 teachers and 496
students in the sample were men. Figure 1 manifests the development model of the
online information system. We have created this website
as a center of media for the instructor to registered. The
domain is then registered for using as a reference to other
website that is on the network. The name www.forstd.net
means networking and collaborative learning for students
and instructors. And by registering for the domain, it also
comes with renting a web server or hosting for storing
files and data under the Linux operating system. This
implies that it has the capability to request and response
efficiently. Meanwhile, the researchers also use PHP
language in developing the system and mySql as a
database for storing data of registered members and other
important data such as subject details, class documents,
FAQ and transferred data, which these database can be
add, remove, edit and display on the website. The
principle of creating the online information system of
each individual may be dissimilar in accordance with the
agenda of the users and must be unique to those who
already have registered. Each instructor will receive a
URL: uniform resource locator’s, according to the user’s
himself. It must not be similar to the registered ones. This is done by creating the folders of each individual for
representing their pictures, documents, Question and
Answers of each person individually, in spite registering
for the same service and website. Science Publications 2.3. Data Analysis Data analysis is the statistical analysis of survey
research data to personalize the percentage of gender,
age, social status and the number of users in information
systems. The questionnaires used in evaluating the
information system in teaching and learning online are
analyzed for the mean (average), standard deviation
(One-Way Analysis of Variance) and the ANOVA (F-test)
where they are used as a query tool with message options
to choose upon the estimation standard (rating scale) up
to 5 levels as described below: Figure 2 is the actual application of the model build
in reference of the first picture. It is open to all
students, teachers and ordinary viewers to test this
system by just registering the user. Users will receive
an extra storage in the website to present varieties of
learning and teaching materials. The second part is the
evaluation of the online information system from guest
that enters the website “www.forstd.net.” Both students
and instructors are required to answer some online
questionnaires. The questionnaires are made directly to 6
JCS
Table 1. The amount of population and sample groups
Status
Population
Sample group
Instructors
133
46
Students
9320
450
Total
9453
496 116 Petcharat Pattanasethanon and Kriangsak Chanthinok / Journal of Computer Science 9 (1): 114-121, 2013 Fig. 1. Information system model for online classroom instructions Fig. 1. Information system model for online classroom instructions
Fig. 2. The outline of the application according to the model Fig. 1. Information system model for online classroom instructions Fig. 1. Information system model for online classroom instructions Fig. 2. The outline of the application according to the model •
Instructors can inspect the number of documents that
to be used and manifested online Science Publications 3. RESULTS AND DISCUSSION The
Contaminant errors of the population displayed in terms
of Sum Square (SS) and Means Square (MS) has a value
of 0.613 and 0.306, respectively. The results of the
variance in the four groups with subgroups are more
likely to agree with the large extent, where “P” is the
index value of 0.06 as the “F” index is 2.7 and for the SS
and MS has a value of 0.055 and 0.013, respectively. Table 5 is the comparison of statistical index for the
acceptability behavior in using the information system
technology in teaching and learning of the instructor’s
group. The index “P” has the value of “0”, which means
that the population in the teacher’s group accepts and
supports the access of information system in teaching
and learning (highly). While the Index “F” is the values
of comparing variance of two groups. Group (df) with
the values of 59.78 implies that the populations in each
group are independent and are directly related to the
evaluation of accepting the information system with
education behavior. The overall performance shows
significant changes in the statistic as 0.5. The
Contaminant errors of the population displayed in terms
of Sum Square (SS) and Means Square (MS) has a value
of 0.613 and 0.306, respectively. The results of the
variance in the four groups with subgroups are more
likely to agree with the large extent, where “P” is the
index value of 0.06 as the “F” index is 2.7 and for the SS
and MS has a value of 0.055 and 0.013, respectively. Table 2 displays the summary from the sample group
of instructors and students that has been handed the
survey. Overall, most of the instructors’ gender is female
that has up to 80.5 percent in total, while the other 19.5
are male. However, the age group in this survey varies
among participation sample. At ages of 31-40 years,
there are about 18 participants or approximately 39.1
percent in total, while from ages 41-50, 9 more than
participants are active in the 30s, which is 58.7 percent. On a contrary, from ages 51-60, only one participant or
2.2 as a percentage is active. In addition, another factor
that is hardly ignored is the number of access in the
system. 3. RESULTS AND DISCUSSION •
Grading papers for students is available 24/7 The result in the first part is about the ability of the
information system through teaching and learning online
as described and listed as follow: g p p
•
Instructors and students can do a live chat through
the system •
Students can view, download and subscribe
documents from their instructors •
The instructor has their own website in the online
information system server within a minute by just
registering into the server and should not be the
same as the previous registered user •
Digital file types are eligible for submission through
the upload section p
•
Students are able to suggest, question and raise
concerns to instructors or expertise in certain fields
to answer it •
Instructors are provided with an opportunity to
publish the documents in the system and create a
course that teaches students diversely •
Students are free to study and revise lessons
independently 117 JCS Petcharat Pattanasethanon and Kriangsak Chanthinok / Journal of Computer Science 9 (1): 114-121, 2013 Table 2. The evaluation of our system through questionnaires
based on the users’ social status, age, gender and
access frequency
Instructor
Student
Social
---------------------
---------------------
status
Amount Percent
Amount Percent
Gender
Male
9
19.50
98
21.73
Female
37
80.50
352
78.27
Age
Under 20
-
-
207
46.00
years old
Age 21-30
-
-
243
54.00
Age 31-40
18
39.10
-
-
Age 41-50
27
58.70
-
-
Age 51-60
1
2.20
-
-
Age above
-
-
-
-
61 years old
Frequency Less than
-
-
-
-
of access
once a week
Daily
-
-
-
-
Once a week
-
-
-
-
Twice a week
-
-
67
14.89
3 days/ week
9
19.57
95
21.11
4 days/ week
12
26.08
88
19.56
5 days/ week
19
41.31
146
32.44
6 days/ week
6
13.04
54
12.00
Total
46
100.00
450
100.00 Table 2. The evaluation of our system through questionnaires
based on the users’ social status, age, gender and
access frequency years of age. Obviously, 32.44% claimed that he
frequency of their access is as much as 5 days per week,
while 12% of students manifest the frequent of their
access at 6 days per week. 3. RESULTS AND DISCUSSION In Table 3, the analysis of accepting behavior of
instructor and student in the public university shows the
standard deviation and satisfactory level according to the
users’ status. It can be implied that the overall
performance is acceptable in a high level of satisfaction. The mean(average) of instructors is 4.25 and the
students’ mean is 4.26, which implies that the
information system for online education has satisfy the
instructor and student profusely and also the recognition
of the online information system helps improving the
education quality effectively. From Table 4, we can interpret the table as a
summary of comparing the behavior of the online
information system acceptance from the survey. The
survey is divided into 5 sections, which considers the
index of “F” (F-test) and index “P” (P-test).The index f
is used to compare the ratio of the variance of each of the
different questions that affect the behavior of the
system’s online activities. The data for each group’s
question will have the “F” index that is between 0.71 to
1.67, which means that the statistic of satisfy opinion is
high. And based on the result of the query, it has the
same direction and independent variables. The index “P”
refers to the probability of the occurrence of each group
is independent of each other with values between 0.06
and 0.267. However, the data considered in this study is
statistically in terms of significance level at 0.05. The second part is an evaluation of the information
system of teaching and learning online with the survey
research. After collecting data, the analyzed results are
as follows. The results obtained from the analyzed data through
system evaluation in the public Universities can be
apprehended as follows. Table 5 is the comparison of statistical index for the
acceptability behavior in using the information system
technology in teaching and learning of the instructor’s
group. The index “P” has the value of “0”, which means
that the population in the teacher’s group accepts and
supports the access of information system in teaching
and learning (highly). While the Index “F” is the values
of comparing variance of two groups. Group (df) with
the values of 59.78 implies that the populations in each
group are independent and are directly related to the
evaluation of accepting the information system with
education behavior. The overall performance shows
significant changes in the statistic as 0.5. Science Publications 3. RESULTS AND DISCUSSION Most people use this system for approximately 5
days per week or 41.31 percent, while the other 26.08
percent of the group accesses 4 days per week and 19.57
percent for 3 days. The least access is denoted as 6 days
per week or only 13.04 percent. Likewise, 78.27 per cent
of the female students actively participate in the survey. Students with the age above 20 have dominantly
participated in this survey. The other 46% are under 20 JCS 118 Petcharat Pattanasethanon and Kriangsak Chanthinok / Journal of Computer Science 9 (1): 114-121, 2013 Petcharat Pattanasethanon and Kriangsak Chanthinok / Journal of Computer Science 9 (1): 114-121, 2013 Petcharat Pattanasethanon and Kriangsak Chanthinok / Journal of Computer Science 9 (1): 114-121, 2013 Table 3. Shows the recognition of information system through online education of students and instructor
Instructor
Student Table 4. Shows data behavior regarding the users’ acceptability of the online information system
Acceptability
F
P
Part1. Expectations regarding the performance of the system
Stability of continuous accesses
1.37
0.221
Stability of file upload and submission
1.19
0.176
Part 2. Expectations for the effort of the user
The menu or category of information in this website
0.71
0.232
Convenient of accessing data
1.05
0.218
Part 3: Attitude towards the use of information technology in the education system
Speed browse for spectacular file
0.87
0.236
Convenient of the site access
1.67
0.060
Part 4. An environmental that provide the process (operation)
Convenience in downloading content
1.12
0.168
Part 5. The user’s confidence of information system in teaching and learning
Monitoring and security
1.07
0.141
Part 6. The satisfactory level for applying information technology into educational system
Satisfaction in online information systems
1.08
0.267 Table 5. Compares behavior of acceptability in using the information system technology in learning and teaching of the
instructor’s group
Acceptability
Mean of square
SS
df
MS
F
Prob
Information
Between groups
0.613
2
0.306
59.78
0.00
system as a tool
Within groups
0.055
4
0.013
2.70
0.06
for education
Total
0.668
6 Table 6. Compares the acceptability behavior of accessing the information system technology in learning and teaching of the
student’s group
Acceptability
Mean of Square
SS
df
MS
F
Prob. Science Publications 3. RESULTS AND DISCUSSION Information
Between groups
0.403
2
0.201
20.63
0.00
technology as a
Within Groups
0.072
4
0.018
1.84
0.16
tool for education
Total
0.475
6 each group are independent and are directly relevant to
the evaluation of accepting the information system with
education behavior. (The overall performance shows
significant changes in the statistic as 0.5) The
Contaminant errors of the population displayed in terms
of Sum Square (SS) and Means Square (MS) has a value
of 0.403 and 0.072, respectively. The results of the
variance in the four groups with subgroups are more
likely to agree with the large extent, where “P” is the Table 6 is the statistic for the acceptability behavior
in terms of accessing this information system technology
in teaching and learning for the student’s group. The
index “P” has obtained the value of “0”, which means
that the populations in the student’s group accept and
feel supportive with the information system in teaching
and learning (highly). While the Index “F” is the values
of comparing variance of two groups. The group (df),
with the value of 20.63, manifests that the populations in JCS JCS 119 Petcharat Pattanasethanon and Kriangsak Chanthinok / Journal of Computer Science 9 (1): 114-121, 2013 encourages lifelong learning (Life Long Learning)
(Collinsa and Halverson, 2010) encourages lifelong learning (Life Long Learning)
(Collinsa and Halverson, 2010) index value of 0.16 as the “F” index is 1.84 and for the
SS and MS has a value of 0.055 and 0.013, respectively. •
Reduce the usage of hard copy submission and
global warming •
Reduce the usage of hard copy submission and
global warming 4. CONCLUSION Therefore, it can lead
student’s
motivation
to
external
knowledge;
Students has an opportunity to see others work,
which can help improve their work better •
Confidence of users in using information system
technology for teaching and learning •
The intention to use ICT in teaching and learn The result of the evaluation of adopting information
system for online education purpose of professor and
students are split into 6 parts. The acceptance was in a
profoundly satisfying level and the variance of opinion in
response to the question is low (Table 3). Information on
the behavior of the online system were evaluated
according to the purpose of the six independent
components and is directly related to the behavior of the
online system by the “F” index value is between 0.71 to
1.67 and “P” index values ranged from 0.06 to 0.267
(Table 4). In conclusion, the behavior of accepting the
information system in teaching online on both instructors
and students are at the large extent. By using analysis of
variance of the data as a whole, the differences are
statistically recognized with a significant level at 0.05. •
It allows opportunities for students to access
through expertize in various field. The student can
ask for any information that they need from
experts directly. Which is hardly possible in the
conventional classroom teaching style. It also
saves time and financial budget for students,
while comparing with inter-communication in the
same manner •
The instructor can reconstruct their course content
easily because the data on the site is dynamically
created, thus the instructor can update their course
content to student at any time. While the student
themselves will also figure out the changes or
movement at all time. In addition, learners can
interact and comment on the content of learning
material, which is more flexible than traditional
learning. Besides, changes that satisfy the needs of
students are important Science Publications 4. CONCLUSION •
Reduce the cost to instructor who wanted to teach
online by not hosting or registering the domain, but
applying it to the RLM (URL) into their own The first part of this evaluation for information
system regarding online teaching of instructors and
students at public universities can be concluded as the
context described below: In the second part of the analysis of query results, we
can summarize the content as follows. Survey
respondents include professors and students. The
majority of the respondents were women and the amount
of individual answering the survey is divided to the
female instructor of 80.5 percent, female student 78.27
percent, while male instructor are 19.5 percent and
students are 21.73 respectively. •
The results of this research lead to the development
of a network and a platform of teaching for
instructors at Mahasarakham University. Every
institution which instructors can share their
knowledge and publication of scholarly works
together can also implement this system to
effectively provide an alternative path for education. The questionnaires for evaluating the behavior of
adopting information technology in teaching and
learning are divided into 6 parts: y p
p
•
It is a dominant medium for those who want to learn
wider than in classrooms and it encourages
interaction such as interaction with learning and/or
teaching and interacting with lesson content and
learning materials in the form of meeting to
discussion and the exchange of ideas (Ellis et al.,
2009) and in the form of teaching other than rigid
teaching. For example, an exercises or tests those
teachers provide to students •
Expectations regarding the performance of the
system
•
Expectations about the effort of the user
•
Attitude towards the exploitation of information
technology in teaching and learning
•
Environment that helps achieving operations
•
Confidence of users in using information system
technology for teaching and learning
•
The intention to use ICT in teaching and learning •
Expectations regarding the performance of the
system •
Expectations about the effort of the user •
Attitude towards the exploitation of information
technology in teaching and learning •
Environment that helps achieving operations p
•
This opens up opportunities for students to display
their work among classmates easily. It can be
expressed worldwide. 5. ACKNOWLEDGEMENT This research is financially supported by the Faculty
of Accountancy and Management, Mahasarakham
University, Mahasarakham, Thailand. •
This is helpful for changing the paradigms of
learning,
learning
is
student-centered
and •
This is helpful for changing the paradigms of
learning,
learning
is
student-centered
and JCS 120 Petcharat Pattanasethanon and Kriangsak Chanthinok / Journal of Computer Science 9 (1): 114-121, 2013 Science Publications 6. REFERENCES Muir-Herzig, R.G., 2004. Technology and its impact in
the classroom. Comput. Educ., 42: 111-131. 10.1016/S0360-1315(03)00067-8 Agre, P.E., 2002. Real-time politics: The internet and the
political process. Inform. Soc., 18: 311-331. Oblinger, D.G. and J.L. Oblinger, 2005. Educating the
Net Generation. 1st Edn., Educause, ISBN-10:
0967285321, pp: 264. AlAwadhi, S. and A. Morris, 2008. The Use of the
UTAUT Model in the adoption of e-Government
services in Kuwait. Proceedings of the 41st Annual
Hawaii
International
Conference
on
System
Sciences, Jan. 7-10, IEEE Xplore Press, Waikoloa,
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Nursing. Master Thesis, Kasetsart University,
Thailand. Sawasdiyakorn, T., 1998. The Survey of Problem and
Need on Instructional Media Utilization of the
Undergraduate Architectural Instructors in the
Higher Educational Institutes. The Ministry of
University Affaires. Collinsa, A. and R. Halverson, 2010. The second
educational revolution: Rethinking education in the
age of technology. J. Comput. Assis. Learn., 26: 18-
27. DOI: 10.1111/j.1365-2729.2009.00339.x Davis, F.D., 1989. Perceived usefulness, perceived ease
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adoption using UTAUT. Proceedings of the 2nd
International Conference on Mobile, Hybrid and On-
Line Learning, Feb. 10-16, IEEE Xplore Press, Saint
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viruses. Euro. Surveill,14: 19230-19230. Gregg, A., J. Mundie and J. Xia, 2009. Overcoming the
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(DTL’ 09), pp: 1-5. Wagner, N., K. Hassanein and M. Head, 2008. Who is
responsible for E-Learning Success in Higher
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Stakeholders’
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Joint Carrier Synchronization Algorithm by Open-Loop Acquisition and Closed-Loop Tracking in High-Dynamic Environments
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西北工业大学学报 西北工业大学学报 西北工业大学学报
ＪｏｕｒｎａｌｏｆＮｏｒｔｈｗｅｓｔｅｒｎＰｏｌｙｔｅｃｈｎｉｃａｌＵｎｉｖｅｒｓｉｔｙ Ｄｅｃ．
Ｖｏｌ．３６
２０１８
Ｎｏ．６ Ｄｅｃ．
Ｖｏｌ．３６
２０１８
Ｎｏ．６ ２０１８年１２月
第３６卷第６期 ＪｏｕｒｎａｌｏｆＮｏｒｔｈｗｅｓｔｅｒｎＰｏｌｙｔｅｃｈｎｉｃａｌＵｎｉｖｅｒｓｉｔｙ ｈｔｔｐｓ：／／ｄｏｉ．ｏｒｇ／１０．１０５１／ｊｎｗｐｕ／２０１８３６６１２３２ 高动态环境下联合开环捕获和闭环跟踪的
载波同步算法 韩春雷
１，白滢钰
２，司江勃
３ 韩春雷
１，白滢钰
２，司江勃
３ １．中国电子科技集团公司第二十研究所，陕西西安 ７１００６８；２．西安邮电大学，陕西西安 ７１０１００；
３．西安电子科技大学，陕西西安 ７１００７１
æ
è
ç
ö
ø
÷ 摘 要：针对高动态环境下载波同步困难和跟踪精度低的问题，提出联合开环捕获与闭环跟踪的高动
态载波同步算法。首先，该算法通过开环捕获得到多普勒频率偏移及多普勒变化率偏移的粗估计，将
剩余多普勒频率偏移和变化率偏移控制在一个较小的范围内。其次，在载波跟踪阶段采用三阶锁相
环（ＰＬＬ）对剩余多普勒频率偏移和变化率偏移进行跟踪。提出的算法同时具有快速捕获与精确跟踪
的良好特性。最后，通过Ｍａｔｌａｂ仿真对同步算法进行验证。仿真结果表明，当信噪比为８ｄＢ，归一化
多普勒频率偏移和多普勒变化率偏移分别在（－０．２５，０．２５）和（－１０
－４，１０
－４）范围内时，该算法的误码率
相比理论值仅损失０．７ｄＢ。 关 键 词：高动态；载波同步；开环捕获；闭环跟踪
中图分类号：ＴＮ９１ 文献标志码：Ａ 文章编 收稿日期：２０１７⁃１２⁃０８
基金项目：国家自然科学基金（５１４０９２１４）与中国电子科技集团公司数据链重点实验室开放基金（ＣＬＤＬ⁃２０１８２４１０）资助
作者简介：韩春雷（１９８２—）中国电子科技集团公司博士、高级工程师，主要从事信息系统总体技术研究。 文章编号：１０００⁃２７５８（２０１８）０６⁃１２３２⁃０４ 文章编号：１０００⁃２７５８（２０１８）０６⁃１２３２⁃０４ 在高动态环境中，通信双方之间的高速相对运
动往往会在接收机接收信号上引入较大的多普勒频
率及其高阶变化率。为了适应高动态环境，捕获和
跟踪接收信号中的多普勒频率及其变化率，就必须
加宽中低动态接收机载波跟踪环路的环路带宽。然
而环路带宽的加大势必降低载波跟踪精度，当跟踪
环路处于低信噪比通信环境中时尤为如此。噪声的
引入甚至会导致载波跟踪环失锁。因此，在动态适
应力和跟踪精度之间的权衡，一直吸引着许多学者
进行深入的研究。高动态环境下常用的跟踪技术包
括基于Ｋａｌｍａｎ滤波器的跟踪技术、基于最大似然的
跟踪环技术以及基于锁频环（ＦＬＬ）辅助的锁相环
（ＰＬＬ）技术等等。其中，基于Ｋａｌｍａｎ滤波器的跟踪
方法通过自适应地调整环路参数对载波相位差、多
普勒频率及其变化率进行估计。然而，由于高动态
环境对系统稳定性的影响较大，该方法无法实现快
速捕获［１⁃２］。基于最大似然辅助的环路跟踪算法是
一种渐进无偏估计，其参数的估计方差接近于 Ｃｒａｍｅｒ⁃Ｒａｏ下界（ＣＲＬＢ），但由于进行二维或三维
搜索所需的存储量和计算量很大，导致算法跟踪速
度变慢［３⁃４］。在ＦＬＬ辅助ＰＬＬ的结构中，ＦＬＬ和
ＰＬＬ分别用作载波参数的同步和细同步，并由ＰＬＬ
决定最终的跟踪精度。这种方法的结构相对简单，
但由于ＦＬＬ环路带宽较大，引入附加的噪声功率，
降低了跟踪精度［５］。为了解决这些问题，本文提出
了一种联合开环捕获和闭环跟踪的高动态载波同步
算法。即通过开环阶段能够实现快速捕获，同时通
过ＰＬＬ实现高精度载波跟踪。该算法不仅具有较
高动态适应能力，也具有较大的多普勒频率及其变
化率的估计范围。本文的贡献有以下２点：①适用
于高动态通信环境的线性频率变化情况；②估计范
围大，精度高。 １ 系统模型 接收机的结构框图如图１所示。该接收系统的 接收机的结构框图如图１所示。该接收系统的 收稿日期：２０１７⁃１２⁃０８
基金项目：国家自然科学基金（５１４０９２１４）与中国电子科技集团公司数据链重点实验室开放基金（ＣＬＤＬ⁃２０１８２４１０）资助
作者简介：韩春雷（１９８２—）中国电子科技集团公司博士、高级工程师，主要从事信息系统总体技术研究。 基金项目：国家自然科学基金（５１４０９２１４）与中国电子科技集团公司数据链重点实验室开放基金（ＣＬＤＬ⁃２０１８２４１０）资助
作者简介：韩春雷（１９８２—）中国电子科技集团公司博士、高级工程师，主要从事信息系统总体技术研究。 韩春雷，等：高动态环境下联合开环捕获和闭环跟踪的载波同步算法
３
３
２
１
· 韩春雷，等：高动态环境下联合开环捕获和闭环跟踪的载波同步算法 第６期 第６期 ·
３
３
２
１
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θ －＾θ －＾ω（ｉＴ）－
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÷·（ｉＴ）２
（８） （６） 勒变化率并解调得到译码信息。
图１ 接收机载波同步系统框图 （７） －
１
２！
æ
è
ç
ö
ø
÷·（ｉＴ）２
（８） （８） ２！
è
ø
结合（６）～（８）式可以得到最终结果：
Δ＾ａ＝２αＮ
Ｔ２∑
Ｎ
ｋ＝１
θ（ｋ）ｋ２－（Ｎ＋１）ｋ＋γＮ
６
é
ë
êê
ù
û
úú
（９）
Δ＾ｆ＝αＮ
Ｔ∑
Ｎ
ｋ＝１
θ（ｋ）·
βＮ
１５ｋ－（２Ｎ＋１）γＮ
１０
－（Ｎ＋１）ｋ２
é
ë
êê
ù
û
úú
（１０）
式中，计算系数αＮ，βＮ，γＮ分别表示如下
αＮ＝
１８０
Ｎ（Ｎ＋１）（Ｎ－１）（Ｎ＋２）（Ｎ－２）（１１）
βＮ＝（２Ｎ＋１）（８Ｎ＋１１）
（１２）
γＮ＝（Ｎ＋１）（Ｎ＋２）
（１３）
由于
式和
式推导出的多普勒频率偏移 ！
è
ø
结合（６）～（８）式可以得到最终结果：
Δ＾ａ＝２αＮ
Ｔ２∑
Ｎ
ｋ＝１
θ（ｋ）ｋ２－（Ｎ＋１）ｋ＋γＮ
６
é
ë
êê
ù
û
úú
（９）
Δ＾ｆ＝αＮ
Ｔ∑
Ｎ
ｋ＝１
θ（ｋ）·
βＮ
１５ｋ－（２Ｎ＋１）γＮ
１０
－（Ｎ＋１）ｋ２
é
ë
êê
ù
û
úú
（１０） 图１ 接收机载波同步系统框图 （９） ２ 开环捕获 在捕获操作中，利用了最小均方误差（ＭＭＳＥ）
方法获得多普勒频率偏移及其变化率偏移粗估计
值。假设接收到的复基带信号可以表示为： 在捕获操作中，利用了最小均方误差（ＭＭＳＥ）
方法获得多普勒频率偏移及其变化率偏移粗估计
值。假设接收到的复基带信号可以表示为：
ｒ（ｔ）＝ｓ（ｔ）·ｅｊ（θ＋Δｆｔ＋０．５Δａｔ２）＋ｎ（ｔ）
（１）
式中，ｓ（ｔ）是已调信号，θ 是载波的初始相位，Δｆ是
多普勒频率偏移，Δａ是多普勒变化率偏移，ｎ（ｔ）表
示高斯白噪声，导频长度为Ｎ，符号周期为Ｔ。Δｆ和
Δａ相应的对数似然函数为［６］ βＮ
１５ｋ－（２Ｎ＋１）γＮ
１０
－（Ｎ＋１）ｋ２
é
ë
êê
ù
û
úú
（１０）
式中，计算系数αＮ，βＮ，γＮ分别表示如下
αＮ＝
１８０
Ｎ（Ｎ＋１）（Ｎ－１）（Ｎ＋２）（Ｎ－２）（１１）
βＮ＝（２Ｎ＋１）（８Ｎ＋１１）
（１２）
γＮ＝（Ｎ＋１）（Ｎ＋２）
（１３） （１０） 接
带信
表
为：
ｒ（ｔ）＝ｓ（ｔ）·ｅｊ（θ＋Δｆｔ＋０．５Δａｔ２）＋ｎ（ｔ）
（１） 式中，ｓ（ｔ）是已调信号，θ 是载波的初始相位，Δｆ是
多普勒频率偏移，Δａ是多普勒变化率偏移，ｎ（ｔ）表 式中，ｓ（ｔ）是已调信号，θ 是载波的初始相位，Δｆ是
多普勒频率偏移，Δａ是多普勒变化率偏移，ｎ（ｔ）表
示高斯白噪声，导频长度为Ｎ，符号周期为Ｔ。Δｆ和
Δａ相应的对数似然函数为［６］ αＮ＝
１８０
Ｎ（Ｎ＋１）（Ｎ－１）（Ｎ＋２）（Ｎ－２）（１１）
βＮ＝（２Ｎ＋１）（８Ｎ＋１１）
（１２）
γＮ＝（Ｎ＋１）（Ｎ＋２）
（１３） （１２） ，
，（）
示高斯白噪声，导频长度为Ｎ，符号周期为Ｔ。Δｆ和
Δａ相应的对数似然函数为［６］ （１３） 由于（９）式和（１０）式推导出的多普勒频率偏移 Λ（ｒ｜Δ
ｆ，Δ
ａ）＝
ｅｘｐＲｅ∫
Ｔ
０ｒ（ｔ）ｓ∗（ｔ）ｅｊ（Δ
ｆｔ＋０．５Δ
ａｔ２）ｄｔ
{
}
(
)
（２）
信
［７］ Λ（ｒ｜Δ
ｆ，Δ
ａ）＝
ｅｘｐＲｅ∫
Ｔ
０ｒ（ｔ）ｓ∗（ｔ）ｅｊ（Δ
ｆｔ＋０．５Δ
ａｔ２）ｄｔ
{
}
(
)
（２）
基带信号相位为［７］
θ（ｔ＋Δｔ）＝
θ（ｔ）＋Δｆ（ｔ）Δｔ＋
Δａ（ｔ）
２！
（Δｔ）２＋Ｒ（ｔ，Δｔ）
（３） Λ（ｒ｜Δｆ，Δａ）＝
ｅｘｐＲｅ∫
Ｔ
０ｒ（ｔ）ｓ∗（ｔ）ｅｊ（Δ
ｆｔ＋０．５Δ
ａｔ２）ｄｔ
{
}
(
)
（２） 和多普勒变化率偏移估计公式是闭式解形式，因此
其复杂性是相当低的，能够实现快速捕获，适应高动
态环境，快速得出多普勒频率偏移和多普勒变化率
偏移粗估计值。 （２） ，
偏移粗估计值。
θ（ｔ＋Δｔ）＝
θ（ｔ）＋Δｆ（ｔ）Δｔ＋
Δａ（ｔ）
２！
（Δｔ）２＋Ｒ（ｔ，Δｔ）
（３） （３） （３） ３ 闭环跟踪 对于剩余项Ｒ（ｔ，Δｔ），当Δｔ→０时，趋向于
ｏ［（Δｔ）３］，其中ｏ［（Δｔ）３］表示Δｔ的三阶无穷小。 对于剩余项Ｒ（ｔ，Δｔ），当Δｔ→０时，趋向于
ｏ［（Δｔ）３］，其中ｏ［（Δｔ）３］表示Δｔ的三阶无穷小。 对于剩余项Ｒ（ｔ，Δｔ），当Δｔ→０时，趋向于
ｏ［（Δｔ）３］，其中ｏ［（Δｔ）３］表示Δｔ的三阶无穷小。
因此在非常短的时间内接收到的基带相位的二次多
项式可以提供多普勒频率偏移和多普勒变化率频移
的近似值。换句话说，当给定在时间点ｔ１，ｔ２，…，
ｔＮ（ｔ１＜ｔＮ）上的相位抽样值
θ（１）
θ（２）…
θ（Ｎ） 虽然经过开环捕获后，剩余多普勒频率偏移与
变化率偏移已经控制在一个很小的范围之内，但相
位误差仍会随着时间推移而增加。由于三阶环拥有
跟踪加速度的能力［８］，因此可以用来跟踪多普勒变
化率。本文的闭环跟踪三阶环路滤波器的结构如图
２所示［９］。 因此在非常短的时间内接收到的基带相位的二次多
项式可以提供多普勒频率偏移和多普勒变化率频移
的近似值
换句话说当给定在时间点 因此在非常短的时间内接收到的基带相位的二次多
项式可以提供多普勒频率偏移和多普勒变化率频移
的近似值。换句话说，当给定在时间点ｔ１，ｔ２，…，
（
）上的相位抽样值
θ（１）
θ（２）
θ（Ｎ） 的近似值。换句话说，当给定在时间点ｔ１，ｔ２，…， ｔＮ（ｔ１＜ｔＮ）上的相位抽样值
θ（１），
θ（２），…，
θ（Ｎ），
并进行相位的差分计算，消除相位模糊后，可得相位
误差计算公式为： 图２ 三阶环路滤波器 ）－＾θ（ｉ）］２
（４）
＋
１
２！
ε（ｉＴ）２
（５）
差，可以分别对＾θ、Δ＾ｆ和
图２ 三阶环路滤波器 ｅ（Ｎ）＝∑
Ｎ
ｉ＝１
［
θ（ｉ）－＾θ（ｉ）］２
（４）
＾θ（ｉ）＝＾θ ＋＾ω（ｉＴ）＋
１
２！
ε（ｉＴ）２
（５） （４） （５） 为了得到最小均方误差，可以分别对＾θ、Δ＾ｆ和
Δ＾ａ求偏导并令其为零，即： 为了得到最小均方误差，可以分别对＾θ、Δ＾ｆ和
Δ＾ａ求偏导并令其为零，即： 图２ 三阶环路滤波器 西 北 工 业 大 学 学 报 第３６卷 ·
４
３
２
１
· 其中，Ｚ
－１表示延时器，ＮＣＯ表示数控振荡器，
Ｃ１，Ｃ２，Ｃ３表示滤波器参数，其值如下所示
Ｃ１＝
８ｂωｎＴ＋２ω３
ｎＴ３
Ｋ０Ｋｄ（８＋４ｂωｎＴ＋２ａω２
ｎＴ２＋ω３
ｎＴ３）
（１４）
Ｃ２＝
８ｂω２
ｎＴ２＋４ω３
ｎＴ３
Ｋ０Ｋｄ（８＋４ｂωｎＴ＋２ａω２
ｎＴ２＋ω３
ｎＴ３）
（１５）
Ｃ３＝
８ｂω３
ｎＴ３
Ｋ０Ｋｄ（８＋４ｂωｎＴ＋２ａω２
ｎＴ２＋ω３
ｎＴ３） 化多普勒频率偏移和多普勒变化率偏移范围分别为
（－０．２５，０．２５）和（－１０
－４，１０
－４）。为验证本文提出的
载波同步算法，分别对相位跟踪曲线和误码率进行
数值仿真。 其中，Ｚ
－１表示延时器，ＮＣＯ表示数控振荡器， Ｃ１，Ｃ２，Ｃ３表示滤波器参数，其值如下所示 在图３中，当环路带宽Ｂｐ＝０．０２时，约在７００个
数据符号之后该系统能稳定跟踪参数。而当Ｂｐ＝
０．０４时，达到稳定跟踪需要约４２０个数据符号长
度。当Ｂｐ＝０．０６时，该系统跟踪载波信号仅需要
３００个数据符号。由该三阶锁相环的相位跟踪曲线
图可以看出，三阶锁相环的环路带宽Ｂｐ越高，系统
进入跟踪阶段的速度越快。 在图３中，当环路带宽Ｂｐ＝０．０２时，约在７００个
数据符号之后该系统能稳定跟踪参数。而当Ｂｐ＝
０．０４时，达到稳定跟踪需要约４２０个数据符号长
度。当Ｂｐ＝０．０６时，该系统跟踪载波信号仅需要
３００个数据符号。由该三阶锁相环的相位跟踪曲线
图可以看出，三阶锁相环的环路带宽Ｂｐ越高，系统
进入跟踪阶段的速度越快。 （１４） （１５） （１６） 图４给出了，在归一化多普勒变化率偏移ΔａＴ２
＝０．０００１，不同的环路带宽Ｂｐ，以及不同的多普勒
频率偏移条件下，系统的误码率随着信噪比变化的
曲线，其中理论值为不存在多普勒频率偏移和多普
率变化率偏移的理想情况。从图４ａ）可以看出，当
Ｅｓ／Ｎ０≥８ｄＢ，Ｂｐ＝０．０４和Ｂｐ＝０．０２时，误码率曲线
和理论值非常接近；同时环路带宽越高，误码率越
高。结合考虑图２中的跟踪时间和图３中的误码率，
我们认为选择环路带宽Ｂｐ＝０．０４更适合于进一步
分析。由图４ｂ）可知，当Ｅｓ／Ｎ０＝８ｄＢ，归一化多普
勒频率偏移ΔｆＴ＝０．２时，误码率相比理论值仅损失
１．１ｄＢ。当归一化多普勒频率偏移ΔｆＴ＝０．１５时，误
码率相比理论值仅损失０．７ｄＢ。 式中，Ｋｄ为鉴相器增益，Ｋ０为ＮＣＯ增益，ａ＝１．１，ｂ＝
２．４为阻尼系数等于０．７０７的经验值，ωｎ为环路固有 式中，Ｋｄ为鉴相器增益，Ｋ０为ＮＣＯ增益，ａ＝１．１，ｂ＝
２．４为阻尼系数等于０．７０７的经验值，ωｎ为环路固有 频率，与环路的等效噪声带宽的关系为 ωｎ＝
４ａｂ－４
ａｂ２＋ａ２－ｂ
ＢＩ
（１７） （１７） ４ 仿真分析 ４ 仿真分析 基于图１所示的系统模型对本文提出的联合开
环捕获闭环跟踪算法进行仿真验证。系统的各个仿
真参数如下：采用ＱＰＳＫ调制，每帧１０００ｂｉｔ数据，
１２８ｂｉｔ导频，信噪比范围设为（６ｄＢ，１２ｄＢ），归一 码率相比理论值仅损失０．７ｄＢ。 图３ 相位跟踪曲线
图４ 误码率曲线
５ 结 论
本文提出了在高动态环境下联合开环捕获和闭
环跟踪的载波同步算法。该算法将两者结合并发挥
两者各自的优势，从而可以实现快速捕获和精确跟
踪。当信噪比为８ｄＢ，归一化多普勒频率偏移ΔｆＴ
＝０．１５和归一化多普勒变化率偏移ΔａＴ２＝１０
－４时，
误码率相比理论值仅损失０．７ｄＢ。 图３ 相位跟踪曲线 两者各自的优势，从而可以实现快速捕获和精确跟
踪。当信噪比为８ｄＢ，归一化多普勒频率偏移ΔｆＴ
＝０．１５和归一化多普勒变化率偏移ΔａＴ２＝１０
－４时，
误码率相比理论值仅损失０．７ｄＢ。 两者各自的优势，从而可以实现快速捕获和精确跟
踪。当信噪比为８ｄＢ，归一化多普勒频率偏移ΔｆＴ
＝０．１５和归一化多普勒变化率偏移ΔａＴ２＝１０
－４时，
误码率相比理论值仅损失０．７ｄＢ。 本文提出了在高动态环境下联合开环捕获和闭
环跟踪的载波同步算法。该算法将两者结合并发挥 韩春雷，等：高动态环境下联合开环捕获和闭环跟踪的载波同步算法 ·
５
３
２
１
· 第６期 参考文献： 参考文献： ［１］ ＳｃａｌａＢＦＬａ，ＢｉｔｍｅａｄＲＲ，ＱｕｉｎｎＢＧ．ＡｎＥｘｔｅｎｄｅｄＫａｌｍａｎＦｉｌｔｅｒＦｒｅｑｕｅｎｃｙＴｒａｃｋｅｒｆｏｒＨｉｇｈ⁃ＮｏｉｓｅＥｎｖｉｒｏｎｍｅｎｔｓ［Ｊ］．ＩＥＥＥ
ＴｒａｎｓｏｎＳｉｇｎａｌＰｒｏｃｅｓｓｉｎｇ，１９９６，４４（２）：４３１⁃４３４ ［２］ ＰａｋＪＭ，ＡｈｎＣＫ，ＬｉｍＭＴ．Ｓｅｌｆ⁃ＲｅｃｏｖｅｒｉｎｇＥｘｔｅｎｄｅｄＫａｌｍａｎＦｉｌｔｅｒｆｏｒＦｒｅｑｕｅｎｃｙＴｒａｃｋｉｎｇ［Ｃ］∥ＩｎｔｅｒｎａｔｉｏｎａｌＣｏｎｆｅｒｅｎｃｅ
ｏｎＣｏｎｔｒｏｌ，ＡｕｔｏｍａｔｉｏｎａｎｄＳｙｓｔｅｍｓ，２０１５ ［３］ 郇浩，陶选如，陶然，等．多普勒频率变化率快速最大似然估计辅助的高动态载波跟踪环路［Ｊ］．电子与信息学报，
２０１４，Ｖ３６（３）：５７７⁃５８２
ＨｕａｎＨａｏ，ＴａｏＸｕａｎｒｕ，ＴａｏＲａｎ，ｅｔａｌ．ＣａｒｒｉｅｒＴｒａｃｋｉｎｇＬｏｏｐｉｎＨｉｇｈＤｙｎａｍｉｃＥｎｖｉｒｏｎｍｅｎｔＡｉｄｅｄｂｙＦａｓｔＭａｘｉｍｕｍＬｉｋｅｌｉ⁃
ｈｏｏｄＥｓｔｉｍａｔｉｏｎｏｆＤｏｐｐｌｅｒＦｒｅｑｕｅｎｃｙＲａｔｅ⁃ｏｆ⁃Ｃｈａｎｇｅ［Ｊ］．ＪｏｕｒｎａｌｏｆＥｌｅｃｔｒｏｎｉｃｓ＆ＩｎｆｏｒｍａｔｉｏｎＴｅｃｈｎｏｌｏｇｙ，２０１４，３６（３）：５７７⁃
５８２（ｉｎＣｈｉｎｅｓｅ） ［３］ 郇浩，陶选如，陶然，等．多普勒频率变化率快速最大似然估计辅助的高动态载波跟踪环路［Ｊ］．电子与信息学报，
２０１４，Ｖ３６（３）：５７７⁃５８２
ＨｕａｎＨａｏ，ＴａｏＸｕａｎｒｕ，ＴａｏＲａｎ，ｅｔａｌ．ＣａｒｒｉｅｒＴｒａｃｋｉｎｇＬｏｏｐｉｎＨｉｇｈＤｙｎａｍｉｃＥｎｖｉｒｏｎｍｅｎｔＡｉｄｅｄｂｙＦａｓｔＭａｘｉｍｕｍＬｉｋｅｌｉ⁃ ［３］ 郇浩，陶选如，陶然，等．多普勒频率变化率快速最大似然估计辅助的高动态载波跟踪环路［Ｊ］．电子与信息学报，
２０１４，Ｖ３６（３）：５７７⁃５８２ ，
（）：
ＨｕａｎＨａｏ，ＴａｏＸｕａｎｒｕ，ＴａｏＲａｎ，ｅｔａｌ．ＣａｒｒｉｅｒＴｒａｃｋｉｎｇＬｏｏｐｉｎＨｉｇｈＤｙｎａｍｉｃＥｎｖｉｒｏｎｍｅｎｔＡｉｄｅｄｂｙＦａｓｔＭａｘｉｍｕｍＬｉｋｅｌｉ⁃
ｈｏｏｄＥｓｔｉｍａｔｉｏｎｏｆＤｏｐｐｌｅｒＦｒｅｑｕｅｎｃｙＲａｔｅ⁃ｏｆ⁃Ｃｈａｎｇｅ［Ｊ］．ＪｏｕｒｎａｌｏｆＥｌｅｃｔｒｏｎｉｃｓ＆ＩｎｆｏｒｍａｔｉｏｎＴｅｃｈｎｏｌｏｇｙ，２０１４，３６（３）：５７７⁃
５８２（ｉｎＣｈｉｎｅｓｅ） ＨｕａｎＨａｏ，ＴａｏＸｕａｎｒｕ，ＴａｏＲａｎ，ｅｔａｌ．ＣａｒｒｉｅｒＴｒａｃｋｉｎｇＬｏｏｐｉｎＨｉｇｈＤｙｎａｍｉｃＥｎｖｉｒｏｎｍｅｎｔＡｉｄｅｄｂｙＦａｓｔＭａｘｉｍｕｍＬｉｋｅｌｉ⁃
ｈｏｏｄＥｓｔｉｍａｔｉｏｎｏｆＤｏｐｐｌｅｒＦｒｅｑｕｅｎｃｙＲａｔｅ⁃ｏｆ⁃Ｃｈａｎｇｅ［Ｊ］．ＪｏｕｒｎａｌｏｆＥｌｅｃｔｒｏｎｉｃｓ＆ＩｎｆｏｒｍａｔｉｏｎＴｅｃｈｎｏｌｏｇｙ，２０１４，３６（３）：５７７⁃ ＨｕａｎＨａｏ，ＴａｏＸｕａｎｒｕ，ＴａｏＲａｎ，ｅｔａｌ．ＣａｒｒｉｅｒＴｒａｃｋｉｎｇＬｏｏｐｉｎＨｉｇｈＤｙｎａｍｉｃＥｎｖｉｒｏｎｍｅｎｔＡｉｄｅ ，
，
，
ｇ
ｐ
ｇ
ｙ
ｙ
ｈｏｏｄＥｓｔｉｍａｔｉｏｎｏｆＤｏｐｐｌｅｒＦｒｅｑｕｅｎｃｙＲａｔｅ⁃ｏｆ⁃Ｃｈａｎｇｅ［Ｊ］．ＪｏｕｒｎａｌｏｆＥｌｅｃｔｒｏｎｉｃｓ＆ＩｎｆｏｒｍａｔｉｏｎＴｅｃｈｎｏｌｏｇｙ，２０１４，３６（３）：５７７⁃
５８２（ｉｎＣｈｉｎｅｓｅ） ［４］ ＷｏｎＪＨ，ＰａｎｙＴ，ＥｉｓｓｆｅｌｌｅｒＢ．ＩｔｅｒａｔｉｖｅＭａｘｉｍｕｍＬｉｋｅｌｉｈｏｏｄＥｓｔｉｍａｔｏｒｓｆｏｒＨｉｇｈ⁃ＤｙｎａｍｉｃＧＮＳＳＳｉｇｎａｌＴｒａｃｋｉｎｇ［Ｊ］．ＩＥＥＥ
ＴｒａｎｓｏｎＡｅｒｏｓｐａｃｅ＆ＥｌｅｃｔｒｏｎｉｃＳｙｓｔｅｍｓ，２０１２，４８（４）：２８７５⁃２８９３ ［５］ ＳｕＪ，ＴａｏＨＨ，ＲａｏＸ，ｅｔａｌ．ＣｏｈｅｒｅｎｔｌｙＩｎｔｅｇｒａｔｅｄＣｕｂｉｃＰｈａｓｅＦｕｎｃｔｉｏｎｆｏｒＭｕｌｔｉｐｌｅＬＦＭＳｉｇｎａｌｓＡｎａｌｙｓｉｓ［Ｊ］．Ｅｌｅｃｔｒｏｎｉｃｓ
Ｌｅｔｔｅｒｓ，２０１５，５１（５）：４１１⁃４１３ ［６］ 邓晓东，孙武．基于ＦＬＬ＋ＰＬＬ的载波跟踪环路设计［Ｊ］．现代防御技术，２０１０，３８（４）：１３７⁃１４１ ［６］ 邓晓东，孙武．基于ＦＬＬ＋ＰＬＬ的载波跟踪环路设计［Ｊ］．现代防御技术，２０１０，３８（４）：１３７⁃１４１
ＤｅｎｇＸｉａｏｄｏｎｇ，ＳｕｎＷｕ．ＤｅｓｉｇｎｏｆＣａｒｒｉｅｒＴｒａｃｋｉｎｇＬｏｏｐＢａｓｅｄｏｎＦＬＬ＋ＰＬＬ［Ｊ］．ＭｏｄｅｒｎＤｅｆｅｎｃｅＴｅｃｈｎｏｌｏｇｙ，２０１０，３８（４）：
１３７⁃１４１（ｉｎＣｈｉｎｅｓｅ） ＤｅｎｇＸｉａｏｄｏｎｇ，ＳｕｎＷｕ．ＤｅｓｉｇｎｏｆＣａｒｒｉｅｒＴｒａｃｋｉｎｇＬｏｏｐＢａｓｅｄｏｎＦＬＬ＋ＰＬＬ［Ｊ］．ＭｏｄｅｒｎＤｅｆｅｎｃｅＴｅｃｈｎｏｌｏｇｙ，２０１０，３８（４）：
１３７⁃１４１（ｉｎＣｈｉｎｅｓｅ） ［７］ ＵｍｂｅｒｔｏＭｅｎｇａｌｉ，ＡｌｄｏＮＤ′Ａｎｄｒｅａ．ＳｙｃｈｒｏｎｉｚａｔｉｏｎＴｅｃｈｎｉｑｕｅｓｆｏｒＤｉｇｉｔａｌＲｅｃｅｉｖｅｒｓ［Ｍ］．ＮｅｗＹｏｒｋ，ＰｌｅｎｕｍＰｒｅｓｓ，１９９７：
８０⁃８１ ［８］ ＳｕＹＴ，ＷｕＲＣ．ＦｒｅｑｕｅｎｃｙＡｃｑｕｉｓｉｔｉｏｎａｎｄＴｒａｃｋｉｎｇｉｎＨｉｇｈＤｙｎａｍｉｃＥｎｖｉｒｏｎｍｅｎｔｓ［Ｊ］．ＩＥＥＥＴｒａｎｓｏｎＶｅｈｉｃｕｌａｒＴｅｃｈｎｏｌｏ⁃
ｇｙ，２０００，４９（６）：２４１９⁃２４２９ ［９］ 吴华明，苏雁泳，刘爱军．锁相环与锁频环在数字Ｃｏｓｔａｓ环中的应用［Ｊ］．科学技术与工程，２０１０，１０（１９）：４６４５⁃４６５０
ＷｕＨｕａｍｉｎｇ，ＳｕＹａｎｙｏｎｇ，ＬｉｕＡｉｊｕｎ．ＡｐｐｌｉｃａｔｉｏｎｏｆＰＬＬａｎｄＦＬＬｉｎｔｈｅＤｉｇｉｔａｌＣｏｓｔａｓＬｏｏｐ［Ｊ］．ＳｃｉｅｎｃｅＴｅｃｈｎｏｌｏｇｙａｎｄＥｎ⁃
ｇｉｎｅｅｒｉｎｇ，２０１０，１０（１９）：４６４５⁃４６５０（ｉｎＣｈｉｎｅｓｅ） ＪｏｉｎｔＣａｒｒｉｅｒＳｙｎｃｈｒｏｎｉｚａｔｉｏｎＡｌｇｏｒｉｔｈｍｂｙＯｐｅｎ⁃ＬｏｏｐＡｃｑｕｉｓｉｔｉｏｎａ
Ｃｌｏｓｅｄ⁃ＬｏｏｐＴｒａｃｋｉｎｇｉｎＨｉｇｈ⁃ＤｙｎａｍｉｃＥｎｖｉｒｏｎｍｅｎｔｓ ＨａｎＣｈｕｎｌｅｉ
１，ＢａｉＹｉｎｇｙｕ
２，ＳｉＪｉａｎｇｂｏ
３
１．Ｎｏ．２０ＲｅｓｅａｒｃｈＩｎｓｔｉｔｕｔｅｏｆＣＥＴＣ，Ｘｉ′ａｎ７１００６８，Ｃｈｉｎａ；
２．Ｘｉ′ａｎＵｎｉｖｅｒｓｉｔｙｏｆＰｏｓｔｓ＆Ｔｅｌｅｃｏｍｍｕｎｉｃａｔｉｏｎｓ，Ｘｉ′ａｎ７１０１００，Ｃｈｉｎａ；
３．ＸｉｄｉａｎＵｎｉｖｅｒｓｉｔｙ，Ｘｉ′ａｎ７１００７１，Ｃｈｉｎａ
æ
è
ç
ç
ö
ø
÷
÷ Ａｂｓｔｒａｃｔ：Ｃｏｎｓｉｄｅｒｉｎｇｔｈｅｄｉｆｆｉｃｕｌｔｙｏｆｃａｒｒｉｅｒｓｙｎｃｈｒｏｎｉｚａｔｉｏｎａｎｄｔｈｅｌｏｗｔｒａｃｋｉｎｇａｃｃｕｒａｃｙｉｎｈｉｇｈ⁃ｄｙｎａｍｉｃｅｎｖｉ⁃
ｒｏｎｍｅｎｔｓ，ｔｈｅｊｏｉｎｔｏｐｅｎ⁃ｌｏｏｐａｃｑｕｉｓｉｔｉｏｎａｎｄｃｌｏｓｅｄ⁃ｌｏｏｐｔｒａｃｋｉｎｇｃａｒｒｉｅｒｓｙｎｃｈｒｏｎｉｚａｔｉｏｎａｌｇｏｒｉｔｈｍｉｓｐｒｅｓｅｎｔｅｄ．
Ｆｉｒｓｔｌｙ，ｔｈｅａｌｇｏｒｉｔｈｍｄｅｒｉｖｅｓｔｈｅｃｏａｒｓｅｅｓｔｉｍａｔｉｏｎｏｆＤｏｐｐｌｅｒｆｒｅｑｕｅｎｃｙａｎｄＤｏｐｐｌｅｒｒａｔｅｖｉａｏｐｅｎ⁃ｌｏｏｐ
ａｃｑｕｉｓｉｔｉｏｎ，ｔｈｅｎｔｈｅｒｅｓｉｄｕａｌＤｏｐｐｌｅｒｆｒｅｑｕｅｎｃｙａｎｄＤｏｐｐｌｅｒｒａｔｅａｒｅｃｏｎｆｉｎｅｄｉｎｔｏａｓｍａｌｌｒａｎｇｅ．Ｓｅｃｏｎｄｌｙ，ｔｈｅｒｅ⁃
ｓｉｄｕａｌＤｏｐｐｌｅｒｒａｔｅｉｓｔｒａｃｋｅｄｂｙｕｓｉｎｇａｔｈｉｒｄ⁃ｏｒｄｅｒｐｈａｓｅ⁃ｌｏｃｋｅｄｌｏｏｐ（ＰＬＬ）．Ｔｈｅｐｒｅｓｅｎｔａｌｇｏｒｉｔｈｍｈａｓｔｈｅａｄｖａｎ⁃
ｔａｇｅｓｏｆｆａｓｔａｃｑｕｉｓｉｔｉｏｎａｎｄｈｉｇｈｔｒａｃｋｉｎｇａｃｃｕｒａｃｙ．Ｆｉｎａｌｌｙ，ｔｈｅｎｕｍｅｒｉｃａｌｓｉｍｕｌａｔｉｏｎｉｓｃｏｎｄｕｃｔｅｄｔｏｖｅｒｉｆｙｔｈｅ
ｐｒｅｓｅｎｔｓｙｎｃｈｒｏｎｉｚａｔｉｏｎａｌｇｏｒｉｔｈｍ．Ｔｈｅｓｉｍｕｌａｔｉｏｎｒｅｓｕｌｔｓｉｎｄｉｃａｔｅｔｈａｔａｔｔｈｅｓｉｇｎａｌｔｏｎｏｉｓｅｒａｔｉｏ（ＳＮＲ）ｏｆａｎｄｎｏｒ⁃
ｍａｌｉｚｅｄＤｏｐｐｌｅｒｆｒｅｑｕｅｎｃｙａｎｄＤｏｐｐｌｅｒｒａｔｅｒａｎｇｉｎｇｆｒｏｍｔｏ，ｔｈｅｂｉｔ⁃ｅｒｒｏｒ⁃ｒａｔｅ（ＢＥＲ）ｐｅｒｆｏｒｍａｎｃｅｄｅｇｒａｄａｔｉｏｎｉｓ
ａｓｌｏｗａｓｃｏｍｐａｒｉｎｇｗｉｔｈｔｈｅｏｒｅｔｉｃａｌｖａｌｕｅ． Ｋｅｙｗｏｒｄｓ：ｈｉｇｈ⁃ｄｙｎａｍｉｃ；ｃａｒｒｉｅｒｓｙｎｃｈｒｏｎｉｚａｔｉｏｎ；ｏｐｅｎ⁃ｌｏｏｐａｃｑｕｉｓｉｔｉｏｎ；ｃｌｏｓｅｄ⁃ｌｏｏｐｔｒａｃｋｉｎｇ
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https://openalex.org/W4391681905
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https://mycokeys.pensoft.net/article/115452/download/pdf/
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Identification of two new species and a new host record of Distoseptispora (Distoseptisporaceae, Distoseptisporales, Sordariomycetes) from terrestrial and freshwater habitats in Southern China
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MycoKeys
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Copyright: © Xue-Mei Chen et al.
This is an open access article distributed under
terms of the Creative Commons Attribution
License (Attribution 4.0 International –
CC BY 4.0). Abstract During our investigation of saprophytic fungi in Guizhou and Hainan provinces, China,
three hyphomycetes were collected from terrestrial and freshwater habitats. Based on
morphological characteristics and phylogenetic analyses of combined ITS, LSU, tef1-α,
and rpb2 sequence data, two new species are introduced: Distoseptispora hainanensis
and D. lanceolatispora. Additionally, one known species, D. tectonae, previously unreport-
ed from Edgeworthia chrysantha, is newly reported. Detailed descriptions, illustrations,
and a phylogenetic tree to show the two new species and the new host record of Dis-
toseptispora are provided. In addition, a checklist of Distoseptispora species with their
locations, lifestyles, habitats, and hosts is provided. Academic editor: Xinlei Fan
Received: 8 November 2023
Accepted: 29 December 2023
Published: 9 February 2024 Key words: 2 new taxa, asexual morph, phylogeny, taxonomy Citation: Chen X-M, Tang X,
Ma J, Liu N-G, Tibpromma S,
Karunarathna SC, Xiao Y-P, Lu Y-Z
(2024) Identification of two new
species and a new host record of
Distoseptispora (Distoseptisporaceae,
Distoseptisporales, Sordariomycetes)
from terrestrial and freshwater
habitats in Southern China. MycoKeys
102: 83–105. https://doi.org/10.3897/
mycokeys.102.115452 Identification of two new species and a new host record of
Distoseptispora (Distoseptisporaceae, Distoseptisporales,
Sordariomycetes) from terrestrial and freshwater habitats
in Southern China Identification of two new species and a new host record of
Distoseptispora (Distoseptisporaceae, Distoseptisporales,
Sordariomycetes) from terrestrial and freshwater habitats
in Southern China Xue-Mei Chen1,2 , Xia Tang3,4 , Jian Ma1,4 , Ning-Guo Liu1 , Saowaluck Tibpromma2 ,
Samantha C. Karunarathna2,5 , Yuan-Pin Xiao1 , Yong-Zhong Lu1,3 1 School of Food and Pharmaceutical Engineering, Guizhou Institute of Technology, Guiyang 550003, China Abstract MycoKeys 102: 83–105 (2024)
DOI: 10.3897/mycokeys.102.115452 Research Article Introduction Distoseptispora K.D. Hyde, McKenzie & Maharachch. was introduced by Su et al. (2016) with D. fluminicola McKenzie, Hong Y. Su, Z.L. Luo & K.D. Hyde, as the type
species. Most Distoseptispora species are reported as saprophytes, typically
found on decaying wood in terrestrial and freshwater habitats (Hyde et al. 2016,
2019; Su et al. 2016; Xia et al. 2017; Yang et al. 2018; Crous et al. 2019; Luo et
al. 2019). The initial descriptions of Distoseptispora are derived from its asex-
ual morphology (Hyde et al. 2016, 2019, 2020; Su et al. 2016; Yang et al. 2018,
2021; Luo et al. 2019; Sun et al. 2020). The first description of a sexual morph
of Distoseptispora was described by Yang et al. (2021). Recently, Konta et al. (2023) identified the second sexual species on dead leaves of Licuala glabra, and
provided detailed explanations, enhancing our understanding of Distoseptispora 83 Xue-Mei Chen et al.: Identification of two new species and a new host record of Distoseptispora sexual morphology. This sexual morph is characterized by solitary or gregarious,
immersed to semi-immersed, subglobose to ellipsoidal, perithecial, dark brown
ascomata with a short neck; 8-spored, cylindrical, short pedicellate asci with
non-amyloid apical annuli; and fusiform, 0–3-septate, hyaline ascospores with
mucilaginous sheaths (Yang et al. 2021; Konta et al. 2023). The asexual morph of
Distoseptispora was recently expanded upon by Yang et al. (2021), incorporating
macronematous, mononematous, solitary or fasciculate conidiophores, blastic,
terminal, percurrent, cylindrical conidiogenous cells; and acrogenous, solitary, ob-
clavate, ellipsoidal, obovoid or fusiform, rostrate or not, euseptate, distoseptate
or rarely muriform conidia with or without a septal pore and mucilaginous sheath. Distoseptispora has been found on various hosts viz. Tectona, Pandanus,
bamboo, Clematis, Carex, Dipterocarpus, Licuala glabra, Cocos nucifera, Phrag-
mites australis, Thysanolaena maxima, Platanus orientalis, and decaying wood
and grasses (Shoemaker and White 1985; McKenzie 1995; Hyde et al. 2016,
2019, 2021, 2023; Su et al. 2016; Tibpromma et al. 2018; Crous et al. 2019;
Phookamsak et al. 2019; Phukhamsakda et al. 2020, 2022; Sun et al. 2020;
Zhai et al. 2022; Afshari et al. 2023; Hu et al. 2023; Konta et al. 2023). Most
Distoseptispora species have been described in Asia, mainly in China, Thailand,
and Malaysia, and only a few have been described in Europe (Shoemaker and
White 1985; McKenzie 1995; Phookamsak et al. 2019; Ma et al. 2022; Zhai et
al. 2022; Zhang et al. 2022; Konta et al. Materials and methods Materials and methods Introduction 2023). Distoseptispora comprises 74
accepted species in Index Fungorum (2024), but there is an ambiguity in the
taxonomic status of D. submersa Z.L. Luo, K.D. Luo et al. (2019) stated that
D. submersa is phylogenetically closely related to D. tectonae, and there are only
minor size differences in conidiophores and conidia between D. tectonae and
D. submersa. Dong et al. (2021) synonymized D. submersa under D. tectonae,
thus, Distoseptispora comprises 73 accepted saprobic species, of which 44
were from freshwater habitats, 29 from terrestrial habitats, and five from both
terrestrial and freshwater environments (Hyde et al. 2016, 2019; Luo et al. 2019;
Monkai et al. 2020; Yang et al. 2021; Ma et al. 2022; Zhang et al. 2022; Afshari
et al. 2023; Hu et al. 2023; Konta et al. 2023; Liu et al. 2023). In this study, three fresh hyphomycetous fungal collections were encountered
during a microfungal investigation in Hainan and Guizhou provinces. Based on
multi-gene phylogeny and morphological comparison, two new species, Dis-
toseptispora hainanensis and D. lanceolatispora are introduced. In addition, a
new host record of D. tectonae from Edgeworthia chrysantha is also reported. DNA extraction, PCR amplification, and sequencing Fresh mycelia were scraped from cultures that were incubated at 25–27 °C for
28 days. Fungal genomic DNA was extracted using the Biospin Fungus Genom-
ic DNA Extraction Kit (BioFlux, Shanghai, China), following the manufacturer’s
instructions. Four gene regions: internal transcribed spacer (ITS), large subunit
ribosomal DNA (LSU), translation elongation factor 1-alpha (tef1-α), and RNA
polymerase II second largest subunit (rpb2) were selected. The primers used in
this study for each gene region were as follows: ITS4 and ITS5 for ITS (White et
al. 1990), LR0R and LR5 for LSU (Vilgalys and Hester 1990; Cubeta et al. 1991),
EF1-983F and EF1-2218R for tef1-α (Rehner and Samuels 1994), and rpb2 with
fRPB2-5F and fRPB2-7cR (Liu et al. 1999).i Polymerase chain reaction (PCR) amplifications were carried out in a 50 µL
reaction volume containing 44 μL of 1.1 × T3 Super PCR Mix (TsingKe Biotech,
Chongqing, China), 2 µL of DNA template, and 2 µL of each forward and reverse
primer. The amplification condition for LSU and ITS consisted of initial denatur-
ation at 94 °C for 3 min, followed by 35 cycles of 45 s at 94 °C, 50 s at 56 °C, and
1 min at 72 °C, and a final extension period of 10 min at 72 °C. The amplification
condition for the tef1-α gene consisted of initial denaturation at 94 °C for 3 min,
followed by 30 cycles of 30 s at 94 °C, 50 s at 56 °C, and 1 min at 72 °C, a final ex-
tension period of 10 min at 72 °C. The amplification condition for the rpb2 gene
consisted of initial denaturation at 95 °C for 5 min, followed by 35 cycles of 15 s
at 95 °C, 50 s at 56 °C, and 1 min at 72 °C, a final extension period of 10 min at
72 °C. The quality of PCR amplification products was examined with 1% agarose
electrophoresis gels stained with ethidium bromide, and the PCR products were
sent to TsingKe Biotech, Chongqing, China for purification and sequencing. Sample collection, isolation, and morphological study Fresh specimens were collected from Hainan and Guizhou provinces in China. Fungal colonies were mounted on a slide with distilled water and were observed
and examined using a stereomicroscope (SMZ 745, Nikon, Tokyo, Japan). Mi-
cro-morphological characteristics were captured with a Nikon EOS 90D digital
camera combined with an ECLIPSE Ni-U compound microscope (Nikon, Tokyo,
Japan). The sizes of the fungal structures were measured using the Tarosoft (R)
Image Frame Work program (IFW 0.97 version), and the photo plates were pro-
cessed with Adobe Photoshop CC 2019 (Adobe Systems, San Jose, CA, USA). MycoKeys 102: 83–105 (2024), DOI: 10.3897/mycokeys.102.115452 84 Xue-Mei Chen et al.: Identification of two new species and a new host record of Distoseptispora Single spore isolations were carried out following the methods described in
Senanayake et al. (2020). Germinated conidia were transferred to fresh potato
dextrose agar (PDA) plates and incubated at 25–27 °C for four weeks. Cul-
ture characteristics, including color, shape, and size, were recorded. Herbarium
specimens were deposited in the herbarium of the Guizhou Academy of Agri-
culture Sciences (GZAAS), Guiyang, China, and the living cultures were depos-
ited at the Guizhou Culture Collection, China (GZCC). Faces of Fungi and Index
Fungorum numbers were obtained following the protocols outlined by Jayasiri
et al. (2015) and Index Fungorum (2024), respectively. Phylogenetic analyses The raw sequences were initially checked with BioEdit v 7.0.5.3 (Hall 1999). Forward and reverse sequences were assembled using SeqMan v. 7.0.0
(DNASTAR, Madison, WI, USA). Sequence data (LSU, ITS, tef1-α, and rpb2) for
Distoseptispora were downloaded from GenBank based on the blast results
and recent publications (Table 1). Each individual gene dataset was aligned
using the online program MAFFT version 7 with the “auto” option (Hall 1999;
Katoh and Standley 2013). These alignments were visually inspected and
manually improved in BioEdit v 7.0.5.3. Multi-gene alignments were combined
by SequenceMatrix (Vaidya et al. 2011). In this study, phylogenetic analyses
were performed using maximum likelihood (ML), maximum parsimony (MP), MycoKeys 102: 83–105 (2024), DOI: 10.3897/mycokeys.102.115452 85 Xue-Mei Chen et al.: Identification of two new species and a new host record of Distoseptispora Table 1. Names, strain numbers, and corresponding GenBank accession numbers of taxa used in this study. Taxa names
Strain
GenBank Accessions
References
LSU
ITS
tef1-α
rpb2
Aquapteridospora aquatica
MFLUCC 17-2371T
MW287767
MW286493
N/A
N/A
Dong et al. (2021)
Distoseptispora adscendens
HKUCC 10820
DQ408561
N/A
N/A
DQ435092
Shenoy et al. (2006)
D. amniculi
MFLU 17-2129T
MZ868761
MZ868770
N/A
MZ892982
Yang et al. (2021)
D. appendiculata
MFLUCC 18-0259T
MN163023
MN163009
MN174866
N/A
Luo et al. (2019)
D. aqualignicola
KUNCC 21-10729T
ON400845
OK341186
OP413480
OP413474
Zhang et al. (2022)
D. aquamyces
KUNCC 21-10731T
OK341199
OK341187
OP413482
OP413476
Zhang et al. (2022)
D. aquatica
MFLUCC 15-0374T
KU376268
MF077552
N/A
N/A
Su et al. (2016)
MFLUCC 18-0646
MK849793
MK828648
N/A
N/A
Luo et al. (2019)
D. aquisubtropica
GZCC 22-0075T
ON527941
ON527933
ON533677
ON533685
Ma et al. (2022)
D. atroviridis
GZCC 20-0511T
MZ868763
MZ868772
MZ892978
MZ892984
Yang et al. (2021)
D. bambusae
MFLUCC 20-0091T
MT232718
MT232713
MT232880
MT232881
Sun et al. (2020)
MFLUCC 14-0583
MT232717
MT232712
N/A
MT232882
Sun et al. (2020)
D. bambusicola
GZCC 21-0667T
MZ474872
MZ474873
N/A
N/A
Hyde et al. (2023)
D. bangkokensis
MFLUCC 18-0262T
MZ518206
MZ518205
N/A
N/A
Shen et al. (2021)
D. cangshanensis
MFLUCC 16-0970T
MG979761
MG979754
MG988419
N/A
Luo et al. (2018)
D. caricis
CPC 36498T
MN567632
MN562124
N/A
MN556805
Crous et al. (2019)
CPC 36442
N/A
MN562125
N/A
MN556806
Crous et al. (2019)
D. chinensis
GZCC 21-0665T
MZ474867
MZ474871
MZ501609
N/A
Hyde et al. (2021)
D. clematidis
MFLUCC 17-2145T
MT214617
MT310661
N/A
MT394721
Phukhamsakda et al. (2020)
D. crassispora
KUMCC 21-10726T
OK341196
OK310698
OP413479
OP413473
Zhang et al. (2022)
D. MycoKeys 102: 83–105 (2024), DOI: 10.3897/mycokeys.102.115452 Phylogenetic analyses curvularia
KUMCC 21-10725T
OK341195
OK310697
OP413478
OP413472
Zhang et al. (2022)
D. cylindricospora
DLUCC 1906T
OK513523
OK491122
OK524220
N/A
Phukhamsakda et al. (2022)
D. dehongensis
KUMCC 18-0090T
MK079662
MK085061
MK087659
N/A
Hyde et al. (2019)
D. dipterocarpi
MFLUCC 22-0104T
OP600052
OP600053
N/A
OP595140
Afshari et al. (2023)
D. effusa
GZCC 19-0532T
MZ227224
MW133916
N/A
N/A
Yang et al. (2021)
D. eusptata
MFLUCC 20-0154T
MW081544
MW081539
N/A
MW151860
Li et al. (2021)
MFLU 20-0568
MW081545
MW081540
MW084994
MW084996
Li et al. (2021)
D. fasciculata
KUMCC 19-0081T
MW287775
MW286501
MW396656
N/A
Dong et al. (2021)
D. fluminicola
MFLUCC 15-0417T
KU376270
MF077553
N/A
N/A
Su et al. (2016)
D. fusiformis
GZCC 20-0512T
MZ868764
MZ868773
MZ892979
MZ892985
Yang et al. (2021)
D. gasaensis
HJAUP C2034T
OQ942891
OQ942896
OQ944455
N/A
Hu et al. (2023)
D. guanshanensis
HJAUP C1063T
OQ942898
OQ942894
OQ944452
OQ944458
Hu et al. (2023)
D. guizhouensis
GZCC 21-0666T
MZ474869
MZ474868
MZ501610
MZ501611
Hyde et al. (2021)
D. guttulata
MFLUCC 16-0183T
MF077554
MF077543
MF135651
N/A
Yang et al. (2018)
DLUCC B43
MN163016
MN163011
N/A
N/A
Luo et al. (2019)
D. hainanensis
GZCC 22-2047T
OR438894
OR427328
OR449122
OR449119
This study
D. hyalina
MFLUCC 17-2128T
MZ868760
MZ868769
MZ892976
MZ892981
Yang et al. (2021)
D. hydei
MFLUCC 20-0481T
MT742830
MT734661
N/A
MT767128
Monkai et al. (2020)
D. jinghongensis
HJAUP C2120T
OQ942893
OQ942897
OQ944456
N/A
Hu et al. (2023)
D. lancangjiangensis
KUN-HKAS 112712T
MW879522
MW723055
N/A
MW882260
Shen et al. (2021)
D. lanceolatispora
GZCC 22-2045T
OR43BB95
OR427329
OR449123
OR449120
This study
D. leonensis
HKUCC 10822
DQ408566
N/A
N/A
DQ435089
Shenoy et al. (2006)
D. licualae
MFLUCC 14-1163AT
ON650675
ON650686
ON734007
N/A
Konta et al. (2023)
MFLUCC 14-1163BT
ON650676
ON650687
ON734008
N/A
Konta et al. (2023) 1. Names, strain numbers, and corresponding GenBank accession numbers of taxa used in this study. 86 Xue-Mei Chen et al.: Identification of two new species and a new host record of Distoseptispora Taxa names
Strain
GenBank Accessions
References
LSU
ITS
tef1-α
rpb2
D. lignicola
MFLUCC 18-0198T
MK849797
MK828651
N/A
N/A
Luo et al. (2019)
D. longispora
HFJAU 0705T
MH555357
MH555359
N/A
N/A
Song et al. (2020)
D. longnanensis
HJAUP C1040T
OQ942886
OQ942887
OQ944451
N/A
Hu et al. (2023)
D. martinii
CGMCC 3.18651T
KX033566
KU999975
N/A
N/A
Xia et al. (2017)
D. meilingensis
JAUCC 4727T
OK562396
OK562390
OK562408
N/A
Zhai et al. (2022)
D. menghaiensis
HJAUP C2045T
OQ942900
OQ942890
N/A
N/A
Hu et al. (2023)
HJAUP C2170T
OQ942888
OQ942899
OQ944457
OQ944461
Hu et al. Phylogenetic analyses (2023)
D. mengsongensis
HJAUP C2126T
OP78784
OP787876
OP961937
N/A
Liu et al. (2023)
D. multiseptata
MFLUCC 16-1044
MF077555
MF077544
MF135652
MF135644
Yang et al. (2018)
MFLUCC 15-0609T
KX710140
KX710145
MF135659
N/A
Hyde et al. (2016)
D. nabanheensis
HJAUP C2003T
OP787877
OP787873
OP961935
N/A
Liu et al. (2023)
D. nanchangensis
HJAUP C1074T
OQ942895
OQ942889
OQ944454
OQ944460
Hu et al. (2023)
D. neorostrata
MFLUCC 18-0376T
MN163017
MN163008
N/A
N/A
Luo et al. (2019)
D. nonrostrata
KUNCC 21-10730T
OK341198
OK310699
OP413481
OP413475
Zhang et al. (2022)
D. obclavata
MFLUCC 18-0329T
MN163010
MN163012
N/A
N/A
Luo et al. (2019)
D. obpyriformis
MFLUCC 17-1694T
MG979764
N/A
MG988422
MG988415
Luo et al. (2018)
DLUCC 0867
MG979765
MG979757
MG988423
MG988416
Luo et al. (2018)
D. pachyconidia
KUMCC 21-10724T
OK341194
OK310696
OP413477
OP413471
Zhang et al. (2022)
D. palmarum
MFLUCC 18-1446T
MK079663
MK085062
MK087660
MK087670
Hyde et al. (2019)
D. phangngaensis
MFLUCC 16-0857T
MF077556
MF077545
MF135653
N/A
Yang et al. (2018)
D. phragmiticola
GUCC 22-0202T
OP749881
OP749888
OP749892
OP752700
Hyde et al. (2023)
D. rayongensis
MFLUCC 18-0415T
MH457137
MH457172
MH463253
MH463255
Hyde et al. (2020)
MFLUCC 18-0417
MH457138
MH457173
MH463254
MH463256
Hyde et al. (2020)
D. rostrata
MFLUCC 16-0969T
MG979766
MG979758
MG988424
MG988417
Luo et al. (2018)
DLUCC 0885
MG979767
MG979759
MG988425
N/A
Luo et al. (2018)
D. saprophytica
MFLUCC 18-1238T
MW287780
MW286506
MW396651
MW504069
Dong et al. (2021)
D. septata
GZCC 22-0078T
ON527947
ON527939
ON533683
ON533690
Ma et al. (2022)
D. sinensis
HJAUP C2044T
OP787875
OP787878
OP961936
N/A
Liu et al. (2023)
D. songkhlaensis
MFLUCC 18-1234T
MW287755
MW286482
MW396642
N/A
Dong et al. (2021)
D. suoluoensis
MFLUCC 17-0224T
MF077557
MF077546
MF135654
N/A
Yang et al. (2018)
MFLUCC 17-1305
MF077558
MF077547
N/A
N/A
Yang et al. (2018)
D. tectonae
MFLUCC 12-0291T
KX751713
KX751711
KX751710
KX751708
Hyde et al. (2016)
MFLU 20-0262
MT232719
MT232714
N/A
N/A
Sun et al. (2020)
MFLUCC 16-0946
MG979768
MG979760
MG988426
MG988418
Dong et al. (2021)
D. tectonae
GZCC 22-2046
OR348896
OR427330
OR449124
OR449121
This study
D. tectonigena
MFLUCC 12-0292T
KX751714
KX751712
N/A
KX751709
Hyde et al. (2016)
D. thailandica
MFLUCC 16-0270T
MH260292
MH275060
MH412767
N/A
Tibpromma et al. (2018)
D. thysanolaenae
KUN-HKAS 102247T
MK064091
MK045851
MK086031
N/A
Phukhamsak et al. (2019)
D. tropica
GZCC 22-0076T
ON527943
ON527935
ON533679
ON533687
Ma et al. (2022)
D. verrucosa
GZCC20-0434T
MZ868762
MZ868771
MZ892977
MZ892983
Yang et al. (2021)
D. wuzhishanensis
GZCC 22-0077T
ON527946
ON527938
ON533682
N/A
Ma et al. (2022)
D. MycoKeys 102: 83–105 (2024), DOI: 10.3897/mycokeys.102.115452 Phylogenetic analyses xishuangbannaensis
KUMCC 17-0290T
MH260293
MH275061
MH412768
MH412754
Tibpromma et al. (2018)
D. yichunensis
HJAUP C1065T
OQ942892
OQ942885
OQ944453
OQ944459
Hu et al. (2023)
D. yongxiuensis
JAUCC 4725T
OK562394
OK562388
OK562406
N/A
Zhai et al. (2022)
D. yunjushanensis
JAUCC 4723T
OK562398
OK562392
OK562410
N/A
Zhai et al. (2022)
D. yunnanensis
MFLUCC 20-0153T
MW081546
MW081541
MW084995
MW151861
Li et al. (2021)
Note: “T” denotes ex-type strain. Newly generated sequences are indicated in black bold. “N/A”: no data available in GenBank. MycoKeys 102: 83–105 (2024), DOI: 10.3897/mycokeys.102.115452 87 87 Xue-Mei Chen et al.: Identification of two new species and a new host record of Distoseptispora and Bayesian posterior probability (BYPP) methods. The analyses were based
on LSU, ITS, tef1-α, and rpb2 combined sequence datasets. The phylogenetic analyses were conducted using the CIPRES Science Gate-
way V. 3.3. “RAxML-HPC v.8 on XSEDE”, “PAUP on XSEDE”, and “MrBayes on
XSEDE (3.2.7a)” were utilized for ML, MP, and BYPP methods, respectively
(Huelsenbeck and Ronquist 2001; Swofford 2002; Stamatakis et al. 2008; Miller
et al. 2010; Ronquist et al. 2012). For the ML analysis, the GTRGAMMA model
of nucleotide evolution was employed, and RAxML rapid bootstrapping with
1,000 bootstrap replicates was obtained (Stamatakis et al. 2008). The MP analysis employed 1,000 random taxa additions to infer trees. Branches of zero length were collapsed, and all multiple parsimonious trees
were saved. The maxtrees value was set to 5,000. For trees generated using
different optimal criteria, parsimony score values were determined for tree
length (TL), consistency index (CI), retention index (RI), and homoplasy in-
dex (HI). To assess clade stability, the bootstrap (BT) method was used with
1,000 iterations, each consisting of 100 trials of random stepwise addition of
taxa (Hillis and Bull 1993). The posterior probabilities (PP) were determined based on Bayesian Markov
chain Monte Carlo sampling (Huelsenbeck and Ronquist 2001). The best nu-
cleotide substitution model for each data partition was determined using the
program MrModeltest 2.2 (Nylander 2004). The GTR + I + G substitution mod-
el with gamma rates and Dirichlet base frequencies was selected for all LSU,
ITS, tef1-α, and rpb2 sequences. To calculate the posterior probabilities, four
simultaneous Markov chains were run for one million generations, with trees
sampled every 100th generation, resulting in a total of 10,000 trees. Phylogenetic analyses A burn-in
parameter of 0.25 was set, indicating that 75% of the trees were remined during
the burn-in phase, and the remaining trees were used for calculating the poste-
rior probabilities in the majority rule consensus tree. FigTree v. 1.4.4. was used for visualizing the phylogenetic trees, and Adobe
Illustrator CC 2019v. 23.1.0 was used to edit trees and figure layout. Phylogenetic analyses results This study utilized a combined multi-gene dataset encompassing ITS, LSU,
tef1-α, and rpb2 sequences to assess the phylogenetic relationships among
Distoseptispora species. The analyses included a total of 90 taxa, designating
Aquapteridospora aquatica X.D. Yu, W. Dong & H. Zhang (MFLUCC 17-2371) as
the outgroup taxon. The combined aligned sequence matrix comprised 3,360
characters, including gaps: LSU (1–840 bp), ITS (841–1406 bp), tef1-α (1407–
2321 bp), and rpb2 (2322–3360 bp). The ML, MP, and Bayesian trees analyzed
exhibited a high degree of similarity in topology and showed no significant con-
flicts. The RAxML analysis yielded a best-scoring tree (ln = -31666.963504),
which is presented in Fig. 1. The matrix encompassed 1572 distinct align-
ment patterns, with 27.15% constituted by undetermined characters or gaps. The estimated base frequencies were as follows: A = 0.239306, C = 0.265297,
G = 0.281926, T = 0.213472; substitution rates AC = 1.429077, AG = 3.512798,
AT = 1.204511, CG = 0.845859, CT = 6.948345, GT = 1.000000; gamma distri-
bution shape parameter α = 0.244431. For the MP analysis, 3360 characters re-
mained unchanged, 330 were variable and parsimoniously uninformative, and MycoKeys 102: 83–105 (2024), DOI: 10.3897/mycokeys.102.115452 88 Xue-Mei Chen et al.: Identification of two new species and a new host record of Distoseptispora etic tree generated from ML analysis based on a combination of LSU, ITS, tef1-a,
pport values of ML and MP equal to or greater than 75%, and PP value equal to o
nodes as ML/PP/MP. The tree is rooted with Aquapteridospora aquatica (MFLUCC
ed by the superscript T. The new collections are in bold red text. Figure 1. Phylogenetic tree generated from ML analysis based on a combination of LSU, ITS, tef1-a, and rpb2 sequence
data. Bootstrap support values of ML and MP equal to or greater than 75%, and PP value equal to or greater than 0.95
are given near the nodes as ML/PP/MP. The tree is rooted with Aquapteridospora aquatica (MFLUCC 17-2371). Ex-type
strains are indicated by the superscript T. The new collections are in bold red text. MycoKeys 102: 83–105 (2024), DOI: 10.3897/mycokeys.102.115452 89 Xue-Mei Chen et al.: Identification of two new species and a new host record of Distoseptispora 1074 were parsimoniously informative. The most parsimonious tree yielded the
following values: TL = 5624, CI = 0.400, RI = 0.738, RC = 0.295, HI = 0.600. Phylogenetic analyses results For
BYPP analysis, Bayesian posterior probabilities from MCMC were evaluated
with a final average standard deviation of split frequencies of 0.009754. In the phylogenetic analyses (Fig. 1), all our newly identified taxa nested with-
in Distoseptispora, affirming their classification within this genus. Distoseptispo-
ra hainanensis (GZCC 22-047) formed a sister clade to D. multiseptata strains
(MFLUCC 16-1044 and MFLUCC 15-0609) with 98% ML, 1.00 PP, and 93% MP
statistical support. Distoseptispora lanceolatispora (GZCC 22-2045) formed a sis-
ter clade to D. neorostrata (MFLUCC 18-0376) with 100% ML, 1.00 PP, and 98% MP
statistical support. In addition, our new collection GZCC 22-2046 clustered togeth-
er with three D. tectonae strains (MFLU 20-0262 and MFLUCC 12-0291) with 98%
ML and 0.96 PP statistical support, indicating they represent the same species. Distoseptispora hainanensis X.M. Chen & Y.Z. Lu, sp. nov.
Index Fungorum: IF900953 Facesoffungi Number: FoF14663 Etymology. The epithet refers to the location “Hainan Province” where the ho-
lotype was collected. Holotype. GZAAS 22-2047. Holotype. GZAAS 22-2047. Description. Saprobic on decaying wood in terrestrial habitat. Sexual
morph: Undetermined. Asexual morph: Colonies on natural substrate superfi-
cial, effuse, dark brown, and hairy. Mycelium mostly immersed, composed of
branched, septate, brown to dark brown, smooth hyphae. Conidiophores 70–
130 × 5–8.5 μm (x– = 103 × 7 μm, n = 20), macronematous, mononematous,
erect, solitary, straight or slightly flexuous, brown to dark brown, paler towards
the apex, cylindrical, 4–6-septate, slightly constricted and darkened at septa,
unbranched, thick-walled. Conidiogenous cells 6–13 × 3.5–6.5 μm (x– = 10
× 5 μm, n = 20), holoblastic, monoblastic, integrated, terminal, indeterminate,
cylindrical, slightly tapering towards the apex, brown, percurrent. Conidia 44–
117 μm × 9–18.5 μm (x– = 90 × 14 μm, n = 20), acrogenous, solitary, obclavate
or obpyriform, rostrate, truncate at the base, straight or slightly curved, up to
22-distoseptate, slightly constricted at septa, brown, verrucose.l Culture characteristics. Colonies grown on PDA circular, dense, fluffy, with
raised center and lobate edge, pale gray in the center, grayish brown in the out-
er ring from the front view, dark brown in the center, and blackish brown in the
outer ring from the reverse view. Material examined. China, Hainan Province, on unidentified decaying wood,
15 May 2021, Xia Tang, HN02 (GZAAS 22-2047, holotype), ex-type living culture,
GZCC 22-2047. Notes. Morphologically, Distoseptispora hainanensis is similar to D. effusa
L.L. Liu & Z.Y. Liu in having macronematous conidiophores, monoblastic conid-
iogenous cells, and acrogenous, obclavate, rostrate conidia (Yang et al. 2021). However, conidia of D. hainanensis are up to 22-distoseptate, whereas those
of D. effusa are only 4–9-distoseptate. In the phylogenetic analyses, D. hainan- 90 MycoKeys 102: 83–105 (2024), DOI: 10.3897/mycokeys.102.115452 90 Xue-Mei Chen et al.: Identification of two new species and a new host record of Distoseptispora
Figure 2. Distoseptispora hainanensis (GZAAS 22-2047, holotype) a, b colonies on substrate c–e conidiophores and
conidia f–h conidiogenous cells bearing conidia i, j conidiophores k–q conidia r, s colony on PDA (r from front s from
reverse). Scale bars: 50 μm (c, d, f–j, l–q); 30 μm (e, k). Xue-Mei Chen et al.: Identification of two new species and a new host record of Distoseptispora Figure 2. Distoseptispora hainanensis (GZAAS 22-2047, holotype) a, b colonies on substrate c–e conidiophores and
conidia f–h conidiogenous cells bearing conidia i, j conidiophores k–q conidia r, s colony on PDA (r from front s from
reverse). Distoseptispora lanceolatispora X.M. Chen & Y.Z. Lu, sp. nov.
Index Fungorum: IF900954 Facesoffungi Number: FoF14664 Holotype. GZAAS 22-2047. Scale bars: 50 μm (c, d, f–j, l–q); 30 μm (e, k). MycoKeys 102: 83–105 (2024), DOI: 10.3897/mycokeys.102.115452 91 Xue-Mei Chen et al.: Identification of two new species and a new host record of Distoseptispora ensis formed a distinct clade sister to D. multiseptata Jiao Yang & K.D. Hyde
with 98% ML, 1 PP, and 93% MP statistical support (Fig. 1). Distoseptispora
hainanensis differs from D. multiseptata in having brown, longer conidiophores
(70–130 μm vs. 23–65 µm) and obclavate or obpyriform, brown, verrucose,
smaller conidia (44–117 μm vs. up to 290 µm) (Hyde et al. 2016). Comparing
DNA sequence data, D. hainanensis diverges from D. multiseptata (MFLUCC 15-
0609) in the ITS by 21/552 bp (3.8% difference), in the LSU by 1/812 bp (0.01%
difference), in tef1-α by 33/912 bp (3.6% difference), and no data is available
for rpb2 of D. multiseptata (MFLUCC 15-0609) in GenBank. Hence, the novel
species, D. hainanensis, is introduced, following the guidelines of Jeewon and
Hyde (2016) and Chethana et al. (2021). Etymology. Referring to the lanceolate conidia.
Holotype. GZAAS 22-2045. Description. Saprobic on submerged decaying wood in freshwater habitat. Sexual morph: Undetermined. Asexual morph: Colonies on substrate effuse,
gregarious, hairy, pale brown to brown. Mycelium mostly immersed, composed
of septate, yellow-brown to brown, smooth hyphae. Conidiophores 120–190
× 4–8 µm (x– = 155 × 6.5 µm, n = 20), macronematous, mononematous, erect,
solitary, straight or slightly flexuous, grayish brown to dark brown, slightly ta-
pering towards the apex, cylindrical, 7–8-septate, unbranched, thick-walled,
smooth-walled. Conidiogenous cells 15–27 × 3–5.5 µm (x– = 20.5 × 4.5 µm,
n = 20), monoblastic, integrated, terminal, cylindrical, slightly tapering towards
the apex, pale brown, percurrent. Conidia 31–90 × 9.5–15 µm (x– = 58.5 × 13 µm,
n = 20), acrogenous, solitary, fusiform or lanceolate, rostrate, truncate at the
base, straight or slightly curved, 5–13-distoseptate, slightly constricted at sep-
ta, olivaceous to olivaceous brown, slightly paler at the apex, verrucous, with or
without apical, hyaline appendages.l Culture characteristics. Colonies grown on PDA circular, dense, flat, dry, gray
to dark gray, radially striated, and a ring in the middle of the colonies with an
entire edge from the front view, dark brown to black with a circular, gray edge
from reverse view, not pigmented. Material examined. China, Hainan Province, on submerged decaying wood
in a freshwater stream, 23 October 2021, Jian Ma, J13 (GZAAS 22-2045, holo-
type), ex-type living culture, GZCC 22-2045. Notes. Distoseptispora lanceolatispora is morphologically similar to D. leonen-
sis (M.B. Ellis) R. Zhu & H. Zhang. However, compared to D. lanceolatispora, D. leonensis has longer conidiophores (120–190 µm vs. 110–130 µm), longer co-
nidiogenous cells (15–27 µm vs. 5–15 µm), and 5–13-distoseptate, fusiform or
lanceolate conidia (Zhang et al. 2022). In the phylogenetic analyses (Fig. 1), D. lanceolatispora forms a unique clade adjacent to D. neorostrata D.F. Bao, Z.L. Luo
& H.Y. Su with 100% ML, 1 PP, and 98% MP support. Based on a pairwise nucle-
otide comparison of ITS and LSU sequences, D. lanceolatispora deviates from 92 MycoKeys 102: 83–105 (2024), DOI: 10.3897/mycokeys.102.115452 92 Xue-Mei Chen et al.: Identification of two new species and a new host record of Distoseptispora Figure 3. Distoseptispora lanceolatispora (GZAAS 22-2045, holotype) a, b colonies on substrate c–e conidiophores and
conidia f, g conidiogenous cells bearing conidia h–k conidia l germinated conidium m, n colony on PDA (m from front
n from reverse). Scale bars: 50 μm (c–g); 30 μm (h–l). Figure 3. Etymology. Referring to the lanceolate conidia.
Holotype. GZAAS 22-2045. Distoseptispora lanceolatispora (GZAAS 22-2045, holotype) a, b colonies on substrate c–e conidiophores and
conidia f, g conidiogenous cells bearing conidia h–k conidia l germinated conidium m, n colony on PDA (m from front
n from reverse). Scale bars: 50 μm (c–g); 30 μm (h–l). MycoKeys 102: 83–105 (2024), DOI: 10.3897/mycokeys.102.115452 93 Xue-Mei Chen et al.: Identification of two new species and a new host record of Distoseptispora D. neorostrata by 39/529 bp (6.8%) for ITS and 14/850 bp (1.6%) for LSU, and
there is no data available for tef1-α and rpb2 for D. neorostrata (MFLUCC 18-0376)
in GenBank. Hence, we introduce the new species, D. lanceolatispora, based on
the criteria established by Jeewon and Hyde (2016) and Chethana et al. (2021). D. neorostrata by 39/529 bp (6.8%) for ITS and 14/850 bp (1.6%) for LSU, and
there is no data available for tef1-α and rpb2 for D. neorostrata (MFLUCC 18-0376)
in GenBank. Hence, we introduce the new species, D. lanceolatispora, based on
the criteria established by Jeewon and Hyde (2016) and Chethana et al. (2021). Description. Saprobic on dead twigs of Edgeworthia chrysantha. Sexual morph:
Undetermined. Asexual morph: Colonies on natural substrate abundant, super-
ficial, dark brown, hairy. Conidiophores 35–80 μm × 4–7.5 μm (x– = 58 × 5.5 μm,
n = 20), macronematous, mononematous, simple, erect to slightly curved, sol-
itary, pale brown to dark brown, cylindrical, 2–4-septate, slightly constricted
at the septa, unbranched, thick-walled. Conidiogenous cells 6–10 μm × 3.5–
6.5 μm (x– = 8 × 4.5 μm, n = 20), holoblastic, monoblastic, integrated, terminal,
cylindrical, slightly tapering towards the apex, brown to reddish brown, percur-
rent. Conidia 190–255 μm × 9.5–16 μm (x– = 220 μm × 13 μm, n = 20), 5–16 μm
(x– = 13 μm, n = 20) wide at the protruding truncate base; 4.5–8 μm (x– = 6.5 μm,
n = 20) wide in the tapering part, acrogenous, solitary, obclavate, elongate, ros-
trate, straight or curved, tapering towards the apex, 9–39-distoseptate, oliva-
ceous-green when young, dark reddish brown at maturity, verrucose. Culture characteristics. Conidia germinating on PDA within 24 h, colonies
circular, dense, umbonate, spreading, fluffy. The surface is slightly rough with
reddish-gray mycelium, colonies somewhat raised in the middle, and with a fili-
form edge. The reverse side is dark gray with a circular, pale reddish-gray edge,
not pigmented. Material examined. Etymology. Referring to the lanceolate conidia.
Holotype. GZAAS 22-2045. China, Guizhou Province, Guiyang City, Guiyang Medic-
inal Botanical Garden, on dead twigs of Edgeworthia chrysantha, 20 August
2022, Xia Tang, JX30 (GZAAS 22-2046), living culture, GZCC 22-2046. Known host and distribution. Tectona grandis (Thailand, Hyde et al. 2016), on
dead stems (Thailand, Sun et al. 2020), on dead, submerged, decaying wood of
unidentified plants (China & Thailand, Luo et al. 2019; Dong et al. 2021; Zhang et
al. 2022), and dead twig and branch of Edgeworthia chrysantha (China, this study).i Known host and distribution. Tectona grandis (Thailand, Hyde et al. 2016), on
dead stems (Thailand, Sun et al. 2020), on dead, submerged, decaying wood of
unidentified plants (China & Thailand, Luo et al. 2019; Dong et al. 2021; Zhang et
al. 2022), and dead twig and branch of Edgeworthia chrysantha (China, this study).i Notes. Distoseptispora tectonae was first isolated from a dead twig of
Tectona grandis in Thailand (Hyde et al. 2016). Since then, this species has
been identified in various countries on different substrates and hosts (Hyde
et al. 2016; Sun et al. 2020; Dong et al. 2021; Zhang et al. 2022). In the phy-
logenetic tree (Fig. 1), our new isolate forms a close lineage to D. tectonae
(GZCC 22-2046) with statistical support of 98% ML and 0.96 PP. Based on
pairwise nucleotide comparisons of ITS, LSU, tef1-α, and rpb2, our new isolate
diverges from D. tectonae (MFLUCC 12-0291, ex-type) by 6/554 bp (1%) for
ITS, 1/852 bp (0.01%) for LSU, 0/980 bp (0%) for tef1-α, and 2/899 bp (0.2%)
for rpb2. In addition, the morphological characteristics of our isolate match
well with the holotype description of D. tectonae (Hyde et al. 2016). This study
reports a new host record of Distoseptispora tectonae on dead twigs of Edge-
worthia chrysantha in China. MycoKeys 102: 83–105 (2024), DOI: 10.3897/mycokeys.102.115452 94 Xue-Mei Chen et al.: Identification of two new species and a new host record of Distoseptispora Figure 4. Distoseptispora tectonae (GZAAS 22-2046) a, b colonies on substrate c, d conidiophores and conidia
e, f conidiophores g–k conidia l germinated conidium m, n colonies on PDA (m from front n from reverse) Scale bars:
50 μm (c, d, g–l); 20 μm (e, f). Figure 4. Discussion Distoseptispora is one of the sporidesmium-like taxa and is well-known for its
asexual morph, which has considerable morphological variations (Su et al. 2016;
Yang et al. 2018, 2021). However, the phylogenetic analyses suggest a lack of cor-
relation between phylogenetic relationships and morphological analyses. For in-
stance, species such as D. appendiculata D.F. Bao, Z.L. Luo & H.Y. Su, D. atroviridis
J. Yang & K.D. Hyde, D. caricis Crous, D. fusiformis J. Yang & K.D. Hyde, D. lanceo-
latispora, D. leonensis, D. neorostrata, D. palmarum S.N. Zhang, K.D. Hyde & J.K. Liu,
and D. saprophytica W. Dong, H. Zhang & K.D. Hyde cluster together as a subclade
in the phylogenetic tree (see Fig. 1). In contrast, morphological analysis reveals
significant differences, especially in the characteristics of conidiophores, conidiog-
enous cells, and conidia (Crous et al. 2019; Hyde et al. 2019; Luo et al. 2019; Dong
et al. 2021; Yang et al. 2021; Zhang et al. 2022). This disparity is common within
the genus. We recommend adopting a combination approach using molecular and
morphological methods for more effective identification within this genus. Worth noting, among the various species of Distoseptispora, D. martinii (J.L. Crane & Dumont) J.W. Xia & X.G. Zhang stands out due to its unique morpho-
logical characteristics, especially its oblate or subglobose conidia, distinguish-
ing it from other species within Distoseptispora (Xia et al. 2017). The species
was initially introduced as Acrodictys martinii J.L. Crane & Dumont by Crane
and Dumont (1975) based on morphological characteristics. Then, it underwent
several taxonomic revisions based solely on morphology (Baker et al. 2002;
Delgado 2009). Later, Xia et al. (2017) reclassified Acrodictys martinii as D. mar-
tinii based on genetic analysis. However, the morphological traits of D. martinii
greatly diverge from typical Distoseptispora features (Crane and Dumont 1975;
Xia et al. 2017). Therefore, we suggest additional collections and analysis of
D. martinii specimens to ensure the reliability of the provided DNA sequence
data. In recent years, Distoseptispora species have been reported worldwide, such
as in China, Hungary, Hawaii, Malaysia, and Thailand (Shoemaker and White
1985; McKenzie 1995; Wu and Zhuang 2005; Zhang et al. 2022). Studies on Dis-
toseptispora have been particularly extensive in China and Thailand (Hyde et al. 2016, 2019, 2020; Su et al. 2016; Yang et al. 2018, 2021; Luo et al. 2019; Sun et
al. 2020; Hu et al. 2023). Etymology. Referring to the lanceolate conidia.
Holotype. GZAAS 22-2045. Distoseptispora tectonae (GZAAS 22-2046) a, b colonies on substrate c, d conidiophores and conidia
e, f conidiophores g–k conidia l germinated conidium m, n colonies on PDA (m from front n from reverse) Scale bars:
50 μm (c, d, g–l); 20 μm (e, f). Figure 4. Distoseptispora tectonae (GZAAS 22-2046) a, b colonies on substrate c, d conidiophores and conidia
e, f conidiophores g–k conidia l germinated conidium m, n colonies on PDA (m from front n from reverse) Scale bars:
50 μm (c, d, g–l); 20 μm (e, f). MycoKeys 102: 83–105 (2024), DOI: 10.3897/mycokeys.102.115452 95 Xue-Mei Chen et al.: Identification of two new species and a new host record of Distoseptispora Xue-Mei Chen et al.: Identification of two new species and a new host record of Distoseptispora MycoKeys 102: 83–105 (2024), DOI: 10.3897/mycokeys.102.115452 Discussion To date, 73 species of Distoseptispora have been doc-
umented, of which 55 have been recorded in China (including known species,
see Table 2). Our collections further highlight the distribution of the genus in Table 2. Distoseptispora species and their locations, lifestyles, habitats, hosts, and corresponding references. Species
Country
Habitat
Host
References
D. adscendens
China; Hungary;
Hawaii
Terrestrial
Decaying wood and decaying branches of
many woody plant species; Platanus orientalis
Shoemaker et al. (1985); McKenzie et al. (1995); Wu et al. (2005); Zhang et al. (2022)
D. amniculi
Thailand
Freshwater
Submerged decaying wood
Yang et al. (2021)
D. appendiculata
Thailand
Freshwater
Submerged decaying wood
Luo et al. (2019)
D. aqualignicola
China
Freshwater
Submerged decaying wood
Zhang et al. (2022)
D. aquamyces
China
Freshwater
Submerged decaying wood
Zhang et al. (2022)
D. aquatica
China
Freshwater
Submerged decaying wood
Su et al. (2016); Luo et al. (2019); Li et al. (2021)
D. aquisubtropica
China
Freshwater
Submerged decaying wood
Ma et al. (2022) s and their locations, lifestyles, habitats, hosts, and corresponding references. MycoKeys 102: 83–105 (2024), DOI: 10.3897/mycokeys.102.115452 96 Xue-Mei Chen et al.: Identification of two new species and a new host record of Distoseptispora 97
MycoKeys 102: 83 105 (2024) DOI: 10 3897/mycokeys 102 115452
Species
Country
Habitat
Host
References
D. atroviridis
China
Freshwater
Submerged decaying wood
Yang et al. (2021)
D. bambusae
China
Terrestrial
Decaying bamboo culms
Sun et al. (2020)
D. bambusicola
China
Freshwater
Submerged bamboo culms
Jayawardena et al. (2022)
D. bangkokensis
Thailand
Freshwater
Submerged decaying wood
Shen et al. (2021)
D. cangshanensis
China
Freshwater
Submerged decaying wood
Luo et al. (2018)
D. caricis
Thailand
Terrestrial
Leaves of Carex sp. Crous et al. (2019)
D. chinensis
China
Freshwater
Submerged decaying wood
Hyde et al. (2021)
D. clematidis
China; Thailand
Freshwater;
Terrestrial
Dried stem of Clematis sikkimensis;
submerged decaying wood
Phukhamsakda et al. (2020); Shen et al. (2021)
D. crassispora
China
Freshwater
Submerged decaying wood
Zhang et al. (2022)
D. curvularia
China
Freshwater
Submerged decaying wood
Zhang et al. (2022)
D. cylindricospora
China
Freshwater
Submerged decaying wood
Phukhamsakda et al. (2022)
D. dehongensis
China; Thailand
Freshwater
Submerged decaying wood
Hyde et al. (2019); Zhang et al. (2022)
D. dipterocarpi
Thailand
Terrestrial
Woody litter of Dipterocarpus sp. Afshari et al. (2023)
D. effusa
China
Freshwater
Submerged decaying wood
Yang et al. (2021)
D. euseptata
China
Freshwater
Submerged decaying wood
Li et al. (2021)
D. Discussion fasciculata
Thailand
Freshwater
Submerged decaying wood
Dong et al. (2021)
D. fluminicola
China
Freshwater
Submerged decaying wood
Su et al. (2016); Luo et al. (2018)
D. fusiformis
China
Freshwater
Submerged decaying wood
Yang et al. (2021)
D. gasaensis
China
Terrestrial
Decaying branches of broadleaf tree
Hu et al. (2023)
D. guanshanensis
China
Terrestrial
Decaying branches of broadleaf tree
Hu et al. (2023)
D. guizhouensis
China
Terrestrial
Decaying wood
Hyde et al. (2021)
D. guttulata
Thailand
Freshwater
Submerged decaying wood
Yang et al. (2018); Luo et al. (2019)
D. hainanensis
China
Terrestrial
Decaying wood
This study
D. hyalina
Thailand
Freshwater
Submerged decaying wood
Yang et al. (2021)
D. hydei
Thailand
Terrestrial
Decaying bamboo culms
Monkai et al. (2020)
D. jinghongensis
China
Terrestrial
Decaying branches of broadleaf tree
Hu et al. (2023)
D. lancangjiangensis
China
Freshwater
Submerged decaying wood
Shen et al. (2021)
D. lanceolatispora
China
Freshwater
Submerged decaying wood
This study
D. leonensis
China; Malaysia
Terrestrial
Decaying culms of grasses or decaying
branches
McKenzie et al. (1995); Wu et al. (2005); Zhang
et al. (2022)
D. licualae
Thailand
Terrestrial
Decaying leaves of Licuala glabra
Konta et al. (2023)
D. lignicola
China; Thailand
Freshwater
Submerged decaying wood
Luo et al. (2019); Yang et al. (2021)
D. longispora
China
Freshwater
Submerged decaying wood
Song et al. (2020)
D. longnanensis
China
Terrestrial
Decaying branches of broadleaf tree
Hu et al. (2023)
D. martinii
China
Terrestrial
Decaying branches
Xia et al. (2017)
D. meilingensis
China
Freshwater
Decaying bamboo culms
Zhai et al. (2022)
D. menghaiensis
China
Terrestrial
Decaying branches of broadleaf tree
Hu et al. (2023)
D. menglunensis
China
Terrestrial
Decaying branches of broadleaf tree
Hu et al. (2023)
D. mengsongensis
China
Terrestrial
Decaying branches
Liu et al. (2023)
D. multiseptata
Thailand
Freshwater
Submerged decaying wood
Hyde et al. (2016); Yang et al. (2018)
D. nabanheensis
China
Terrestrial
Decaying branches
Liu et al. (2023)
D. nanchangensis
China
Terrestrial
Decaying branches of broadleaf tree
Hu et al. (2023)
D. neorostrata
Thailand
Freshwater
Submerged decaying wood
Luo et al. (2019)
D. nonrostrata
China
Freshwater
Submerged decaying wood
Zhang et al. (2022)
D. obclavata
Thailand
Freshwater
Submerged decaying wood
Luo et al. (2019)
D. obpyriformis
China
Freshwater
Submerged decaying wood
Luo et al. (2018)
D. pachyconidia
China
Freshwater;
Terrestrial
Submerged decaying wood; decaying wood
Ma et al. (2022); Zhang et al. (2022)
D. palmarum
Thailand
Terrestrial
Rachis of Cocos nucifera
Hyde et al. (2019)
D. MycoKeys 102: 83–105 (2024), DOI: 10.3897/mycokeys.102.115452 Funding This work was funded by the National Natural Science Foundation of China (NSFC
32360011). Conflict of interest The authors have declared that no competing interests exist. Discussion phangngaensis
Thailand
Freshwater
Submerged decaying wood
Yang et al. (2018)
D. phragmiticola
China
Terrestrial
Decaying Phragmites australis
Hyde et al. (2023)
D. rayongensis
Thailand
Freshwater
Submerged decaying wood
Hyde et al. (2020)
D. rostrata
China
Freshwater
Submerged decaying wood
Luo et al. (2018)
D. saprophytica
Thailand
Freshwater
Submerged decaying wood
Dong et al. (2021) 97 Xue-Mei Chen et al.: Identification of two new species and a new host record of Distoseptispora Species
Country
Habitat
Host
References
D. septata
China
Freshwater
Submerged decaying wood
Ma et al. (2022)
D. sinensis
China
Terrestrial
Decaying branches
Liu et al. (2023)
D. songkhlaensis
Thailand
Freshwater
Submerged decaying wood
Dong et al. (2021)
D. suoluoensis
China
Freshwater
Submerged decaying wood
Yang et al. (2018)
D. tectonae
China; Thailand
Terrestrial;
Freshwater
Decaying twig of Tectona grandis; stems
of dead wood; submerged decaying wood;
decaying twigs of Edgeworthia chrysantha
Hyde et al. (2016); Luo et al. (2018);
Sun et al. (2020); Dong et al. (2021); Li et al. (2021); Zhang et al. (2022); This study
D. tectonigena
Thailand
Terrestrial
Decaying twig of Tectona grandis
Hyde et al. (2016)
D. thailandica
Thailand
Terrestrial
Decaying leaves of Pandanus sp. Tibpromma et al. (2018)
D. thysanolaenae
China
Terrestrial;
Freshwater
Decaying culms of Thysanolaena maxima;
Submerged decaying wood
Phookamsak et al. (2019); Shen et al. (2021)
D. tropica
China
Terrestrial
Decaying wood
Ma et al. (2022)
D. verrucosa
China
Freshwater
Submerged decaying wood
Yang et al. (2021)
D. wuzhishanensis
China
Freshwater
Submerged decaying wood
Ma et al. (2022)
D. xishuangbannaensis
China
Terrestrial;
Freshwater
Decaying leaves of Pandanus utilis;
submerged decaying wood
Tibpromma et al. (2018); Ma et al. (2022)
D. yichunensis
China
Terrestrial
Decaying branches of broadleaf tree
Hu et al. (2023)
D. yongxiuensis
China
Freshwater
Decaying bamboo culms
Zhai et al. (2022)
D. yunjushanensis
China
Freshwater
Decaying bamboo culms
Zhai et al. (2022)
D. yunnanensis
China
Freshwater
Submerged decaying wood
Li et al. (2021) China, and we speculate that the country may harbor a greater diversity of the
genus. Thus, future studies are needed to validate this hypothesis. Acknowledgments The authors thank Shaun Pennycook, Manaaki Whenua – Landcare Research,
New Zealand, for his guidance on the fungal nomenclature and the suggestion
on naming the new taxa. The authors also thank the Guizhou Institute of Tech-
nology for its support of the experiment. Samantha Chandranath Karunarathna
thanks the National Natural Science Foundation of China (Numbers 32260004)
and the High-Level Talent Recruitment Plan of Yunnan Province (“High-End For-
eign Experts” program) for their support. Additional information Ethical statement No ethical statement was reported. Author ORCIDs Xue-Mei Chen https://orcid.org/0009-0004-8631-0735
Xia Tang https://orcid.org/0000-0003-2705-604X
Jian Ma https://orcid.org/0009-0008-1291-640X
Ning-Guo Liu https://orcid.org/0000-0002-9169-2350
Saowaluck Tibpromma
https://orcid.org/0000-0002-4706-6547
Samantha C. Karunarathna https://orcid.org/0000-0001-7080-0781
Yuan-Pin Xiao https://orcid.org/0000-0003-1730-3545
Yong-Zhong Lu
https://orcid.org/0000-0002-1033-5782 Author contributions Conceptualization - Xue-Mei Chen and Yong-Zhong Lu; data curation - Xue-Mei Chen, Xia
Tang, Jian Ma, Ning-Guo Liu; formal analysis - Yuan-Pin Xiao, Xue-Mei Chen, Xia Tang,
Jian Ma; funding acquisition - Yong-Zhong Lu; investigation - Saowaluck Tibpromma, Sa- MycoKeys 102: 83–105 (2024), DOI: 10.3897/mycokeys.102.115452 98 Xue-Mei Chen et al.: Identification of two new species and a new host record of Distoseptispora mantha C. Karunarathna, Yuan-Pin Xiao, Yong-Zhong Lu; methodology - Xue-Mei Chen,
Yong-Zhong Lu; project administration - Yuan-Pin Xiao, Yong-Zhong Lu; resources -
Yong-Zhong Lu, Saowaluck Tibpromma, Samantha C. Karunarathna; software - Xue-Mei
Chen; supervision - Yong-Zhong Lu, Saowaluck Tibpromma, Samantha C. Karunarathna;
validation - Xue-Mei Chen, Xia Tang, Jian Ma, Ning-Guo Liu; visualization - Saowaluck
Tibpromma, Samantha C. Karunarathna; writing original draft - Xue-Mei Chen; writing,
review and editing - Xue-Mei Chen, Xia Tang, Jian Ma, Ning-Guo Liu, Saowaluck Tibprom-
ma, Samantha C. Karunarathna, Yuan-Pin Xiao, Yong-Zhong Lu. All authors have read
and agreed to the published version of the manuscript. Data availability All of the data that support the findings of this study are available in the main text. References Afshari N, Gomes de Farias AR, Bhunjun CS, Phukhamsakda C, Hyde KD, Lumyong S
(2023) Distoseptispora dipterocarpi sp. nov. (Distoseptisporaceae), a lignicolous fun-
gus on decaying wood of Dipterocarpus in Thailand. Current Research in Environmen-
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Biology Centre, Uppsala University, Uppsala. Phookamsak R, Hyde KD, Jeewon R, Bhat DJ, Jones EBG, Maharachchikumbura SSN, Raspé O, Karunarathna SC, Wanasinghe DN, Hongsanan S, Doilom M, Tennakoon DS, Machado AR, Firmino AL, Ghosh A, Karunarathna A, Mešić A, Dutta AK, Thongbai B, Devadatha B, Norphanphoun C, Senwanna C, Wei DP, Pem D, Ackah FK, Wang GN, Ji- ang HB, Madrid H, Lee HB, Goonasekara ID, Manawasinghe IS, Kušan I, Cano J, Gené J, Li JF, Das K, Acharya K, Raj KNA, Latha KPD, Chethana KWT, He MQ, Dueñas M, Jadan M, Martín MP, Samarakoon MC, Dayarathne MC, Raza M, Park MS, Telleria MT, Chaiwan N, Matočec N, Silva NI, Pereira OL, Singh PN, Manimohan P, Uniya P, Shang QJ, Bhatt RP, Perera RH, Alvarenga RLM, Nogal-Prata S, Singh SK, Vadthanarat S, Oh SY, Huang SK, Rana S, Konta S, Paloi S, Jayasiri SC, Jeon SJ, Mehmood T, Gibertoni TB, Nguyen TTT, Singh U, Thiyagaraja V, Sarma VV, Dong W, Yu XD, Lu YZ, Lim YW,
Chen Y, Tkalčec Z, Zhang ZF, Luo ZL, Daranagama DA, Thambugala KM, Tibpromma
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00448-4 Phukhamsakda C, Nilsson RH, Bhunjun CS, Farias ARGD, Sun YR, Wijesinghe SN, Raza
M, Bao DF, Lu L, Tibpromma S, Dong W, Tennakoon DS, Tian XG, Xiong YR, Karunarath-
na SC, Cai L, Luo ZL, Wang Y, Manawasinghe IS, Camporesi I, Kirk PM, Promputtha I,
Kuo CH, Su HY, Doilom M, Li Y, Fu YP, Hyde KD (2022) The numbers of fungi: Contri- MycoKeys 102: 83–105 (2024), DOI: 10.3897/mycokeys.102.115452 103 Xue-Mei Chen et al.: Identification of two new species and a new host record of Distoseptispora butions from traditional taxonomic studies and challenges of metabarcoding. Fungal
Diversity 114: 327–386. https://doi.org/10.1007/s13225-022-00502-3 butions from traditional taxonomic studies and challenges of metabarcoding. Fungal
Diversity 114: 327–386. https://doi.org/10.1007/s13225-022-00502-3 Rehner SA, Samuels GJ (1994) Taxonomy and phylogeny of Gliocladium analysed from
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634. https://doi.org/10.1016/S0953-7562(09)80409-7 Ronquist F, Teslenko M, Van Der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 32: Efficient Bayesian Phylogenet-
ic Inference and model choice across a large model space. Systematic Biology 61:
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Distoseptispora longispora sp. Nov. from freshwater habitats in China. Mycotaxon
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Qeios · Definition, February 7, 2020 Open Peer Review on Qeios 4: 76912750-76861526 National Cancer Institute Qeios ID: ZCH5UD · https://doi.org/10.32388/ZCH5UD Source National Cancer Institute. 4: 76912750-76861526. NCI Thesaurus. Code C41935. National Cancer Institute. 4: 76912750-76861526. NCI Thesaurus. Code C41935. Physical location of CDKL2_Gene Qeios ID: ZCH5UD · https://doi.org/10.32388/ZCH5UD 1/1
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https://www.mdpi.com/1422-8599/2002/1/M282/pdf?version=1238585648
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en
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(1-(Phenanthrolo[5,6-d]imidazol-2-yl)-3-(4,5-diphenylimidazol-2-yl)benzene
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Molbank
| 2,003
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cc-by
| 361
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1-(Phenanthrolo[5,6-d]imidazol-2-yl)-3-(4,5-diphenylimidazol-2-yl)benzene
1 of 2
http://mdpi.org/molbank/molbank2002/m0282.htm
Molbank 2002, M282
www.molbank.org
(1-(Phenanthrolo[5,6-d]imidazol-2-yl)-3-(4,5-diphenylimidazol-2-yl)benzene
Hui Chao*, Cai-Wu Jiang, Hao Zhang and Liang-Nian Ji**
State Key Laboratory of Optoelectronic Materials and Technologies / Department of Chemistry, Zhongshan
University, Guangzhou 510275, P. R. China
E-mail: *ceschh@zsu.edu.cn, **cesjln@zsu.edu.cn
Received: 2 April 2002 / Accepted: 16 May 2002 / Published: 20 February 2003
Keywords: polypyridine ligands, phenanthroline, benzil
2-(3-Formylphenyl)imidazo[4,5-f][1,10]phenanthroline was prepared by a previously published method [1]. A
mixture of 2-(3-formylphenyl)imidazo[4,5-f][1,10]phenanthroline (0.25 g, 0.77 mmol), benzil (0.16 g, 0.77
mmol), ammonium acetate (1.19 g, 15.4 mmol) and glacial acetic acid (10 cm3) was refluxed for about 2 h.
The cooled solution was filtered, diluted with water (about 60 cm3) and neutralized with concentrated
aqueous ammonia. The precipitate was collected and purified by column chromatography on alumina with
ethanol-toluene (1:2 v/v) as eluent to give the title compound as a pale yellow powder. Yield 0.27 g, 67.7%.
1
H NMR (500 MHz, d6-DMSO): 13.95 (s, 1H), 13.07 (s, 1H), 9.24 (s, 1H), 9.05 (d, 2H, J = 8), 9.00 (d, 2H, J
= 8), 8.29 (d, 1H, J = 8), 8.25 (d, 1H, J = 8), 7.93 (d, 2H, J = 6.5), 7.83 (m, 3H), 7.74 (t, 1H, J = 8), 7.57 (d,
2H, J = 7), 7.57 (t, 1H, J = 8), 7.36 (t, 2H, J = 8), 7.28 (t, 2H, J = 7.0).
13
C NMR (125 MHz, d6-DMSO): 150.3, 147.8, 145.0, 143.7, 137.4, 135.7, 135.1, 131.1, 130.9, 130.5, 129.6,
129.3, 128.5, 128.1, 127.7, 127.2, 126.6, 126.1, 125.9, 123.7, 123.3, 123.0, 119.3.
IR (KBr. cm-1): 3430, 1629, 1564, 1481, 1451, 1412, 1385, 1352, 1259, 1077, 1026, 1001, 968, 809, 766,
737, 698.
UV-Vis (l, nm, in ethanol): 288, 304.
FAB-MS ([M+1]+): 515.
Acknowledgement: Support for this research provided by the National Natural Science Foundation of China,
the National Science Foundation of Guangdong Province and Research Fund of Royal Society of Chemistry
U.K. is gratefully acknowledged.
References and Notes
1. Chao, H.; Ye, B.-H.; Li, H.; Li, R.-H.; Zhou, J.-Y.; Ji, L.-N. Polyhedron 2000, 19, 1975.
Sample availabylitiy: available from the authors
4/1/2009 1:33 PM
1-(Phenanthrolo[5,6-d]imidazol-2-yl)-3-(4,5-diphenylimidazol-2-yl)benzene
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http://mdpi.org/molbank/molbank2002/m0282.htm
© 2002 MDPI. All rights reserved.
4/1/2009 1:33 PM
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Amharic
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PENERJEMAHAN DOKUMEN INGGRIS-INDONESIA MENGGUNAKAN MESIN PENERJEMAH STATISTIK DENGAN WORD REORDERING DAN PHRASE REORDERING
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Jurnal ilmu komputer dan informasi (Journal of computer science and information)/Jurnal Ilmu Komputer dan Informasi
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|
https://openalex.org/W4214525354
|
https://link.springer.com/content/pdf/10.1007/s13202-022-01462-9.pdf
|
English
| null |
A 2-D analytical solution to infinite conductivity fracture injection pressure transient analysis in non-Newtonian/Newtonian composite infinite reservoirs
|
Journal of petroleum exploration and production technology
| 2,022
|
cc-by
| 9,300
|
Abstractl Two fluid regions are usually created during either water or polymer injection into an oil reservoir for enhanced oil recov-
ery. The two fluid regions create a composite reservoir system that requires analytical solution that can be used to evaluate
the injection performance. In this study, we present a 2-D solution for non-Newtonian fluid in the inner zone coupled with
infinite boundary in the outer zone using power law model. The outer zone fluid can either be Newtonian or non-Newtonian. Moreover, the mathematical model is formulated such that either zone can be with non-Newtonian power law fluid or New-
tonian fluid. The infinite conductivity fractured solution obtained was compared with the ones obtained using the radial line
source solution that had already been developed in the literatures, and a perfect match was obtained. An application was
made to three examples that have been presented in a previous work, and satisfactory results were obtained. The dimension-
less pressure drops during the early linear flow regime for all values of flow indices are the same, but with deviation during
radial flow regime. Keywords Power-law (shear thinning) fluid · Well test (Pressure transient) analysis · Infinite conductivity hydraulic
fracture · Non-Newtonian · Newtonian fluid interface · Polymer front List of symbols
B
Formation volume factor, rm3/stm3
CD
Dimensionless wellbore storage
ct
Total compressibility Pa−1
h
Formation thickness, m
k
Formation permeability, m2
m, n
Flow behavior index (power-law parameter),
dimensionless
PD'
Dimensionless wellbore pressure derivative
PD
Dimensionless wellbore pressure drop
Pi
Initial pressure, Pa
PwD
Dimensionless wellbore flowing pressure
PD
Dimensionless pressure drop in Laplace space
PWD
Dimensionless pressure drop in Laplace space
q
Volumetric flow rate,m3/sec
gw
G Radial distance at wellbore, m
RD
Dimensionless radial distance at any point, (r/xf)
S
Skin factor, dimensionless
t
Time, s
t*ΔP'
Pressure derivative, Pa
tD
Dimensionless time
x, y
Length in x and y directions, respectively
xf
Fracture half length
l
Matrix characteristic length
j
Number of normal sets of fractures; j = 1, 2, 3. Journal of Petroleum Exploration and Production Technology (2022) 12:2453–2465
https://doi.org/10.1007/s13202-022-01462-9 Journal of Petroleum Exploration and Production Technology (2022) 12:2453–2465
https://doi.org/10.1007/s13202-022-01462-9 ORIGINAL PAPER-PRODUCTION ENGINEERING A 2‑D analytical solution to infinite conductivity fracture injection
pressure transient analysis in non‑Newtonian/Newtonian composite
infinite reservoirs Mojeed Olawale Oluogun1 · Alpheus O. Igbokoyi1 Received: 8 October 2021 / Accepted: 17 January 2022 / Published online: 1 March 2022
© The Author(s) 2022 * Mojeed Olawale Oluogun
moluogun@aust.edu.ng 1
African University of Science and Technology, Abuja,
Nigeria Introduction Injecting foreign immiscible fluid into a reservoir or porous
media will create a composite feature within the reservoir. In the oil exploitation, polymer, water or waste slurry can
be injected into a reservoir for the purpose of enhanced oil
recovery or waste disposal. Monitoring the injected fluid’s
front is therefore necessary. Lund and Ikoku (1981) pre-
sented a numerical radial model for a composite reservoir
with non-Newtonian fluid in the inner region (Zone 1)
and Newtonian fluid in the outer region (Zone 2). Martha
and Ertekin (1983) presented a Numerical Simulation of
a Power-Law Fluid Flow in a Vertically Fractured Reser-
voir with finite conductivity. Ertekin et al. (1987) also pre-
sented another numerical method with similar composite
reservoir as in Lund and Ikoku, but used finite conductivity
hydraulic fracture. Prior to Ertekin et al.’s work, Ikoku and
Ramey (1979), and Odeh and Yang (1979) have presented
radial analytical line source solutions for a non-Newtonian
fluid flow in porous media using power law model. The
two solutions are similar and essentially the same in ana-
lytical approach. Vongvuthipornchai and Raghavan (1987)
presented a numerical method for evaluating infinite con-
ductivity hydraulic fracture with non-Newtonian power law
fluid flow in porous media. They observed that the dimen-
sionless pressure drops during early linear flow regime are
the same for all flow behavior indices. Raghavan and Chen
(2017) also presented a new analytical line source solution
for non-Newtonian fluid, which was used to compute the
dimensionless pressure drop for infinite conductivity hydrau-
lic fracture. The early linear flow is not present in the work
of Raghavan and Chen (2017) as observed in the numerical
work of Vongvuthipornchai and Raghavan (1987). Fig. 1 Reservoir conceptual model not provide a matching in their field examples to capture the
entire behavior of the pressure transient data. We compared our 2-D analytical solution with the results
obtained using Ikoku and Ramey (1979) and Odeh and Yang
line source solutions to obtain infinite conductivity hydraulic
fracture solution; we obtained a good match. However, our
result is at variant with that of Raghavan and Chen (2017)
when the flow behavior index is less than 1.0. We also used
the 2-D solution we obtained, applying Cinco-Ley and
Meng’s (1988) method to compute the dimensionless pres-
sure drop for finite conductivity fracture and compared the
results with that of Ertekin et al. Abstractl Γ(x)
Gamma (factorial) function
ϕ
Porosity, fraction of bulk volume
λ
Interporosity flow parameter, dimensionless
μ
Viscosity, pa-s
θ
Effective mobility ratio for a two-region compos-
ite reservoir
ω
Storage capacity ratio, dimensionless
π
3.142
Subscripts
1
Referring to Zone 1, inner region
2
Referring to Zone 2, outer region for two-region
composite reservoir
D
Dimensionless quantity
m
Matrix
f
Fracture
* Mojeed Olawale Oluogun
moluogun@aust.edu.ng
1
African University of Science and Technology, Abuja,
Nigeria 23456789)
3 Journal of Petroleum Exploration and Production Technology (2022) 12:2453–2465 2454 x
y
-y
-x
Zone 2
Zone 1
In-situ Fluid
Polymer
Fracture
As an injector, fluid flows from
Zone 1 to Zone 2. Direc on of flow
Fig. 1 Reservoir conceptual model Introduction (1987); we obtained a fairly
perfect match that gave us confidence in our solution. van
den Hoek et al. (2012) presented three field examples which
practically demonstrated the problem we are addressing in
this study. We used our 2-D model to match the pressure
transient data and a good match was obtained. Thus, our
2-D model presented a new analytical method for analyzing
fractured polymer or waste/drilling cuttings slurry injection
well where two fluid zones are created due to the mobility
and diffusivity ratios, and different flow behavior index in
the two zones. Essentially, the major contribution of this
study is the presentation of analytical solution that can be
used to evaluate pressure behavior in a composite reservoir
with two zones of different flow behavior index. It could be
either non-Newtonian/Newtonian or non-Newtonian/non-
Newtonian as the case may be. In Well Test Analysis, meth-
ods of estimating formation parameters are usually derived
from the analytical solution. In this study, we developed a 2-D analytical solution for
infinite conductivity hydraulic fracture in a composite reser-
voir with non-Newtonian fluid in the inner region (Zone 1)
and Newtonian fluid in the outer region (Zone 2) as shown
in Fig. 1. It is a general solution which can also be used for
non-Newtonian fluid with different flow behavior index in
Zone 1 and Zone 2. To the best of our knowledge, this ana-
lytical solution for a composite reservoir is appearing in the
literature for the first time. Previous analytical works (such
as Ambastha (1988) and, Abbaszadeh and Kamal (1989)) are
on composite reservoir with Newtonian fluids in both Zone1
and Zone 2. van den Hoek et al. (2012) provided a method of
analyzing pressure transient behavior in fractured polymer
injection well. van den Hoek’s method of evaluation is to
identify the non-Newtonian radial flow regime in the inner
region (Zone 1) and fixed a straight line of the radial flow
regime to estimate the flood front. van den Hoek et al. did The analytical solution in Laplace space is numeri-
cally converted to real-time space using Stehfest (1970)
Algorithm 368. 1 3 Journal of Petroleum Exploration and Production Technology (2022) 12:2453–2465 2455 Mathematical model We have assumed homogeneous isotropic formation;
hence, the relative permeability in both directions is taken
to be unity.i The linearized partial differential equation for naturally frac-
tured reservoir (NFR) is The solution for an infinite system (Carslaw and Jaeger,
1959) is: (1)
xn−1
D
휕2PDf
휕x2
D
+ yn−1
D
휕2PDf
휕y2
D
−sf(s)PDf = 0
(2)
f(s) = 휔(1 −휔)s + 휆
(1 −휔)s + 휆 The governing equation for Zone 2 in an infinite system is
(7)
PD1
xw, yw ≤xD, yD, ≤∞ = CgvKv
g
√
sfs
(1)
(7)
PD1
xw, yw ≤xD, yD, ≤∞ = CgvKv
g
√
sfs
(1) (7) The governing equation for Zone 2 in an infinite system is (2) (8)
PD2
xDi, yDi ≤xD, yD ≤∞ = C3gv2KV2
g
√
훼sfs
(8) All the dimensionless variables and derivations are in
“Appendix A”. Wellbore storage CD and skin S can be incorporated in the
wellbore solutions in the Laplace space as shown in Eq. 9. (3)
휔=
(휑ct)f
(휑ct)f + (휑ct)m
(4)
λ = 훿
kmx2
f
kf (9)
PwD
(1, s, CD, S) =
sPwD(1, s) + S
{s + s2CD
(sPwD(1, s) + S)} (3) (9) (4) Type curve and validation The assumptions used in developing this model are: We used the radial line source solutions developed by
Ikoku and Ramey (1979), and Odeh and Yang (1979) to
develop the solution for an infinite conductivity hydraulic
fracture and compared the results with our 2-D analytical
solution. We also used Eq. 34 in the work of Rghavan and
Chen (2017) which is the analytical solution developed in
their work for an infinite conductivity hydraulic fracture to
obtain similar curve for flow behavior index n of 0.2, 0.6 and
1.0. The results obtained are shown in Fig. 2. As shown in
Fig. 2a, b, Raghavan and Chen (2017) solution did not match
other solutions at flow behavior index n of 0.2 and 0.6 except
for 1.0. The reason for this discrepancy could be due to the
line source solution used by Raghavan and Chen (2017) or
in the conversion of their line source solution to that of an
infinite conductivity hydraulic fracture. 1. Fluid flow in the inner region (Zone 1) is governed by
the infinite conductivity hydraulic fracture model in
Eq. 1.i 2. The reservoir is infinite.l i
3. Both regions could exhibit non-Newtonian flow.l l
4. Laminar and Darcy’s flow is valid. 4. Laminar and Darcy’s flow is valid. l
5. Isothermal, single-phase and slightly compressible
fluid with constant properties. l
6. Homogenous and isotropic system.l l
6. Homogenous and isotropic system.l 7. Pseudoplastic (shear-thinning) fluid obeys Ostwald de
Waele power-law relationship.f 8. Steady state effective viscosity. 8. Steady state effective viscosity. f
9. The mathematical model also assumed there is no tran-
sition region. i
We also compared our results with that of Ertekin et al
(1987) for a finite dimensionless conductivity value of π. The
result is shown in Fig. 3. Ertekin et al (1987) used numerical
method which probably may have contributed to the observed
variation. However, the pattern remained the same. 10. Warren and Root (1963) matrix pseudo-steady state
transfer function is used in the mathematical model. The solution to Eq. 1, applying to the inner region (Zone
1), after using a g-transformation as shown in “Appendix
A” is The dimensionless pressure drop for various values of n
computed with the infinite reservoir described in Eq. 7 for
both homogeneous and naturally fractured reservoir shown
in Fig. 4a, b, respectively. Type curve and validation (5)
PD1
xD, yD, ≤xDi, yDi
= C1gv1Iv1
g
√
sfs
+ C2gv1Kv1
g
√
sfs
(6)
g2 =
4
(3 −n)2
[
x3−n
D
krx
+
y3−n
D
kry
]
(PolyaninandZaitsev, 2004) (5)
PD1
xD, yD, ≤xDi, yDi
= C1gv1Iv1
g
√
sfs
+ C2gv1Kv1
g
√
sfs
(5)
The plot of the dimensionless pressure drop of non-
Newtonian fluid with different flow behavior index (Fig. 4)
reveals that the early pressure behavior is practically the
same and exhibits linear flow except in the late radial flow
regime. Having validated our solution for a non-composite
reservoir, we proceed to develop the solution for a composite
reservoir. (6)
g2 =
4
(3 −n)2
[
x3−n
D
krx
+
y3−n
D
kry
]
(PolyaninandZaitsev, 2004) (6)
g2 =
4
(3 −n)2
[
x3−n
D
krx
+
y3−n
D
kry
]
(PolyaninandZaitsev, 2004) (6) xDi and yDi is the coordinate at the flood front. When n = 1 xDi and yDi is the coordinate at the flood front. When n = 1 xDi and yDi is the coordinate at the flood front. When n = 1
in Eq. 6, a Newtonian radial coordinate is obtained. l
in Eq. 6, a Newtonian radial coordinate is obtained. l
in Eq. 6, a Newtonian radial coordinate is obtained. 1 3 Journal of Petroleum Exploration and Production Technology (2022) 12:2453–2465 2456 0.01
0.1
1
10
0.0001
0.001
0.01
0.1
1
10
100
1000
10000
Dimensionless PD & tDxPD'
Dimensionless me tD
Ikoku & Ramey
Odeh & Yang
Raghavan & Chen
A & M 2-D
0.01
0.1
1
10
0.0001
0.001
0.01
0.1
1
10
100
1000
10000
Dimensionless PD & tDxPD'
Dimensionless me tD
Ikoku & Ramey
Odeh & Yang
Raghavan & Chen
A & M 2-D
0.01
0.1
1
10
0.0001
0.001
0.01
0.1
1
10
100
1000
10000
Dimensionless PD & tDxPD'
Dimensionless me tD
Ikoku & Ramey
Odeh & Yang
Raghavan & Chen
A & M 2-D
Linear flow
n = 0.2
n = 1.0
n = 0.6
a
b
c
Fig. Type curve and validation 2 Comparison of infinite conductivity hydraulic fracture solutions developed from various line source solutions for flow behavior index
n = 0.2, 0.6 and 1.0 (A&M: Alpheus and Mojeed) 0.01
0.1
1
10
0.0001
0.001
0.01
0.1
1
10
100
1000
10000
Dimensionless PD & tDxPD'
Dimensionless me tD
Ikoku & Ramey
Odeh & Yang
Raghavan & Chen
A & M 2-D
0.01
0.1
1
10
0.0001
0.001
0.01
0.1
1
10
100
1000
10000
Dimensionless PD & tDxPD'
Dimensionless me tD
Ikoku & Ramey
Odeh & Yang
Raghavan & Chen
A & M 2-D
Linear flow
n = 0.2
n = 0.6
a
b 0.01
0.1
1
10
0.0001
0.001
0.01
0.1
1
10
100
1000
10000
Dimensionless PD & tDxPD'
Dimensionless me tD
Ikoku & Ramey
Odeh & Yang
Raghavan & Chen
A & M 2-D
n = 0.6
b 0.01
0.1
1
10
0.0001
0.001
0.01
0.1
1
10
100
1000
10000
Dimensionless PD & tDxPD'
Dimensionless me tD
Ikoku & Ramey
Odeh & Yang
Raghavan & Chen
A & M 2-D
Linear flow
n = 0.2
a Dimensionless PD & tDxPD' Dimensionless PD & tDxPD' 0.01
0.1
1
10
0.0001
0.001
0.01
0.1
1
10
100
1000
10000
Dimensionless PD & tDxPD'
Dimensionless me tD
Ikoku & Ramey
Odeh & Yang
Raghavan & Chen
A & M 2-D
n = 1.0
c
Fig. 2 Comparison of infinite conductivity hydraulic fracture solutions developed from various line source solutions for flow behavior index
0 2 0 6 and 1 0 (A&M: Alphe s and Mojeed) 10 0.01
0.1
1
10
0.0001
0.001
0.01
0.1
1
10
100
1000
10000
Dimensionless PD & tDxPD'
Dimensionless me tD
Ikoku & Ramey
Odeh & Yang
Raghavan & Chen
A & M 2-D
n = 1.0
c Fig. 2 Comparison of infinite conductivity hydraulic fracture solutions developed from various line source solutions for flow behavior index
n = 0.2, 0.6 and 1.0 (A&M: Alpheus and Mojeed) Fig. 3 Match with Ertekin et al. Dimensionless conductivity π and
constant pressure boundary Composite reservoir Figure 6 is for homogeneous reservoir
and naturally fractured reservoir. The pressure derivatives
at the late radial flow in Zone 2 is less than 0.5 because of
the value of theta (mobility ratio) used which is less than
1.0. As shown in Fig. 5, when the mobility ratio is less than
1.0, the dimensionless pressure derivative of the late radial
flow regime will be less than 0.5. When the mobility ratio
is greater than 1.0, the dimensionless pressure derivative of
the late radial flow regime will be greater than 0.5. As shown
in the work of Abbaszadeh and Kamal (1989), the mobil-
ity ratio has a stronger influence on the pressure derivative
behavior.i
0.001
0.01
0.1
1
10
100
0.0001 0.001
0.01
0.1
1
10
100
1000
10000
Dimensionless PD & tDxPD'
Dimensionless me tD
alpha/teta=1/0.1
alpha/teta=1/1
alpha/teta=1/10
Linear Flow
tDxPD'=0.5
Fig. 5 XD = 1.0, ω = 0.01, λ = 10–1, RD = 2.5 (n = 1 for Zone 1) and
(m = 1 for Zone 2); infinite reservoir
0.001
0.01
0.1
1
10
100
0.0001 0.001
0.01
0.1
1
10
100
1000
10000
Dimensionless PD & tDxPD'
Dimensionless me tD
n=0.2/m=1
n=0.6/m=1
Linear Flow
0.001
0.01
0.1
1
10
100
0.0001 0.001
0.01
0.1
1
10
100
1000
10000
Dimensionless PD & tDxPD'
Dimensionless me tD
n=0.2/m=0.8
n=0.6/m=0.8
Linear Flow
a
b
Fig. 6 XD = 0.732 (n for Zone 1) and (6a: m = 1.0 for Zone 2, 6b: m = 0.8 for Zone 2); infinite homogeneous reservoir 0.001
0.01
0.1
1
10
100
0.0001 0.001
0.01
0.1
1
10
100
1000
10000
Dimensionless PD & tDxPD'
Dimensionless me tD
alpha/teta=1/0.1
alpha/teta=1/1
alpha/teta=1/10
Linear Flow
tDxPD'=0.5 Figures 6 and 7 show the behavior of the composite res-
ervoir when different flow behavior indices are used in the
inner and outer zones. Figure 6 is for homogeneous reservoir
and naturally fractured reservoir. The pressure derivatives
at the late radial flow in Zone 2 is less than 0.5 because of
the value of theta (mobility ratio) used which is less than
1.0. As shown in Fig. 5, when the mobility ratio is less than
1.0, the dimensionless pressure derivative of the late radial
flow regime will be less than 0.5. When the mobility ratio
is greater than 1.0, the dimensionless pressure derivative of
the late radial flow regime will be greater than 0.5. Composite reservoir The two equations used in the modeling of the composite
system with an infinite conductivity hydraulic fracture in the
inner zone are Eqs. 5 and 8. The details of the derivation are
in “Appendix A” under composite system. The final wellbore
dimensionless pressure drop equation in Laplace space is
Eq. 56 in “Appendix A”. Figures 6 and 7 are generated with
RD = 2.5, alpha = 0.001 and theta = 0.1. Figure 5 demonstrates
the effect of mobility and diffusivity ratios as in the work of
Ambastha (1988) and Abbaszadeh and Kamal (1989). The
value of n and m in Fig. 5 is 1.0 and the value of the pres-
sure derivative must be 0.5 at the longtime radial flow when
theta value is unity (1.0). As defined in “Appendix”, alpha
and theta represent, respectively, the diffusivity and mobil-
ity ratios of Zone 1 to Zone 2 as shown in Eqs. 50 and 51. Since Eq. 56 is a general solution, it must replicate the case
of Newtonian fluid in both zones and this is shown in Fig. 5. Fig. 3 Match with Ertekin et al. Dimensionless conductivity π and
constant pressure boundary 1 3 Journal of Petroleum Exploration and Production Technology (2022) 12:2453–2465 2457 0.001
0.01
0.1
1
10
100
0.0001 0.001
0.01
0.1
1
10
100
1000
10000
Dimensionless PD & tDxPD'
Dimensionless me tD
n=0.2
n=0.4
n=0.6
n=0.8
n=1.0
Linear Flow
0.001
0.01
0.1
1
10
100
0.0001 0.001
0.01
0.1
1
10
100
1000
10000
Dimensionless PD & tDxPD'
Dimensionless me tD
n=0.2
n=0.4
n=0.6
n=0.8
n=1.0
Linear Flow
a
b
Fig. 4 Dimensionless pressure drop for an infinite conductivity hydraulic fracture in homogeneous (4a) and naturally fractured (4b) reservoirs 0.001
0.01
0.1
1
10
100
0.0001 0.001
0.01
0.1
1
10
100
1000
10000
Dimensionless PD & tDxPD'
Dimensionless me tD
n=0.2
n=0.4
n=0.6
n=0.8
n=1.0
Linear Flow
a 0.001
0.01
0.1
1
10
100
0.0001 0.001
0.01
0.1
1
10
100
1000
10000
Dimensionless PD & tDxPD'
Dimensionless me tD
n=0.2
n=0.4
n=0.6
n=0.8
n=1.0
Linear Flow
b 100 Dimensionless PD & tDxPD' Dimensionless PD & tDxPD' Fig. 4 Dimensionless pressure drop for an infinite conductivity hydraulic fracture in homogeneous (4a) and naturally fractured (4b) reservoirs Figures 6 and 7 show the behavior of the composite res-
ervoir when different flow behavior indices are used in the
inner and outer zones. Limitation of model Example #1 Fig. 9 shows the type curve match for the fal-
loff data in a polymer injection well after few months of
injection. During radial flow, the dimensionless wellbore
pressure drop and its pressure derivative for a non-composite
reservoir are given by Eqs. 11 and 12, respectively. 1. There are many non-Newtonian viscosity models
(Savins (1969)). Our mathematical model is only devel-
oped for OstWaal-de-Waele power law model. However,
it is applicable to Newtonian model by setting the flow
behavior index to unity. (11)
PWDf =
(1
2 × 1
v
)
tv
D (11)
PWDf =
(1
2 × 1
v
)
tv
D
(12)
P
1 v (11) 2. The model assumed no transition region. 3. Warren and Root (1963) matrix pseudo-steady state flow
into the fracture is assumed. This may not be applicable to
matrix region of high natural fracture intensity where com-
plex models like trilinear and triple-porosity models exist. (12)
tD × P
wDf = 1
2tv
D (12) On a log–log scale, a straight line can be fixed on the
pressure and pressure derivative data. The slope of such
straight line on the pressure derivative data is v from which
the value of n can be determined Composite reservoir 7 XD = 0.732, ω = 0.01, λ = 10–1, (n for Zone 1) and (7a: m = 1.0 for Zone 2, 7b: m = 0.8 for Zone 2); Fig. 7 XD = 0.732, ω = 0.01, λ = 10–1, (n for Zone 1) and (7a: m = 1.0 for Zone 2, 7b: m = 0.8 for Zone 2); infinite naturally fractured reservoir Composite reservoir As shown
in the work of Abbaszadeh and Kamal (1989), the mobil-
ity ratio has a stronger influence on the pressure derivative
behavior. Dimensionless PD & tDxPD' Fig. 5 XD = 1.0, ω = 0.01, λ = 10–1, RD = 2.5 (n = 1 for Zone 1) and
(m = 1 for Zone 2); infinite reservoir 0.001
0.01
0.1
1
10
100
0.0001 0.001
0.01
0.1
1
10
100
1000
10000
Dimensionless PD & tDxPD'
Dimensionless me tD
n=0.2/m=1
n=0.6/m=1
Linear Flow
0.001
0.01
0.1
1
10
100
0.0001 0.001
0.01
0.1
1
10
100
1000
10000
Dimensionless PD & tDxPD'
Dimensionless me tD
n=0.2/m=0.8
n=0.6/m=0.8
Linear Flow
a
b
Fig. 6 XD = 0.732 (n for Zone 1) and (6a: m = 1.0 for Zone 2, 6b: m = 0.8 for Zone 2); infinite homogeneous reservoir 0.001
0.01
0.1
1
10
100
0.0001 0.001
0.01
0.1
1
10
100
1000
10000
Dimensionless PD & tDxPD'
Dimensionless me tD
n=0.2/m=1
n=0.6/m=1
Linear Flow
a 0.001
0.01
0.1
1
10
100
0.0001 0.001
0.01
0.1
1
10
100
1000
10000
Dimensionless PD & tDxPD'
Dimensionless me tD
n=0.2/m=0.8
n=0.6/m=0.8
Linear Flow
b Dimensionless PD & tDxPD' Dimensionless PD & tDxPD' Fig. 6 XD = 0.732 (n for Zone 1) and (6a: m = 1.0 for Zone 2, 6b: m = 0.8 for Zone 2); infinite homogeneous reservoir 3 Journal of Petroleum Exploration and Production Technology (2022) 12:2453–2465 2458 0.001
0.01
0.1
1
10
100
0.0001 0.001
0.01
0.1
1
10
100
1000
10000
Dimensionless PD & tDxPD'
Dimensionless me tD
n=0.2/m=1.0
n=0.6/m=1.0
Linear Flow
0.001
0.01
0.1
1
10
100
0.0001 0.001
0.01
0.1
1
10
100
1000
10000
Dimensionless PD & tDxPD'
Dimensionless me tD
n=0.2/m=0.8
n=0.6/m=0.8
Linear Flow
a
b
Fig. 7 XD = 0.732, ω = 0.01, λ = 10–1, (n for Zone 1) and (7a: m = 1.0 for Zone 2, 7b: m = 0.8 for Zone 2); infinite naturally fractured reservoir 0.001
0.01
0.1
1
10
100
0.0001 0.001
0.01
0.1
1
10
100
1000
10000
Dimensionless PD & tDxPD'
Dimensionless me tD
n=0.2/m=0.8
n=0.6/m=0.8
Linear Flow
b 0.001
0.01
0.1
1
10
100
0.0001 0.001
0.01
0.1
1
10
100
1000
10000
Dimensionless PD & tDxPD'
Dimensionless me tD
n=0.2/m=1.0
n=0.6/m=1.0
Linear Flow
a Dimensionless PD & tDxPD' Dimensionless PD & tDxPD' Fig. Application to practical example (2012) example #1 (15)
tD × P
wDf = A × 1
2tv
D
(16)
t × ΔP
= A × 1
2
( q
2휋h
) 1+n
3−n
(휇eff
kh
)
n
3−n
(
1
n(∅ct
)
t
) 1−n
3−n
t
1−n
3−n (15)
tD × P
wDf = A × 1
2tv
D (15) (16)
t × ΔP
= A × 1
2
( q
2휋h
) 1+n
3−n
(휇eff
kh
)
n
3−n
(
1
n(∅ct
)
t
) 1−n
3−n
t
1−n
3−n (16) Equations 64–66 show that A is a function of the position
of the flood front, mobility and diffusivity ratios and flow
behavior index n and m. We fixed Eq. 15 on the dimension-
less pressure derivative curve by computing the dimension-
less pressure derivative from the values of tD, using Eq. 12. The numerical value of A is determined by trial and error
until the log–log straight line is perfectly on the non-Newto-
nian radial flow region of the dimensionless pressure deriva-
tives. For example, Fig. 8 is the type curve for example #1. The straight line with the red color was drawn with Eq. 12,
while the straight line with the green color was drawn with
Eq. 15 with the A value of 0.8. Figure 9 shows the complete
type curve match, though without the red line. The reason
for Fig. 8 is to pictorially show the workflow. The value of A
for the examples #2 and #3 is 0.95. This is because we used
the same value of flow behavior index for the examples #2
and #3. Fig. 9 van den Hoek et al. (2012) example #1 zone in this case is before the estimated flood front. One
parameter in the van den Hoek et al.’s (2012) formula for
estimating the transition zone is the cumulative pumping
time. A low estimate of the pumping time can put the transi-
tion zone before the actual flood front. l
Refer to Table 1 for the parameters used to generate the
type curve. In this example, both the type curve match and
van den Hoek et al. (2012) methods have the same value of
flow behavior index n in Zone 1. Hence the green straight
line from the dimensionless pressure derivative coincided
with that of van den Hoek et al. (2012) when a perfect match
is obtained. van den Hoek et al. Application to practical example (13)
v = 1 −n
3 −n Van den Hoek et al. (2012) presented three practical exam-
ples that we used to practically validate our solution. Table 1
shows the parameters used in Eqs. 56 and 9 to generate the
type curves in obtaining the match for the three examples. The injected polymer is in Zone 1 with flow behavior index
n, while Zone 2 contains the in situ fluid with flow behavior
index m equal to 1.0. This is a case of non-Newtonian and
Newtonian fluids in Zone 1 and Zone 2, respectively.l Van den Hoek et al. (2012) presented three practical exam-
ples that we used to practically validate our solution. Table 1
shows the parameters used in Eqs. 56 and 9 to generate the
type curves in obtaining the match for the three examples. (13) Using the dimensionless definition in Eqs. 21 and 22 in
“Appendix”, Eq. 12 becomes: The injected polymer is in Zone 1 with flow behavior index
n, while Zone 2 contains the in situ fluid with flow behavior
index m equal to 1.0. This is a case of non-Newtonian and
Newtonian fluids in Zone 1 and Zone 2, respectively.l (14)
t × ΔP
= 1
2
( q
2휋h
) 1+n
3−n
(휇eff
kh
)
n
3−n
(
1
n(∅ct
)
t
) 1−n
3−n
t
1−n
3−n (14) l
If RD is the radial distance of the flood front, assuming a
square model, then, Equations 11–14 are derived from Eq. 43. The factoriza-
tion and inversion of Eq. 56 for a longtime case is presented
in “Appendix”. As shown in Eq. 64 in the “Appendix”, (10)
xDi,yDi
= RD
√
휋 xDi,yDi
= RD
√
휋 (10) Example
Zone 1
flow index
n
Zone 2
flow index
m
Dimensionless
polymer front RD
Wellbore
storage CD
Skin factor S
Alpha (α)
Theta (θ)
#1
0.6
1.0
2.5
0.005
3.7
0.001
0.1
#2
0.8
1.0
3.0
0.3
0.0
0.001
0.1
#3
0.8
1.0
5.5
1.0
4.6
0.001
0.1 Table 1 Match parameters for
the three examples 1 3 Journal of Petroleum Exploration and Production Technology (2022) 12:2453–2465 2459 Eq. 12 cannot be applied without a correction factor. By
applying the method of van den Hoek et al. (2012) to Eqs. 12
and 14, we can obtain similar expression for a composite
reservoir. Thus: Fig. 9 van den Hoek et al. Discussion of the results The examples #1 and #3 are from the same well. Therefore,
we do not change the values of alpha and theta. We, how-
ever, maintained the same values for example #2. p
Having developed the analytical 2-D mathematical
model, our goal is to use the model in evaluating polymer
flood front. van den Hoek et al.’s (2012) examples provide
appropriate case for practical validation. Formation param-
eters can be evaluated from both zones if injection and
necessary formation data are available. Using Tiab Direct
Synthesis (TDS) for evaluation has been discussed exten-
sively by Katime-Meindl and Tiab (2001), Igbokoyiand
Tiab (2007) and, Omosebi and Igbokoyi(2015). However,
as indicated in Eq. 64 in “Appendix”, a correction factor
is needed for correcting the pressure derivative equation
of non-composite system to that of a composite system. A
major highlight from this study is the fact that the pressure
derivative data in a composite system during non-Newtonian
radial flow are strictly numerically not equal to its equivalent
in the non-composite system. As earlier indicated by van den
Hoek et al. (2012), a correction factor is needed to adjust
the pressure derivative log–log straight line equation for
non-composite system to that of a composite system before
Eq. 12 can be used in the analysis or application of the TDS
formulae from non-composite system. Fig. 11 van den Hoek et al. (2012) example #3 before the falloff test. The same procedure discussed in the
example #1 was used to determine the flood front. van den
Hoek et al. (2012) computed flow behavior index n of 0.6
from the straight line as described in example #1. We are
able to obtain good match using the type curve method with
a flow behavior index n of 0.8. We attributed the difference
in the flow behavior index n to the fact that there is possibil-
ity of polymer degradation after it must have been injected
into the reservoir for a considerable period of time. Moreo-
ver, the type curve match is to capture the entire behavior
of the pressure data as much as possible. In this case, the
computed transition zone by van den Hoek et al. (2012) is
after the flood front. Our green straight line does not match
that of van den Hoek et al. (2012) because of the difference
in the flow behavior index. Application to practical example (2012) used n value of 0.6 which
was derived from the straight line (dotted straight lines on
Figs. 9, 10, 11) drawn by using pressure derivative equa-
tion equivalent to Eq. 16. The point at which the pressure
derivative curve falls off from the straight line is considered
to be the flood front. van den Hoek et al. (2012) developed
an equation for estimating the transition zone. The transition Example #2 Fig. 10 shows the type curve match for this
example. The pressure data are from different injection well,
and the polymer injection has been on for about nine months 0.01
0.1
1
10
100
0.001
0.01
0.1
1
10
100
1000
Dimensionless PD & tDxPD'
Dimensionless me tD
Fig. 8 The type curve used for example #1
Fig. 10 van den Hoek et al. (2012) example #2 0.01
0.1
1
10
100
0.001
0.01
0.1
1
10
100
1000
Dimensionless PD & tDxPD'
Dimensionless me tD Dimensionless PD & tDxPD' Fig. 10 van den Hoek et al. (2012) example #2 Fig. 8 The type curve used for example #1 1 3 Journal of Petroleum Exploration and Production Technology (2022) 12:2453–2465 2460 zone. The flood front radial dimensionless distance used in
the type curves is provided in Table 1. Fig. 11 van den Hoek et al. (2012) example #3 Discussion of the results From the three examples, we observed that as n tends
to 1.0, the value of A in Eqs. 15 and 16 tends to 1.0. We
have used the available field data to prove the validity of our
mathematical model for the case of reservoir flooding that
result in two flow behavior indices in the reservoir. Even
though the flow behavior index we used in example #2 and
#3 is different from that of van den Hoek et al.’s (2012), the
point of intersection of the two straight lines with the pres-
sure derivative curve on the match is practically the same in
each of the examples #2 and #3. As earlier mentioned, both
indicated the same transition zone in each case. Example #3 This injector is the same as in example #1. The
type curve match is shown in Fig. 11. As indicated by van
den Hoek et al. (2012), using the workflow for examples #1
and #2 is not possible because the large wellbore storage has
eliminated the non-Newtonian radial flow in Zone 1 (poly-
mer zone). van den Hoek et al. (2012) assumed a flow behav-
ior index of 0.6 to draw the straight line. This might lead to
uncertainty in the estimation of the flood front. Using a type
curve match method should give a better result with lower
degree of uncertainty. The flow behavior index we used is
0.8. The reason for not being able to match the early part of
the pressure data could be due to the drop in the liquid level
during the falloff test, a phenomenon usually experienced in
the phase segregation case. Conclusions A 2-D general solution for evaluating fractured injection
well performance with non-Newtonian fluid in a compos-
ite reservoir is developed. The computed dimensionless
pressure drop is favorably compared with the wellbore infi-
nite conductivity dimensionless fractured pressure devel-
oped from the line source solutions of Ikoku and Ramey
(1979), and Odeh and Yang (1979). We also used the work The point of the intersection of the straight line with the
pressure derivative curve at best can only be the transition 3 2461 Journal of Petroleum Exploration and Production Technology (2022) 12:2453–2465 of Ertekin et al (1987) to validate our solution for a finite
conductivity case.i (23)
휔=
(휙ct
)
f
(휙ct
)
f + (휙ct
)
m (23) The composite mathematical model in an infinite sys-
tem was applied to solve the examples presented by van den
Hoek et al. (2012). Although the match obtained in exam-
ples #2 and #3 is with higher values of flow behavior index
n, we have confidence in the overall results because of the
consistency in the flood front that agreed with the duration
of injection. (24)
λ = 훿
kmx2
f
kf (24) The dimensionless form of Eq. 18 is given as: The dimensionless form of Eq. 18 is given as: From Warren and Root (1963),
(25)
krxxn−1
D
휕2PDf
휕x2
D
+ kryyn−1
D
휕2PDf
휕y2
D
= 휔
휕PDf
휕tD
+ (1 −휔)휕PDm
휕tD (25
krxxn−1
D
휕2PDf
휕x2
D
+ kryyn−1
D
휕2PDf
휕y2
D
= 휔
휕PDf
휕tD
+ (1 −휔)휕PDm
휕tD The 2-D solution is applicable to naturally fractured res-
ervoirs and also the case of non-Newtonian/non-Newtonian
composite system as in polymer flooding of a heavy crude
reservoir where the heavy crude exhibits non-Newtonian
behavior. As indicated by van den Hoek et al. (2012) and
the longtime inversion of the analytical solution for the com-
posite system in this study, a correction factor is needed to
apply the TDS formulae developed for non-Newtonian radial
flow from the non-composite system. (25) From Warren and Root (1963), From Warren and Root (1963), (25a)
n
( q
2휋h
)n−1(휙ct
)
m
휕Pm
휕t
= 훿km
휇e
(Pf −Pm
) In dimensionless form, (25b)
(1 −휔)휕PDm
휕tD
= 훿
kmx3−n
f
kh
(PDf −PDm
)
(25c)
훿= 4m(m + 2)
l2
m (25b)
(1 −휔)휕PDm
휕tD
= 훿
kmx3−n
f
kh
(PDf −PDm
) (25b) I: Infinite conductivity hydraulic fracture 33 in an infinite system is:
(33)
v = (1 −n)
(3 −n)
⎪
⎪
⎨
⎪
⎪⎩
2
3−n 1.732
2 √sfs
∫
0
1
2
1
2 u
−v
Γ(v)휕u +
2
3−n
2
3−n 0.268
2 √sfs
∫
0
1
2
1
2u
−v
Γ(v)휕u
⎪
⎪
⎬
⎪
⎪⎭ (33) Solution to Eq. 33 in an infinite system is: (41)
⎭ (34)
PDf
xw, yw ≤xD, yD, ≤∞, s = CgvKv
g
√
sfs
And (34)
PDf
xw, yw ≤xD, yD, ≤∞, s = CgvKv
g
√
sfs
And
(41) PDf
xw, yw ≤xD, yD, ≤∞, s = CgvKv
g
√
sfs
(34)
PDf
xw, yw ≤xD, yD, ≤∞, s = CgvKv
g
√
sfs
And
(41) (34)
And (42)
PWDf =
1
4s√sfs
2
3 −n
1−n
3−n 1
2
−v
Γ(v)
⎧
⎪
⎨
⎪⎩
u−v+1
−v + 1
2
3−n 1.732
3−n
2 √sfs
0
+
u−v+1
−v + 1
2
3−n 0.268
3−n
2 √sfs
0
⎫
⎪
⎬
⎪⎭ PWDf =
1
4s√sfs
2
3 −n
1−n
3−n 1
2
−v
Γ(v) For infinite conductivity hydraulic fracture: For infinite conductivity hydraulic fracture: (3
PWDf = 1
2s
2
3 −n
1−n
3−n
1
∫
−1
√0.732 −xD
2 3−n
2
K 1−n
3−n
2
3 −n
√0.732 −xD
2 3−n
2
√
(sfs)휕xD (42) The factorization of Eq. 42 gave: (35) (43)
PWDf =
(1
2 × 1
v
)
tv
D (43) By making By making
(36)
u =
2
3 −n
0.732 −xD
3−n
2 √sfs
(37)
PWDf =
1
2s
√
sfs
2
3 −n
1−n
3−n
⎧
⎪
⎪
⎨
⎪
⎪⎩
2
3−n 1.732
3−n
2 √sfs
∫
0
Kv(u)휕u +
2
3−n 0.268
3−n
2 √sfs
∫
0
Kv(u)휕u
⎫
⎪
⎪
⎬
⎪
⎪⎭ Equation 43 is the radial flow regime approximation. (36) (36) I: Infinite conductivity hydraulic fracture (25c)
훿= 4m(m + 2)
l2
m 훿= 4m(m + 2)
l2
m (25c) The general 2-D non-Newtonian fluid flow equation for the
proposed model is (25d)
λ = 4j(j + 2)
l2
m
kmx3−n
f
kh (
1
n
(kfx
휇e
)(
q
2휋xh
)1−n 휕2Pf
휕x2 + 1
n
(kfy
휇e
)(
q
2휋yh
)1−n 휕2Pf
휕y2
= (휙ct
)
f
휕Pf
휕t + (휙ct
)
m
휕Pm
휕t (25d) In Eq. 25d, we have taken kf = kh. Initial condition (17) (26)
PDf
(xD, yD;tD = 0) = 0 (26) (kfx
휇e
)(1
x
)1−n 휕2Pf
휕x2 +
(kfy
휇e
)(
1
y
)1−n 휕2Pf
휕y2
= n
( q
2휋h
)n−1{(휙ct
)
f
휕Pf
휕t + (휙ct
)
m
휕Pm
휕t
} Outer boundary condition (27)
PDf
(xD, yD ≤∞tD ≥0) = 0 (27) (18) The Laplace transformation of Eq. 25 is The Laplace transformation of Eq. 25 is (28)
krxxn−1
D
휕2PDf
휕x2
D
+ kryyn−1
D
휕2PDf
휕y2
D
= sf(s)PDf (19) (19) (28) (20)
where (20)
where (20) sf(s) = s휔(1 −휔)s + λ
(1 −휔)s + λ (29)
sf(s) = s휔(1 −휔)s + λ
(1 −휔)s + λ (29) (21) (21)
We defined g-transformation as (21)
We defined g-transformation as (21)
We defined g-transformation as (22)
(30)
g2 =
4
(3 −n)2
[
x3−n
D
krx
+
y3−n
D
kry
] (30) (22) 1 3 1 3 3 Journal of Petroleum Exploration and Production Technology (2022) 12:2453–2465 2462 (31)
휕2PDf
휕x2
D
= 휕2g
휕x2
D
휕PDf
휕g
+
( 휕g
휕xD
)2 휕2PDf
휕g2
For a small value of u
(39a)
PWDf = √ 휋tD (31)
휕2PDf
휕x2
D
= 휕2g
휕x2
D
휕PDf
휕g
+
( 휕g
휕xD
)2 휕2PDf
휕g2
For a small value
PWDf = √ 휋tD (31)
(39a)
PWDf = √ 휋tD (39a)
PWDf = √ 휋tD (39a) (31) For a small value of u For a small value of u Using the same derivative transformation for yD, apply to
Eq. 28 with relative permeability of unity gave:
(40)
Kv(u) ≈1
2
(1
2u
)−v
Γ(v) (40)
Kv(u) ≈1
2
(1
2u
)−v
Γ(v) Using the same derivative transformation for yD, apply to
Eq. 28 with relative permeability of unity gave: (40) (32)
휕2PDf
휕g2
+
(
1 + 2(1 −n)
(3 −n)
)
1
g
휕PDf
휕g
= sf(s)PDf
Therefore, for a small of u Eq. I: Infinite conductivity hydraulic fracture 37 becomes:
P
1
2
1−n
3−n 휕2PDf
휕g2
+
(
1 + 2(1 −n)
(3 −n)
)
1
g
휕PDf
휕g
= sf(s)PDf (32)
휕2PDf
휕g2
+
(
1 + 2(1 −n)
(3 −n)
)
1
g
휕PDf
휕g
= sf(s)PDf Therefore, for a small of u Eq. 37 becomes: (32) (32)
(33)
(34)
A d
(41)
PWDf =
1
2s
√
sfs
2
3 −n
1−n
3−n
⎧
⎪
⎪
⎨
⎪
⎪⎩
2
3−n 1.732
3−n
2 √sfs
∫
0
1
2
1
2 u
−v
Γ(v)휕u +
2
3−n
2
3−n 0.268
3−n
2 √sfs
∫
0
1
2
1
2u
−v
Γ(v)휕u
⎫
⎪
⎪
⎬
⎪
⎪⎭ PWDf =
1
2s
√
sfs
2
3 −n
1−n
3−n Define v as:
Solution to Eq. 33 in an infinite system is:
휕g
(
(3
n)
)
g 휕g
(33)
v = (1 −n)
(3 −n)
(41)
PWDf =
1
2s
√
sfs
2
3 −n
1 n
3−n
⎧
⎪
⎪
⎨
⎪
⎪⎩
2
3−n 1.732
3−n
2 √sfs
∫
0
1
2
1
2 u
−v
Γ(v)휕u +
2
3−n
2
3−n 0.268
3−n
2 √sfs
∫
0
1
2
1
2u
−v
Γ(v)휕u
⎫
⎪
⎪
⎬
⎪
⎪⎭ Define v as: Define v as: m is:
(33)
(34)
√
sfs
And
(41)
⎧
⎪
⎪
⎨
⎪
⎪⎩
2
3−n 1.732
3−n
2 √sfs
∫
0
1
2
1
2 u
−v
Γ(v)휕u +
2
3−n
2
3−n 0.268
3−n
2 √sfs
∫
0
1
2
1
2u
−v
Γ(v)휕u
⎫
⎪
⎪
⎬
⎪
⎪⎭ (33)
v = (1 −n)
(3 −n)
⎧
⎪
⎪
⎨
⎪
⎪
2
3−n 1.732
3−n
2 √sfs
∫
0
1
2
1
2 u
−v
Γ(v)휕u +
2
3−n
2
3−n 0.268
3−n
2 √sfs
∫
0
1
2
1
2u
−v
Γ(v)휕u
⎫
⎪
⎪
⎬
⎪
⎪ v = (1 −n)
(3 −n) i
(33)
= (1 −n)
(3 −n)
⎧
⎪
⎪
⎨
⎪
⎪
2
3−n 1.732
3 n
2 √sfs
∫
0
1
2
1
2 u
−v
Γ(v)휕u +
2
3−n
2
3−n 0 Solution to Eq. where n and m are the flow behavior indices in the inner At the interface PD1 = PD2.Therefore, At the interface PD1 = PD2.Therefore, At the interface PD1 = PD2.Therefore, At the interface PD1 = PD2.Therefore, C3 =
gv1
i
C1Iv1
gi
√
(sfs)
+C2Kv1
gi
√
(sfs)
gv2
i Kv2
gi
√
(훼sfs) (52) where n and m are the flow behavior indices in the inner Substituting Eq. 55 in Eq. 44, the infinite conductivity
hydraulic fracture dimensionless pressure drop in a com-
posite system becomes: l
and outer region, respectively. By considering g spatial vari-
able as an equivalent radial distance (Vongvuthipornchai and
Raghavan 1987; Equation B-9). (48)
(49)
(50)
(56)
PWDf = 1
2s
2
3 −n
1−n
3−n
⎧
⎪
⎨
⎪⎩
휃g−m−v1
i
Kv2
gi
√
(훼sfs)
√
(훼sfs)Kv2−1
gi
√
(훼sfs)
Iv1
gi
√
(sfs)
−
Kv1
gi
√
(sfs)
Iv1
gi
√
(sfs)
⎫
⎪
⎬
⎪⎭
1
∫
−1
√0.732 −xD
2 3−n
2 I 1−n
3−n
2
3 −n
√0.732 −xD
2 3−n
2
√
(sfs)휕xD + 1
2s
2
3 −n
1−n
3−n
1
∫
−1
√0.732 −xD
2 3−n
2
K 1−n
3−n
2
3 −n
√0.732 −xD
2 3−n
2
√
(sfs)휕xD (48)
휕PD2
휕g
= −휃1
sgm
i
(49)
휕PD1
휕g
= −1
sgn
i
(50)
훼=
m
( 휑ct휇eff
k
)
2
(
2휋gwh
q
)n−m
n
( 휑ct휇eff
k
)
1
(51)
휃= k1
휇1
휇2
k2
(
q
2휋gwh
)m−n
Observation of Eq. 56 revealed that the Bessel terms in
(56)
PWDf = 1
2s
2
3 −n
3−n
⎧
⎪
⎨
⎪⎩
휃g−m−v1
i
Kv2
gi
√
(훼sfs)
√
(훼sfs)Kv2−1
gi
√
(훼sfs)
Iv1
gi
√
(sfs)
−
Kv1
gi
√
(sfs)
Iv1
gi
√
(sfs)
⎫
⎪
⎬
⎪⎭
1
∫
−1
√0.732 −xD
2 3−n
2 I 1−n
3−n
2
3 −n
√0.732 −xD
2 3−n
2
√
(sfs)휕xD + 1
2s
2
3 −n
1−n
3−n
1
∫
−1
√0.732 −xD
2 3−n
2
K 1−n
3−n
2
3 −n
√0.732 −xD
2 3−n
2
√
(sfs)휕xD (48)
휕PD2
휕g
= −휃1
sgm
i
(49)
휕PD1
휕g
= −1
sgn
i (48) (49) 훼=
m
( 휑ct휇eff
k
)
2
(
2휋gwh
q
)n−m
n
( 휑ct휇eff
k
)
1 (50) (51)
휃= k1
휇1
휇2
k2
(
q
2휋gwh
)m−n (56) (56)
(51) (51) Observation of Eq. 56 revealed that the Bessel terms in
the bracket of the coefficient of the first integral tend to zero
at the large value of sfs which corresponds to the early flow
regime. Hence, we are left with the second integral which
will give the early linearly flow regime as that of Eq. 39. Longtime evaluation of Eq. 56 휕PD2
휕g
= −C3gv2√
(훼sfs)Kv2−1
g√
(훼sfs) (53)
휕PD2
휕g
= −C3gv2√
(훼sfs)Kv2−1
g√
(훼sfs) II: Infinite Conductivity Hydraulic Fracture
in a Composite Reservoir The two equations for the composite system in an infinite
system are: (37) (44)
PD1
xD, yD, ≤xDi, yDi
= C1gv1Iv1
g
√
sfs
+ C2gv1Kv1
g
√
sfs
(44)
for the inner region with infinite conductivity hydraulic
fracture and, At the early linear flow, sfs is large and is approximately
sω. By using the expression in Raghavan and Chen (2017)
in Eq. 40 of their paper, we can show that (45)
PD2
xDi, yDi ≤xD, yD ≤∞ = C3gv2KV2
g
√
훼sfs
(45) for the outer region. for the outer region. (38)
PWDf =
1
2s√sfs
2
3 −n
1−n
3−n 휋sec
휋1−n
3−n
2
(38) (46)
v1 = (1 −n)
(3 −n) v1 = (1 −n)
(3 −n) (46) At early time, the expression in Eq. 38 for all values of n
0 ≤n ≤1 gave: and (39)
(47)
v2 = (1 −m)
(3 −m) (39)
v2 = (1 −m)
(3 −m) (39)
PWDf = √
휋tD
휔
v2 = (1 −m)
(3 −m) (47) (39) For homogeneous formation when ω = 1; For homogeneous formation when ω = 1; For homogeneous formation when ω = 1; 1 3 2463 Journal of Petroleum Exploration and Production Technology (2022) 12:2453–2465 where n and m are the flow behavior indices in the inner References (62) Ambastha AK (1988) Pressure transient analysis for composite system. Study done at Stanford University, USA y
y
Carslaw HS, Jaeger JC (1959) Conductor of Heat in solids, 2nd edn. Clarendon Press, Oxford, p 397 (63) p
Cinco-Ley H, PEMEX and University of Mexico, Meng HZ (1988)
Dowell Schluberger. Pressure transient analysis of wells with
finite conductivity vertical fractures in Double porosity reservoirs. SPE 18172. Annual Technical Exhibition of SPE held in Houston,
TX, October 2–5 Therefore, Therefore, (64)
tD × P
wDf = A1tv2
D −A2t
v1
2
D + 1
2tv1
D (64)
tD × P
wDf = A1tv2
D −A2t
v1
2
D + 1
2tv1
D
SPE 18172. Annual Technical Exhibition of SPE held i
TX, October 2–5
Ertekin T, Adewumi MA, Daud ME (1987) Pressure transi
iour of non-Newtonian/Newtonian fluid composite tD × P
wDf = A1tv2
D −A2t
v1
2
D + 1
2tv1
D (64) Ertekin T, Adewumi MA, Daud ME (1987) Pressure transient behav-
iour of non-Newtonian/Newtonian fluid composite systems in A1 =
휃g−m−2v1−2v2+1
i
훼−v2(v2 −1)v2
(
1
2
)−2v2+2(
2
3−n
)2v1+1(1.7322−n + 0.2682−n)
(v1 + 1)Γ(v1 + 1)Γ(v2 + 1) (65) porous media with a finite conductivity vertical fracture, SPE
17053. Soc Pet Eng J. https://doi.org/10.2118/17053-PA (66)
A2 =
Γ
(v1
)(
2
3−n
)2v1+1(1.7322−n + 0.2682−n)
22−v1g2v1
i
(v1 + 1)Γ
(
v1
2
) (66) Igbokoyi AO, Tiab D (2007) New type curves for the analysis of pres-
sure transient data dominated by skin and wellbore storage—non-
Newtonian fluid, SPE 106997. Soc Pet Eng J. https://doi.org/10.
2118/106997-MS Following van den Hoek et al.’s (2012) method and when
m = 1.0, we can represent Eq. 64 as; Ikoku CU, Ramey HJ (1979) Transient flow of non-Newtonian power-
law fluids in porous media, SPE 7139. Soc Pet Eng J 19(3):164–
174. https://doi.org/10.2118/7139-PA (67)
tD × P
wDf = A × 1
2tv1
D Katime-Meindl I, Tiab D (2001) Analysis of pressure transient test of
non-newtonian fluids in infinite reservoir and in the presence of
a single linear boundary by the direct synthesis technique, SPE
71587. Soc Pet Eng J. https://doi.org/10.2118/71587-MS (67) Note that when m = 1.0, v2 is zero and A1 vanishes. A is
a factor to be determined by trial and error as described in
the three examples. Lund O, Ikoku CU (1981) Pressure transient behaviour of non-New-
tonian/Newtonian fluid composite reservoirs, SPE 9401. References Soc Pet
Eng J 271–280. https://doi.org/10.2118/9401-PAl Martha JA, Ertekin T (1983) Numerical simulation of power law fluid
flow in a vertically fractured reservoir, SPE 12011 Acknowledgements We acknowledged the initial contribution of Dr. Obinna Ezulike of University of Alberta, Canada and Dr. Omotayo
Omosebi of Beckley Laboratory, USA, who started this work with Dr. Alpheus Igbokoyi. l
Abbaszadeh M, Kamal M (1989) Pressure_transient testing of water-
injection wells. SPE 16744l Odeh AS, Yang HT (1979) Flow of non-Newtonian power-law fluids
through porous media SPEJf Omosebi O, Igbokoyi A (2015) Analysis of pressure falloff tests of
non-Newtonian power-law fluids in naturally-fractured bounded
reservoirs. KeAi Pet 1(2015):318–341. https://doi.org/10.1016/j.
petlm.2015.10.006 Funding The authors received no financial support for the research,
authorship, and/ or publication of this article. (53)
For a small value of u References
Ambastha AK (1988) Pressure transient analysis for composite system. Study done at Stanford University, USA
Carslaw HS, Jaeger JC (1959) Conductor of Heat in solids, 2nd edn. Clarendon Press, Oxford, p 397
Cinco-Ley H, PEMEX and University of Mexico, Meng HZ (1988)
Dowell Schluberger. Pressure transient analysis of wells with
finite conductivity vertical fractures in Double porosity reservoirs. SPE 18172. Annual Technical Exhibition of SPE held in Houston,
TX, October 2–5
Ertekin T, Adewumi MA, Daud ME (1987) Pressure transient behav-
i
f
N
t
i
/N
t
i
fl id
it
t
i 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/. (53)
For a small value of u (53) (53) (53)
For a small value of u For a small value of u (57)
Iv(u) ≈
(1
2u
)v
∕Γ(v + 1) Combining Eqs. 48 and 53, at the interface; Combining Eqs. 48 and 53, at the interface; (57) (54)
C3 =
휃g−m−v2
i
s
√
(훼sfs)Kv2−1
gi
√
(훼sfs) At longtime when the Laplace variable s is small, sfs
approximates to s. (54) (54) By applying Eqs. 36, 40 on 56, the longtime approxima-
tion of Eq. 56 in real dimensionless quantity is: Combining Eqs. 52 and 54. (58)
PWDf = B1 + B2 + B3 (55)
C1 =
휃g−m−v1
i
Kv2
gi
√
(훼sfs)
s
√
(훼sfs)Kv2−1
gi
√
(훼sfs)
Iv1
gi
√
(sfs)
−
C2Kv1
gi
√
(sfs)
Iv1
gi
√
(sfs)
where. PWDf = B1 (58) where. (59)
B1 =
휃g−m−2v1−2v2+1
i
훼−v2(v2 −1)(
1
2
)−2v2+2(
2
3−n
)2v1+1(1.7322−n + 0.2682−n)
(v1 + 1)Γ(v1 + 1)s1+v2 (59)
B1 =
휃g−m−2v1−2v2+1
i
훼−v2(v2 −1)(
1
2
)−2v2+2(
2
3−n
)2v1+1(1.7322−n + 0.2682−n)
(v1 + 1)Γ(v1 + 1)s1+v2
(60)
휃g−m−2v1−2v2+1
i
훼−v2(v2 −1)(
1
2
)−2v2+2(
2
3
)2v1+1(1.7322−n + 0.2682−n) (59) (60)
B1 =
휃g−m−2v1−2v2+1
i
훼−v2(v2 −1)(
1
2
)−2v2+2(
2
3−n
)2v1+1(1.7322−n + 0.2682−n)
(v1 + 1)Γ(v1 + 1)Γ(v2 + 1)
tv2
D (60)
B1 =
휃g−m−2v1−2v2+1
i
훼−v2(v2 −1)(
1
2
)−2v2+2(
2
3−n
)2v1+1(1.7322−n + 0.2682−n)
(v1 + 1)Γ(v1 + 1)Γ(v2 + 1)
tv2
D (60) 1 3 2464 Journal of Petroleum Exploration and Production Technology (2022) 12:2453–2465 B2 = −
Γ
(v1
)(
2
3−n
)2v1+1(1.7322−n + 0.2682−n)
22−v1g2v1
i
(v1 + 1)s
v1
2 +1 Therefore,
(61)
B2 = −
Γ
(v1
)(
2
3−n
)2v1+1(1.7322−n + 0.2682−n)
22−v1g2v1
i
(v1 + 1)s
v1
2 +1
(62)
B2 = −
Γ
(v1
)(
2
3−n
)2v1+1(1.7322−n + 0.2682−n)
22−v1g2v1
i
v1
2
(v1 + 1)Γ
(
v1
2
)
t
v1
2
D
(63)
B3 =
1
2v1
tv1
D
(64)
tD × P
wDf = A1tv2
D −A2t
v1
2
D + 1
2tv1
D
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need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Warren JE, Root PJ (1963) The behaviour of naturally fractured res-
ervoirs, SPE 426. Soc Pet Eng J 3(03):245–255. https://doi.org/
10.2118/426-PA Van den Hoek PJ, Mahani H, Sorop TG, Brooks AD, Zwaan M, Sen
S, Shell Global Solutions International BV, Shuaili K, Aadi F
(2012) Petroleum Development Oman LLC. Application of Injec-
tion Fall-Off Analysis in Polymer Flooding SPE 154376 SPE
EUROPEC 2012 4–7f Declaration Polyanin AD, Zaitsev VF (2004) Handbook of nonlinear partial dif-
ferential equations, 1. CRC Press, Boca Raton, pp 442–443 Conflict of interest On behalf of all authors, the corresponding author
states that there is no conflict of interest. Raghavan R, Raghavan Inc R, Chen C (2017) Kappa engineering. Fractured-injection-well performance under non-Newtonian,
power-law fluids. SPE Reservoir Evaluation and Engineering. SPE 187955l Open Access This article is licensed under a Creative Commons Attri-
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included in the article's Creative Commons licence, unless indicated Savins JG (1969) Non-Newtonian flow through porous media. Ind Eng
Chem 61(10):18–47. https://doi.org/10.1021/ie507/8005 Stehfest H (1970) Algorithm 368: numerical inversion of laplace trans-
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S, Shell Global Solutions International BV, Shuaili K, Aadi F
(2012) Petroleum Development Oman LLC. Application of Injec-
tion Fall-Off Analysis in Polymer Flooding SPE 154376 SPE
EUROPEC 2012 4–7f Vongvuthipornchai S, Raghavan R (1987) Pressure falloff behavior
in vertically fractured wells: Non-Newtonian power-law fluids. SPEFE 1 3 1 3 3
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English
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A fruit firmness QTL identified on linkage group 4 in sweet cherry (Prunus avium L.) is associated with domesticated and bred germplasm
|
Scientific reports
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cc-by
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A fruit firmness QTL identified on linkage group 4 in
sweet cherry (Prunus avium L.) is associated with
domesticated and bred germplasm. n Cai, José Quero-Garcia, Teresa Barreneche, Elisabeth Dirlewanger,
Christopher Saski, Amy Iezzoni To cite this version: Lichun Cai, José Quero-Garcia, Teresa Barreneche, Elisabeth Dirlewanger, Christopher Saski, et al.. A fruit firmness QTL identified on linkage group 4 in sweet cherry (Prunus avium L.) is associated
with domesticated and bred germplasm.. Scientific Reports, 2019, 9 (1), pp.5008. 10.1038/s41598-
019-41484-8. hal-02627402 A fruit firmness QTL identified on linkage group 4 in
sweet cherry (Prunus avium L.) is associated with
domesticated and bred germplasm. Lichun Cai, José Quero-Garcia, Teresa Barreneche, Elisabeth Dirlewanger,
Christopher Saski, Amy Iezzoni Distributed under a Creative Commons Attribution 4.0 International License A fruit firmness QTL identified on
linkage group 4 in sweet cherry
(Prunus avium L.) is associated with
domesticated and bred germplasm Received: 15 August 2018
Accepted: 8 March 2019
Published: xx xx xxxx Received: 15 August 2018
Accepted: 8 March 2019
Published: xx xx xxxx Fruit firmness is an important market driven trait in sweet cherry (Prunus avium L.) where the desirable
increase in fruit firmness is associated with landrace and bred cultivars. The aim of this work was to
investigate the genetic basis of fruit firmness using plant materials that include wild cherry (syn. mazzard), landrace and bred sweet cherry germplasm. A major QTL for fruit firmness, named qP-FF4.1,
that had not previously been reported, was identified in three sweet cherry populations. Thirteen
haplotypes (alleles) associated with either soft or firm fruit were identified for qP-FF4.1 in the sweet
cherry germplasm, and the “soft” alleles were dominant over the “firm” alleles. The finding that sweet
cherry individuals that are homozygous for the “soft” alleles for qP-FF4.1 are exclusively mazzards
and that the vast majority of the bred cultivars are homozygous for “firm” alleles suggests that this
locus is a signature of selection. Candidate genes related to plant cell wall modification and various
plant hormone signaling pathways were identified, with an expansin gene being the most promising
candidate. These results advance our understanding of the genetic basis of fruit firmness and will help to
enable the use of DNA informed breeding for this trait in sweet cherry breeding programs. Sweet cherry (Prunus avium L.) is an important fruit crop in temperate regions and fresh fruit is highly valued. Sweet cherries for the fresh market are hand harvested, often mechanically sorted and frequently in transit for
several weeks to distant markets. Because of the harvesting, handling and marketing practices, fresh market sweet
cherries need to be firm when harvested. Consumers also prefer fresh market sweet cherries that are firm1. Sour
cherry (Prunus cerasus L.), a tetraploid relative of the diploid sweet cherry, is primarily used for processed prod-
ucts such as jam, juice and pie filling, and therefore neither the supply chain nor the consumer requires the level
of firmness necessary for sweet cherry. In addition, the softer texture of sour cherry is a positive attribute for their
use in cooked products and beverages. However, very soft sour cherries are rejected by the processors as mechan-
ical pit removal is problematic.h p
p
The sweet cherries grown for fruit production were domesticated from wild cherry (syn. HAL Id: hal-02627402
https://hal.inrae.fr/hal-02627402v1
Submitted on 26 May 2020 L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d’enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés. HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License www.nature.com/scientificreports www.nature.com/scientificreports Received: 15 August 2018
Accepted: 8 March 2019
Published: xx xx xxxx Scientific Reports | (2019) 9:5008 | https://doi.org/10.1038/s41598-019-41484-8 A fruit firmness QTL identified on
linkage group 4 in sweet cherry
(Prunus avium L.) is associated with
domesticated and bred germplasm mazzard), which is
believed to have originated around the Caspian and Black Seas and subsequently spread throughout Europe and
south to Iran2. Mazzards have extremely small fruit and are grown for their high value lumber. The three mazzards
used in this study also have very soft fruit. Increases in fruit size and firmness are the main fruit traits associated
with the domestication of sweet cherry from its wild relatives. Sour cherry was formed from the hybridization
between sweet cherry and the tetraploid ground cherry (P. fruticosa Pall.), a wild bush species native to Eastern
Europe2. Sour and ground cherry have fruit that is significantly softer than that of the commercial sweet cherry.hi it
The importance of fruit firmness for sweet cherry can be traced back to early records of cherry cultivation. For instance, sweet cherry cultivation in the Jerte Valley, Extremadura, Spain, reported for the first time in 1352,
was based on farmer’s selection of local cultivars with improved quality attributes, including firmer fruit3. Cherry
cultivation in this region grew significantly during the XIXth century, and was mainly based on four very firm
cultivars harvested stemless, which were traditionally known as ‘Picotas’ including ‘Ambrunés’, ‘Pico Negro’, ‘Pico 1Department of Horticulture, Michigan State University, East Lansing, MI, 48824, USA. 2UMR 1332 BFP, INRA,
Université de Bordeaux, F-33140, Villenave d’Ornon, France. 3Department of Plant and Environmental Sciences,
Clemson University, Clemson, SC, 29634, USA. Correspondence and requests for materials should be addressed to
A.I. (email: iezzoni@msu.edu) 1 www.nature.com/scientificreports/ Populationa
Size
Mean ± SD
Range
σ2
g
σ2
e
Heritability
INRA ‘Fercer’ × ‘X’ F1 populationb
67
55 ± 12
32–80
139
30
0.97
INRA germplasm collectionc
193
65 ± 11
35–86
95
72
0.73
RosBREED pedigreed populationd
528
243 ± 47
137–351
2535
244
0.91
Table 1. Summary of fruit firmness data for three sweet cherry populations. Note: aDifferent methods for
phenotyping were used for the INRA and RosBREED populations. bPhenotypic data is from seven years
(2009–2013, 2015–2016). cPhenotypic data is from two years (2014–2015). dPhenotypic data is from two years
(2011–2012). Table 1. Summary of fruit firmness data for three sweet cherry populations. Note: aDifferent methods for
phenotyping were used for the INRA and RosBREED populations. bPhenotypic data is from seven years
(2009–2013, 2015–2016). cPhenotypic data is from two years (2014–2015). dPhenotypic data is from two years
(2011–2012). Limon Negro’ and ‘Pico Colorado’. A fruit firmness QTL identified on
linkage group 4 in sweet cherry
(Prunus avium L.) is associated with
domesticated and bred germplasm At a period when no modern transportation systems existed in Spain, cher-
ries were transported with mules from this valley to the country’s capital, Madrid, a several day journey, and
consumer demand suggested that the fruit still kept acceptable quality. Another example of a relatively old firm
cultivar is ‘Bing’, which was selected from a seedling of ‘Black Republican’ in 1875 in Oregon, USA4. Today, ‘Bing’
still remains the most important cultivar of the Pacific Northwest, USA, and has also been fundamental in the
extremely fast development of sweet cherry cultivation in Chile, which is a country that directs most of its pro-
duction to long-distance export markets. One of the first goals of sweet cherry breeding programs was to select
hybrids with large and firm fruits. As an example, the INRA breeding program released during the 1980’s the
cultivar ‘Fercer’ (Arcina®), obtained from an open pollination of ‘Stark Hardy Giant’, which was one of the first
cultivars producing very large fruit (up to 15 g) with a high level of firmness as well. Subsequently, this cultivar
was heavily used as a parent in the INRA breeding program, leading to many new cultivars, such as ‘Folfer’,
‘Ferdouce’, ‘Fertille’ or ‘Ferdiva’5.i Little is known about the genetic control of fruit firmness in either sweet or sour cherry. In a study of two F1
populations derived from three sweet cherry cultivars (‘Regina’ × ‘Lapins’, ‘Regina’ × ‘Garnet’), the phenotypic
data of the progeny fit normal distributions suggesting that the trait was quantitatively inherited6. Multiple quan-
titative trait loci (QTLs) for firmness were identified, the largest one on linkage group (LG) 5, but none of the QTL
explained more than 24.1% of the phenotypic variance. A second QTL study was done using the sweet cherry
population ‘Ambrunés’ × ‘Sweetheart’ that also exhibited a continuous distribution for firmness7. In this case, a
previously undetected QTL was identified on LG 1 along with a previously identified QTL on LG 6. No QTL for
fruit firmness have yet to be identified in sour cherry.i i
yi
y
Genes controlling fruit firmness have been well investigated in many species including tomato, peach and
apple. In these species, the physiological modifications of the cell wall organization were considered important
components of tissue firmness8. Many enzymes, which are capable of altering cell wall texture, have been pro-
posed9,10. A fruit firmness QTL identified on
linkage group 4 in sweet cherry
(Prunus avium L.) is associated with
domesticated and bred germplasm For example, endopolygalacturonase (endoPG), encoded by a multiple-gene family, is well established
as one of the major enzymes involved in pectin disassembly in tomato and kiwifruit11,12. In apple, endoPG was
also shown to be involved in fruit softening process and its regulation was found to be ethylene dependent13. Copy number variation of a gene cluster encoding endoPG was also found to mediate flesh texture in peach14. In
sweet cherry, genome-wide transcriptional dynamics from developing fruit between flowering and maturity at
14 time points were investigated and the results suggested tight developmental regulation of genes functioning in
diverse processes such as sugar transport, lipid metabolism and cell wall rearrangement related to changes in fruit
firmness15. To date, no genes have been identified that control the variation for fruit firmness in sweet cherry, but
candidate genes underlying the firmness QTL identified on LG 5 in sweet cherry have been proposed6. In sour
cherry, expansin genes were found to be upregulated during ripening (also the period of fruit softening)16.hi y
p
g
p g
g
p
g
pt
g
The objectives of this study were to (1) identify and characterize the QTL(s) for fruit firmness segregating in
an F1 sweet cherry population, (2) explore whether the QTL identified is associated with the firmness that accom-
panied sweet cherry domestication and breeding, and the presence of softer fruit exhibited by sour cherry, and (3)
identify candidate genes for fruit firmness within the QTL region. Scientific Reports | (2019) 9:5008 | https://doi.org/10.1038/s41598-019-41484-8 Results
Ph Phenotypic variation for fruit firmness. The progeny in the ‘Fercer’ × ‘X’ population exhibited a wide
range of fruit firmness; however, the distribution was bimodal with more individuals exhibiting soft fruit (Table 1,
Figs 1a and S1). ‘X’ was assigned as the paternal parent of this population since the recorded paternal parent was
found incorrect based on genotype data and the correct parent is unknown. ‘Fercer’ had a multi-year fruit firm-
ness mean of ~67 g/mm2 (Min 56, Max 82) which is aligned with the firm-fruited progeny group. A wide range of
variation for fruit firmness was also observed for individuals from the INRA sweet cherry germplasm collection
and RosBREED germplasm (Fig. 1b,c). In the INRA sweet cherry germplasm collection, the majority of soft-
fruited individuals were characterized as landraces as opposed to bred cultivars. In the RosBREED germplasm,
the majority of soft-fruited individuals were either mazzards or hybrids with mazzards. When the two INRA
populations were compared for firmness for the one year that they were both phenotyped, the sweet cherry ger-
mplasm collection exhibited a wider phenotypic distribution compared to the F1 population (Fig. 1). Within the
three populations, the ANOVA analysis revealed highly significant effects for the different genotypes (Table 1). In
all three populations, the broad-sense heritabilities were high (0.73–0.97), indicating that much of the phenotypic
variation in these populations is genetically controlled (Table 1). The highest heritability for fruit firmness (0.97)
was obtained from the multi-year data for the ‘Fercer’ × ‘X’ population, likely because the environmental varia-
tion was low among years compared to the genetic variation as suggested by the bimodal phenotypic distribution.hi g y
p
g
gg
y
p
yp
The firmness of the RosBREED sweet cherry materials and sour cherry materials could be directly compared
as they were phenotyped using the same instrumentation. Results
Ph Fruit from the sour cherry individuals exhibited a Scientific Reports | (2019) 9:5008 | https://doi.org/10.1038/s41598-019-41484-8 2 www.nature.com/scientificreports/ 0
5
10
15
20
25
31-35
36-40
41-45
46-50
51-55
56-60
61-65
66-70
71-75
76-80
81-85
sla
u
d
ivi
d
n
i f
o r
e
b
m
u
N
Fruit firmness
0
5
10
15
20
25
30
35
26-30
31-35
36-40
41-45
46-50
51-55
56-60
61-65
66-70
71-75
76-80
81-85
86-90
91-95
sla
u
d
ivi
d
n
i f
o r
e
b
m
u
N
Fruit firmness
Bred cultivars
Landrace
0
10
20
30
40
50
60
70
80
121-140 141-160 161-180 181-200 201-220 221-240 241-260 261-280 281-300 301-320 321-340 341-360
sla
u
d
ivi
d
n
i f
o r
e
b
m
u
N
Fruit firmness
Landrace, bred cultivar or its offspring
Wild × Cultivar F1 offspring or wild cherry
a
b
c
Figure 1. Frequency distributions of fruit firmness for three sweet cherry panels. (a) INRA ‘Fercer’ × ‘X’ F1
population; (b) INRA germplasm collection; (c) RosBREED pedigreed germplasm. Data from 2015 was used
for (a,b) since it is the only common year in which firmness data was collected for the two INRA sweet cherry
populations. Data summaries and frequency distributions using multi-year data are presented in Table 1 and
Fig. S1, respectively. 0
5
10
15
20
25
31-35
36-40
41-45
46-50
51-55
56-60
61-65
66-70
71-75
76-80
81-85
sla
u
d
ivi
d
n
i f
o r
e
b
m
u
N
Fruit firmness
a Fruit firmness
0
5
10
15
20
25
30
35
26-30
31-35
36-40
41-45
46-50
51-55
56-60
61-65
66-70
71-75
76-80
81-85
86-90
91-95
sla
u
d
ivi
d
n
i f
o r
e
b
m
u
N
Fruit firmness
Bred cultivars
Landrace
b b c 0
10
20
30
40
50
60
70
80
121-140 141-160 161-180 181-200 201-220 221-240 241-260 261-280 281-300 301-320 321-340 341-360
sla
u
d
ivi
d
n
i f
o r
e
b
m
u
N
Fruit firmness
Landrace, bred cultivar or its offspring
Wild × Cultivar F1 offspring or wild cherry
c Figure 1. Frequency distributions of fruit firmness for three sweet cherry panels. (a) INRA ‘Fercer’ × ‘X’ F1
population; (b) INRA germplasm collection; (c) RosBREED pedigreed germplasm. Results
Ph Year
LGa
LODa
CI (cM)a
Peak (cM)
Da
PVE (%)a
2009
Xa4
10.7
28.4–38.0
33.2
−17.6
62.8
2010
X4
23.3
30.2–38.0
34.1
−26.3
82.4
2011
X4
18.1
30.0–40.1
35.0
−22.5
70.2
2012
X4
22.4
31.4–39.1
35.2
−22.9
78.3
2013
X4
11.3
24.9–42.8
33.9
−18.7
54.0
2015
X4
25.9
30.5–38.0
34.2
−25.7
84.6
2016
X4
17.6
30.8–39.0
34.9
−21.7
70.8
MYb
Fa4
20.6
10.3–67.7
39.0
−12.1
20.1
MYb
X4
125.3
33.1–36.0
34.5
−21.8
70.2 Year
LGa
LODa
CI (cM)a
Peak (cM)
Da
PVE (%)a
2009
Xa4
10.7
28.4–38.0
33.2
−17.6
62.8
2010
X4
23.3
30.2–38.0
34.1
−26.3
82.4
2011
X4
18.1
30.0–40.1
35.0
−22.5
70.2
2012
X4
22.4
31.4–39.1
35.2
−22.9
78.3
2013
X4
11.3
24.9–42.8
33.9
−18.7
54.0
2015
X4
25.9
30.5–38.0
34.2
−25.7
84.6
2016
X4
17.6
30.8–39.0
34.9
−21.7
70.8
MYb
Fa4
20.6
10.3–67.7
39.0
−12.1
20.1
MYb
X4
125.3
33.1–36.0
34.5
−21.8
70.2 Table 2. Summary of QTLs for fruit firmness identified on linkage group (LG) 4 in the ‘Fercer’ × ‘X’ F1
population. Note: aLinkage group (LG), LG of ‘X’ (X), LG of ‘Fercer’ (F); Logarithm of odds ratio (LOD);
Confidence interval (CI); Difference between the two homozygotes at the marker loci (d); Percentage of
variation explained by the QTL (PVE). bSignificant QTLs are presented for each year as well as significant QTL
identified when data was combined over multiple years (MY). Table 2. Summary of QTLs for fruit firmness identified on linkage group (LG) 4 in the ‘Fercer’ × ‘X’ F1
population. Note: aLinkage group (LG), LG of ‘X’ (X), LG of ‘Fercer’ (F); Logarithm of odds ratio (LOD);
Confidence interval (CI); Difference between the two homozygotes at the marker loci (d); Percentage of
variation explained by the QTL (PVE). bSignificant QTLs are presented for each year as well as significant QTL
identified when data was combined over multiple years (MY). Table 2. Summary of QTLs for fruit firmness identified on linkage group (LG) 4 in the ‘Fercer’ × ‘X’ F1
population. Note: aLinkage group (LG), LG of ‘X’ (X), LG of ‘Fercer’ (F); Logarithm of odds ratio (LOD);
Confidence interval (CI); Difference between the two homozygotes at the marker loci (d); Percentage of
variation explained by the QTL (PVE). bSignificant QTLs are presented for each year as well as significant QTL
identified when data was combined over multiple years (MY). smaller range of firmness (98–197) compared to sweet cherry (137–397) (Figs 1 and S2). Results
Ph Data from 2015 was used
for (a,b) since it is the only common year in which firmness data was collected for the two INRA sweet cherry
populations. Data summaries and frequency distributions using multi-year data are presented in Table 1 and
Fig. S1, respectively. Year
LGa
LODa
CI (cM)a
Peak (cM)
Da
PVE (%)a
2009
Xa4
10.7
28.4–38.0
33.2
−17.6
62.8
2010
X4
23.3
30.2–38.0
34.1
−26.3
82.4
2011
X4
18.1
30.0–40.1
35.0
−22.5
70.2
2012
X4
22.4
31.4–39.1
35.2
−22.9
78.3
2013
X4
11.3
24.9–42.8
33.9
−18.7
54.0
2015
X4
25.9
30.5–38.0
34.2
−25.7
84.6
2016
X4
17.6
30.8–39.0
34.9
−21.7
70.8
MYb
Fa4
20.6
10.3–67.7
39.0
−12.1
20.1
MYb
X4
125.3
33.1–36.0
34.5
−21.8
70.2
Table 2. Summary of QTLs for fruit firmness identified on linkage group (LG) 4 in the ‘Fercer’ × ‘X’ F1
population. Note: aLinkage group (LG), LG of ‘X’ (X), LG of ‘Fercer’ (F); Logarithm of odds ratio (LOD);
Confidence interval (CI); Difference between the two homozygotes at the marker loci (d); Percentage of
variation explained by the QTL (PVE). bSignificant QTLs are presented for each year as well as significant QTL
identified when data was combined over multiple years (MY). Year
LGa
LODa
CI (cM)a
Peak (cM)
Da
PVE (%)a
2009
Xa4
10.7
28.4–38.0
33.2
−17.6
62.8
2010
X4
23.3
30.2–38.0
34.1
−26.3
82.4
2011
X4
18.1
30.0–40.1
35.0
−22.5
70.2
2012
X4
22.4
31.4–39.1
35.2
−22.9
78.3
2013
X4
11.3
24.9–42.8
33.9
−18.7
54.0
2015
X4
25.9
30.5–38.0
34.2
−25.7
84.6
2016
X4
17.6
30.8–39.0
34.9
−21.7
70.8
MYb
Fa4
20.6
10.3–67.7
39.0
−12.1
20.1
MYb
X4
125.3
33.1–36.0
34.5
−21.8
70.2
Table 2. Summary of QTLs for fruit firmness identified on linkage group (LG) 4 in the ‘Fercer’ × ‘X’ F1
population. Note: aLinkage group (LG), LG of ‘X’ (X), LG of ‘Fercer’ (F); Logarithm of odds ratio (LOD);
Confidence interval (CI); Difference between the two homozygotes at the marker loci (d); Percentage of
variation explained by the QTL (PVE). bSignificant QTLs are presented for each year as well as significant QTL
identified when data was combined over multiple years (MY). Results
Ph As the QTL confidence intervals over-
lapped, the QTLs derived from ‘X’ and ‘Fercer’ were considered to be the same and this QTL was named qP-FF4.1. INRA Sweet Cherry Germplasm Collection: In the INRA sweet cherry germplasm collection, variation in fruit
firmness significantly associated with SNPs located on chromosome four (Fig. 3a). The SNP most significantly
associated with fruit firmness, ss490552928, has a peach physical map position (v.2) of 11,472,398 bp. Fruit firm-
ness was significantly different among the three SNP genotypes, as illustrated for ss490552928 and the second
most significant SNP on chromosome 4, ss490552906, located at 10,880,163 bp (Fig. 3b,c). For ss490552928, mean
fruit firmness for the SNP genotype AB, was intermediate to that of BB and AA, with increased firmness associ-
ated with BB (Fig. 3b). For ss490552906, mean fruit firmness for the SNP genotype AB was significantly less than
that of the most firm genotypic class (AA) and not significantly different from the softest class (BB) (Fig. 3c). In
addition to the SNPs on chromosome 4, an additional SNP on chromosome 1 (ss490546759, 23,455,434 bp) was
significantly associated with fruit firmness (Fig. 3a). This SNP is within a region where a fruit firmness QTL was
previously identified in four of six years in a ‘Regina’ × ‘Lapins’ population6.h and estimated to only cover about one Mbp (10,335,393–11,216,807 bp) based on the peach physical map v.2. The
peak position of this QTL was located at 34.5 cM, which was estimated to be approximately 10.76 Mbp on the
peach physical map v.2 (Table 2). One QTL was identified segregating from ‘Fercer’, but this QTL was only sig-
nificant over multiple years, explaining 20.1% of the phenotypic variance. As the QTL confidence intervals over-
lapped, the QTLs derived from ‘X’ and ‘Fercer’ were considered to be the same and this QTL was named qP-FF4.1. and estimated to only cover about one Mbp (10,335,393–11,216,807 bp) based on the peach physical map v.2. The
peak position of this QTL was located at 34.5 cM, which was estimated to be approximately 10.76 Mbp on the
peach physical map v.2 (Table 2). One QTL was identified segregating from ‘Fercer’, but this QTL was only sig-
nificant over multiple years, explaining 20.1% of the phenotypic variance. As the QTL confidence intervals over-
lapped, the QTLs derived from ‘X’ and ‘Fercer’ were considered to be the same and this QTL was named qP-FF4.1. Results
Ph The fruit firmness of
95% of the sour cherry individuals was less than 170, indicating that almost all the sour cherries were softer than
the sweet cherry landraces, bred cultivars, and their offspring. smaller range of firmness (98–197) compared to sweet cherry (137–397) (Figs 1 and S2). The fruit firmness of
95% of the sour cherry individuals was less than 170, indicating that almost all the sour cherries were softer than
the sweet cherry landraces, bred cultivars, and their offspring. QTL analysis. ‘Fercer’ × ‘X’: The two parental maps constructed consisted of 110 SNPs for ‘Fercer’ and 87
SNPs for ‘X’ with an average coverage of one marker every 6.7 and 7.5 cM, respectively. QTL analysis from the
‘Fercer’ × ‘X’ population identified four QTL in the multi-year analysis (LGs 4, 5, 6 and 8); however, the major
stable QTL was located on LG 4 (Table 2, Fig. 2 and Table S1). This QTL segregating from the ‘X’ parent was sig-
nificant in all seven years evaluated and across years, and the percentage of variation explained by the QTL ranged
from 54.0% to 84.6%. The QTL confidence interval based on multiple years’ analysis was small (33.1–36.0 cM) Scientific Reports | (2019) 9:5008 | https://doi.org/10.1038/s41598-019-41484-8 3 www.nature.com/scientificreports/ p
p
(
n
oitis
o
p
cit
e
n
e
G
cM)
Figure 2. Summary of QTLs for fruit firmness (FF) identified in the INRA ‘Fercer’ × ‘X’ F1 population. QTLs
are shown for each year when detection was significant as well as by combining multiple years of data (MY)
using the ‘multi-environment’ option of MultiQTL software. Explained percentage of variance is given for each
QTL. Figure 2. Summary of QTLs for fruit firmness (FF) identified in the INRA ‘Fercer’ × ‘X’ F1 population. QTLs
are shown for each year when detection was significant as well as by combining multiple years of data (MY)
using the ‘multi-environment’ option of MultiQTL software. Explained percentage of variance is given for each
QTL. and estimated to only cover about one Mbp (10,335,393–11,216,807 bp) based on the peach physical map v.2. The
peak position of this QTL was located at 34.5 cM, which was estimated to be approximately 10.76 Mbp on the
peach physical map v.2 (Table 2). One QTL was identified segregating from ‘Fercer’, but this QTL was only sig-
nificant over multiple years, explaining 20.1% of the phenotypic variance. Results
Ph The solid gray areas on the right side correspond to
regions with positive evidence for the presence of QTLs. (a) Fruit firmness in 2011; (b) Fruit firmness in 2012. Year
Interval
(cM)
Peak
(cM)
BFa
Peak
(cM)
Effecta
PVE
(%)a
2011
31–47
33
4.8
33
48
16.4
2012
31–37
33
32.1
33
83
83.5
Table 3. Summary of the fruit firmness QTL identified on linkage group (LG) 4 in the RosBREED germplasm. Note: aSignificance was presented by Bayes Factor (BF); Additive effect (Effect); Percentage of variation
explained by the QTL (PVE). Year
Interval
(cM)
Peak
(cM)
BFa
Peak
(cM)
Effecta
PVE
(%)a
2011
31–47
33
4.8
33
48
16.4
2012
31–37
33
32.1
33
83
83.5
Table 3. Summary of the fruit firmness QTL identified on linkage group (LG) 4 in the RosBREED germplasm. Note: aSignificance was presented by Bayes Factor (BF); Additive effect (Effect); Percentage of variation
explained by the QTL (PVE). Table 3. Summary of the fruit firmness QTL identified on linkage group (LG) 4 in the RosBREED germplasm. Note: aSignificance was presented by Bayes Factor (BF); Additive effect (Effect); Percentage of variation
explained by the QTL (PVE). Table 3. Summary of the fruit firmness QTL identified on linkage group (LG) 4 in the RosBREED germplasm. Note: aSignificance was presented by Bayes Factor (BF); Additive effect (Effect); Percentage of variation
explained by the QTL (PVE). This difference is probably due to the high number of missing values in 2011 compared to 2012 (219 vs. 126,
respectively). The peak genetic map position of this QTL was 33 cM and the peak physical map position was
estimated to be ~10.8 Mbp. As this is similar to the peak position of the QTL identified in the ‘Fercer’ × ‘X’ pop-
ulation, this QTL was considered to be qP-FF4.1. Predictions for the genotypes of qP-FF4.1 for the RosBREED
germplasm were calculated by FlexQTL, as QQ, Qq and qq, where Q and q represent the “firm” and “soft” alleles,
respectively. FlexQTL used a bi-allelic model denoted by Q and q to estimate the QTL genotypes. The only indi-
viduals in this germplasm set predicted to be qq (and therefore soft) were three mazzard accessions, MIM 17,
MIM 23, and NY 54 (Table S2). Likewise the only individuals in this germplasm set predicted to be Qq were
offspring from these three mazzard accessions plus ‘Moreau’ and ‘Cristobalina’, old landrace cultivars, and their
offspring. Results
Ph INRA Sweet Cherry Germplasm Collection: In the INRA sweet cherry germplasm collection, variation in fruit
firmness significantly associated with SNPs located on chromosome four (Fig. 3a). The SNP most significantly
associated with fruit firmness, ss490552928, has a peach physical map position (v.2) of 11,472,398 bp. Fruit firm-
ness was significantly different among the three SNP genotypes, as illustrated for ss490552928 and the second
most significant SNP on chromosome 4, ss490552906, located at 10,880,163 bp (Fig. 3b,c). For ss490552928, mean
fruit firmness for the SNP genotype AB, was intermediate to that of BB and AA, with increased firmness associ-
ated with BB (Fig. 3b). For ss490552906, mean fruit firmness for the SNP genotype AB was significantly less than
that of the most firm genotypic class (AA) and not significantly different from the softest class (BB) (Fig. 3c). In
addition to the SNPs on chromosome 4, an additional SNP on chromosome 1 (ss490546759, 23,455,434 bp) was
significantly associated with fruit firmness (Fig. 3a). This SNP is within a region where a fruit firmness QTL was
previously identified in four of six years in a ‘Regina’ × ‘Lapins’ population6.h p
yi
y
g
p
p p
RosBREED Sour cherry germplasm: The sour cherry germplasm was also segregating for ss490552928; how-
ever, no sour cherry individuals had the BBBB genotype (Fig. S3a). The majority of the individuals were AAAB,
followed by AAAA, AABB and then ABBB. The association of SNP genotype and fruit firmness was investigated
within two segregating sour cherry populations and found to be not significant (Fig. S3b,c). This is in contrast
to the INRA sweet cherry germplasm collection where BB was the most prevalent genotype for this SNP and BB
individuals had significantly firmer fruit than AB or AA individuals (Fig. 3b).ii gi
yi
g
RosBREED sweet cherry germplasm: Two fruit firmness QTLs were identified in both years for the RosBREED
pedigree germplasm, one small effect QTL on LG 2 and one large effect QTL on LG 4 (Table 3, Fig. 4 and Table S1). The QTL on LG 4 explained 16.4% and 83.5% of the variation for fruit firmness in 2011 and 2012, respectively. Results
Ph Scientific Reports | (2019) 9:5008 | https://doi.org/10.1038/s41598-019-41484-8 4 www.nature.com/scientificreports/ 50
60
70
80
BB
AB
AA
ss490552928
s
s
e
n
m
ri
F
Genotype
BB(n=89, 68a)
AB(n=55, 62b)
AA(n=16, 55c)
50
60
70
80
AA
AB
BB
ss490552906
s
s
e
n
m
ri
F
Genotype
AA(n=103, 68a)
AB(n=49, 60b)
BB(n=6, 56b)
a
b
c
ss490552928
ss490552906
ss490546759
Figure 3. Association of genome-wide SNP markers with fruit firmness in the INRA sweet cherry germplasm
collection. (a) P values for all the SNP markers across the sweet cherry genome. The red dash line indicates the
threshold value for significant SNPs after Bonferroni correction (0.05/1215). (b) Phenotypic means based on
the genotype of the most significant SNP marker ss4905522928, and (c) the second most significant SNP marker
ss490552906. Phenotypic means that were significantly different (P < 0.05) are identified by different letters. a
ss490552928
ss490552906
ss490546759 50
60
70
80
BB
AB
AA
ss490552928
s
s
e
n
m
ri
F
Genotype
BB(n=89, 68a)
AB(n=55, 62b)
AA(n=16, 55c)
b 50
60
70
80
AA
AB
BB
ss490552906
s
s
e
n
m
ri
F
Genotype
AA(n=103, 68a)
AB(n=49, 60b)
BB(n=6, 56b)
c Figure 3. Association of genome-wide SNP markers with fruit firmness in the INRA sweet cherry germplasm
collection. (a) P values for all the SNP markers across the sweet cherry genome. The red dash line indicates the
threshold value for significant SNPs after Bonferroni correction (0.05/1215). (b) Phenotypic means based on
the genotype of the most significant SNP marker ss4905522928, and (c) the second most significant SNP marker
ss490552906. Phenotypic means that were significantly different (P < 0.05) are identified by different letters. Figure 4. QTL mapping result for RosBREED sweet cherry population illustrated by trace plots and posterior
probability of QTL positions along the genome exported from software FlexQTL. The beginning and the end of
the linkage groups are represented by vertical dashed lines. The solid gray areas on the right side correspond to
regions with positive evidence for the presence of QTLs. (a) Fruit firmness in 2011; (b) Fruit firmness in 2012. Figure 4. QTL mapping result for RosBREED sweet cherry population illustrated by trace plots and posterior
probability of QTL positions along the genome exported from software FlexQTL. The beginning and the end of
the linkage groups are represented by vertical dashed lines. Results
Ph Haplotypes for the fruit firmness QTL, qP-FF4.1, and their physical positions on the peach Genome
v2.039. In sweet cherry, haplotypes only identified in mazzard are marked as blue. Haplotypes only identified
in sour cherry are marked as green. The full list of plant materials exhibiting the 16 haplotypes are listed in
Table S3. The haplotypes for eight sour cherry parents are presented in Table S4. The haplotypes were deduced
to be associated with soft or firm fruit based on diplotype analysis in sweet cherry (Fig. 6). Marker
SNP
ss490552883
ss490559054
ss490552906
ss490552912
ss490552928
Physical position (bp)
10,241,247
10,414,884
10,880,163
11,044,975
11,472,398
Genetic position (cM)
33.5
33.6
33.7
33.7
33.8
Haplotype
H1 (firm)
B
B
A
B
B
H2 (soft)
A
B
B
A
A
H3 (soft)
B
B
B
A
A
H4 (firm)
A
A
A
B
B
H5 (firm)
A
B
A
B
B
H6 (firm)
B
B
A
B
A
H7 (firm)
B
A
A
B
B
H8 (soft)
A
A
B
B
A
H9 (soft)
A
B
B
B
A
H10 (soft)
A
B
A
B
A
H11 (soft)
B
A
B
B
A
H12 (soft)
A
A
B
B
B
H13 (soft)
B
B
B
B
A
H14 (?)
A
A
A
B
A
H15 (?)
A
A
B
A
B
H16 (?)
A
B
B
B
B
Figure 5. Haplotypes for the fruit firmness QTL, qP-FF4.1, and their physical positions on the peach Genome
v2.039. In sweet cherry, haplotypes only identified in mazzard are marked as blue. Haplotypes only identified
in sour cherry are marked as green. The full list of plant materials exhibiting the 16 haplotypes are listed in
Table S3. The haplotypes for eight sour cherry parents are presented in Table S4. The haplotypes were deduced
to be associated with soft or firm fruit based on diplotype analysis in sweet cherry (Fig. 6). Figure 5. Haplotypes for the fruit firmness QTL, qP-FF4.1, and their physical positions on the peach Genome
v2.039. In sweet cherry, haplotypes only identified in mazzard are marked as blue. Haplotypes only identified
in sour cherry are marked as green. The full list of plant materials exhibiting the 16 haplotypes are listed in
Table S3. The haplotypes for eight sour cherry parents are presented in Table S4. The haplotypes were deduced
to be associated with soft or firm fruit based on diplotype analysis in sweet cherry (Fig. 6). Results
Ph All other individuals were predicted to be QQ. This suggests that homozygosity for the firm Q allele at
qP-FF4.1 is a signature of selection exhibited by domesticated and bred sweet cherries. Haplotype analysis. To further trace and evaluate the allele effects of qP-FF4.1, five SNPs that span the peak
physical map QTL location were chosen for haplotype (allele) construction (Fig. 5). These five SNPs spanned
a ~1.23 Mbp and ~0.3 cM region of LG 4. Using these five SNPs, 13 haplotypes (H1 to H13) were identified
in the RosBREED sweet cherry germplasm and an additional three haplotypes were identified in sour cherry
(H14 to H16) (Tables S3 and S4). As the QTL haplotypes were based on SNP marker composition spanning the
QTL region and not variation in underlying genes, it is possible that the haplotypes identified over-represent the Haplotype analysis. To further trace and evaluate the allele effects of qP-FF4.1, five SNPs that span the peak
physical map QTL location were chosen for haplotype (allele) construction (Fig. 5). These five SNPs spanned
a ~1.23 Mbp and ~0.3 cM region of LG 4. Using these five SNPs, 13 haplotypes (H1 to H13) were identified
in the RosBREED sweet cherry germplasm and an additional three haplotypes were identified in sour cherry
(H14 to H16) (Tables S3 and S4). As the QTL haplotypes were based on SNP marker composition spanning the
QTL region and not variation in underlying genes, it is possible that the haplotypes identified over-represent the Scientific Reports | (2019) 9:5008 | https://doi.org/10.1038/s41598-019-41484-8 5 www.nature.com/scientificreports/ Marker
SNP
ss490552883
ss490559054
ss490552906
ss490552912
ss490552928
Physical position (bp)
10,241,247
10,414,884
10,880,163
11,044,975
11,472,398
Genetic position (cM)
33.5
33.6
33.7
33.7
33.8
Haplotype
H1 (firm)
B
B
A
B
B
H2 (soft)
A
B
B
A
A
H3 (soft)
B
B
B
A
A
H4 (firm)
A
A
A
B
B
H5 (firm)
A
B
A
B
B
H6 (firm)
B
B
A
B
A
H7 (firm)
B
A
A
B
B
H8 (soft)
A
A
B
B
A
H9 (soft)
A
B
B
B
A
H10 (soft)
A
B
A
B
A
H11 (soft)
B
A
B
B
A
H12 (soft)
A
A
B
B
B
H13 (soft)
B
B
B
B
A
H14 (?)
A
A
A
B
A
H15 (?)
A
A
B
A
B
H16 (?)
A
B
B
B
B
Figure 5. Results
Ph 40
50
60
70
80
H1H1
H2H3
H1H3
H1H2
Diplotype
s
s
e
n
m
ri
F
Diplotype
H1H1(n=17, 62a)
H2H3(n=9, 56ab)
H1H3(n=29, 53b)
H1H2(n=9, 51b)
150
200
250
300
350
H1H5
H1H1
H1H4
H1H6
H4H5
H4H4
H4H6
H2H4
H1H2
H4H10
H4H8
H4H11 H4H12 H1H13
Diplotype
s
s
e
n
m
ri
F
Diplotype
H1H5(n=15, 275a)
H1H1(n=47, 269a)
H1H4(n=104, 263ab)
H1H6(n=16, 261ab)
H4H5(n=48, 256abc)
H4H4(n=63, 255bc)
H4H6(n=6, 226cd)
H2H4(n=15, 224d)
H1H2(n=6, 212de)
H4H10(n=15, 196def)
H4H8(n=17, 192ef)
H4H11(n=10, 180efg)
H4H12(n=9, 174fg)
H1H13(n=18, 162g)
a
b
Figure 6. Comparison of fruit firmness (g/mm2) for progeny based on their diplotypes for qP-FF4.1 from (a)
the ‘Fercer’ × ‘X’ F1 population; and (b) the RosBREED pedigreed population. Only diplotypes represented
by six or more individuals were included. Phenotypic means that were significantly different (P < 0.05) are
identified by different letters. 150
200
250
300
350
H1H5
H1H1
H1H4
H1H6
H4H5
H4H4
H4H6
H2H4
H1H2
H4H10
H4H8
H4H11 H4H12 H1H13
Diplotype
s
s
e
n
m
ri
F
Diplotype
H1H5(n=15, 275a)
H1H1(n=47, 269a)
H1H4(n=104, 263ab)
H1H6(n=16, 261ab)
H4H5(n=48, 256abc)
H4H4(n=63, 255bc)
H4H6(n=6, 226cd)
H2H4(n=15, 224d)
H1H2(n=6, 212de)
H4H10(n=15, 196def)
H4H8(n=17, 192ef)
H4H11(n=10, 180efg)
H4H12(n=9, 174fg)
H1H13(n=18, 162g)
b 40
50
60
70
80
H1H1
H2H3
H1H3
H1H2
Diplotype
s
s
e
n
m
ri
F
Diplotype
H1H1(n=17, 62a)
H2H3(n=9, 56ab)
H1H3(n=29, 53b)
H1H2(n=9, 51b)
a Figure 6. Comparison of fruit firmness (g/mm2) for progeny based on their diplotypes for qP-FF4.1 from (a)
the ‘Fercer’ × ‘X’ F1 population; and (b) the RosBREED pedigreed population. Only diplotypes represented
by six or more individuals were included. Phenotypic means that were significantly different (P < 0.05) are
identified by different letters. number of functional alleles. Of the thirteen haplotypes exhibited by sweet cherry, four were only identified in
the mazzards (H8, H11, H12 and H13) (Tables S2, S3). The haplotypes most frequent in the RosBREED sweet
cherry germplasm and also not present in any mazzards were H4 (49.1%) and H1 (28.2%), suggesting that these
haplotypes are associated with firm fruit possibly due to the influence of human selection and breeding. However,
as only three mazzards were used in this study, it is possible that other mazzards might also possess a “firm” allele. Two of the commercially dominant cultivars notable for their fruit firmness, ‘Bing’ and ‘Ambrunés’, are H1H1 and
H4H4, respectively. Results
Ph These
effects were based on pairings with the “firm” haplotypes H1 or H4, indicating that these “soft” haplotypes present
in wild cherry are dominant to the “firm” haplotypes that are found in bred cultivars. It was not possible to deter-
mine if the three haplotypes identified in sour cherry (H14-16) were associated with firm or soft fruit due to the
dominance of soft compared to firm fruit (Table S4). In silico candidate genes. The qP-FF4.1 interval identified from both the INRA F1 and RosBREED pedi-
greed populations was used for candidate gene identification. This ~ 1.8 Mbp interval was between SNPs located
at 10,156,468 and 11,956,655 bp on chromosome 4 of the peach genome v2.0 and the same SNPs located between
12,928,603 and 14,860,789 bp on the sweet cherry genome (Fig. 7). In this region, 241 genes were predicted in the
sweet cherry genome (Table S5). From these genes, 25 were selected as candidate genes based on their potential
to be involved in the control of fruit firmness (Table 4, Fig. 7). The most promising candidate gene identified was
Pav_sc0002828.1_g410.1.mk which encodes an expansin protein related to plant cell wall metabolism. This gene
is very close to the QTL peak and an expansin gene with homology to Pav_sc0002828.1_g410.1.mk was found to
be expressed in sour cherry fruit and associated with tissue softening16. Of the three expansin genes identified that
were upregulated during softening in sour cherry fruit, the expansin gene PcEXP4 had the highest similarity to the
expansin gene in the sweet cherry genome as evidenced by their placement on a distal lineage, relative to the other
sour cherry expansins (Fig. S5a,b). The candidate expansin gene contains two functional domains (Expansin
EG45 and Expansin CBD) and three encoded signal peptide regions (H, N, and C) located on the N-terminus
region (Fig. S5c). Nine other candidate genes were predicted to encode plant cell wall modifying enzymes which
have been found to be potentially involved in regulating fruit firmness in peach and apple17,18. Fourteen candidate
genes were included as they are potentially involved in various plant hormone signaling pathways well known to
be involved in fruit maturation and ripening in non-climacteric and climacteric fruits and in sweet cherry firm-
ness19,20. Among these candidate genes, two are predicted to be NAC (NAM/ATAF1, 2/CUC2) transcription fac-
tors involved in the ethylene signaling pathway. Discussion
i d Genetic determinism and signature of selection for fruit firmness in sweet cherry. The bimodal
segregation for fruit firmness in the ‘Fercer’ × ‘X’ population provided the opportunity to identify a major QTL for
fruit firmness in sweet cherry that was also identified in a wide range of genetic backgrounds represented by the
INRA sweet cherry germplasm collection and the RosBREED pedigreed population. This is the first report of a
major QTL for fruit firmness identified on LG 4 in sweet cherry. However, due to the small size of the ‘Fercer’ × ‘X’
population, the QTL interval would be affected by potential errors in phenotyping and genotyping as well as the
environmental conditions. Despite this population size limitation, the QTL region estimated for all seven years was
stable and consistent, possibly due to the large effect of this QTL in this population as suggested by the bimodal
phenotypic distribution. However, future fine mapping is needed to more precisely define the QTL interval. In a
prior study of two sweet cherry populations between bred cultivars, ‘Regina’ × ‘Lapins’ and ‘Regina’ × ‘Garnet’,
QTLs for fruit firmness detected in at least three of the six years of study, were identified on LG 1, 2 and 56. On LG
4, a QTL was detected in only two of the six years analyzed in one of the two populations (‘Regina’ × ‘Lapins’), and
this QTL was located on the upper region of chromosome 4 in a region that does not overlap with that for qP-FF4.1. It is possible that the LG 4 QTL, qP-FF4.1, was not identified in these two populations, because all three parents
only had “firm” alleles for this locus. Indeed, all three haplotypes present in ‘Regina’ (H4H5) and ‘Lapins’ (H1H4)
were identified as “firm” alleles in this study. In contrast, the plant materials used in this study resulted in the iden-
tification of qP-FF4.1 because of the presence of “soft” alleles in the plant materials.hit it
The finding that sweet cherry individuals that are homozygous for the “soft” alleles for qP-FF4.1 are exclusively
mazzards and that the vast majority of bred cultivars are homozygous for “firm” alleles suggests that this locus
was a signature of selection during domestication and modern breeding. Results
Ph The haplotypes most frequent in the sour cherry selections were H3 (46.9%) followed by H11
(18.8%) and H10 (12.8%).h The qP-FF4.1 genotypes (diplotypes) for ‘Fercer’ and ‘X’ were H1H2 and H1H3, respectively (Fig. 6a). As
‘Early Burlat’ is the only sweet cherry founder known to have H3, it is likely that ‘Early Burlat’ is an ancestor of ‘X’. When the fruit firmness of the ‘Fercer’ × ‘X’ progeny were compared based on their qP-FF4.1 diplotypes, those
progeny that were H1H1, had significantly firmer fruit than progeny that were H1H3 or H1H2 (Fig. 6a). This is
consistent with the high relative frequency of H1 in bred germplasm. Furthermore, it suggests that H1 is reces-
sive to H3 and H2. In other words, for this QTL, firm fruit appears to be recessive to soft fruit. H1 and H2 were
deduced to be “firm” and “soft” alleles, respectively, as H1H2 individuals had significantly softer fruit than H1H1 Scientific Reports | (2019) 9:5008 | https://doi.org/10.1038/s41598-019-41484-8 6 www.nature.com/scientificreports/ individuals. The inheritance of H3, uniquely present in ‘Early Burlat’ and not present in any other RosBREED
germplasm, was further followed through breeding using this germplasm. Five U.S. cultivars have ‘Early Burlat’
in their ancestry and all five inherited the ‘Early Burlat’ H6, and not H3 (Fig. S4).hfi yi
y
g
The effects on fruit firmness associated with 14 qP-FF4.1 diplotypes were compared for the RosBREED ger-
mplasm. These 14 diplotypes, representing 10 haplotypes (H1, H2, H4 to H6, H8, H10 to H13), each consisted
of firmness data from six to 104 individuals (Fig. 6b). Fruit firmness ranged from a mean of 275 down to 162 for
the softest fruit. The four diplotypes that had the firmest fruit all had one or two copies of H1, paired either with
itself or with H4, H5 or H6. This suggests that in addition to H1 and H4, H5 and H6 can also be considered “firm”
alleles. However, when H1 or H4 were paired with H2, the mean fruit firmness was reduced significantly. This is
consistent with the dominant ‘soft’ effect of H2 observed in the ‘Fercer’ × ‘X’ progeny where H1H2 progeny had
significantly softer fruit than H1H1 progeny. Progeny with H8 and H10-13, only present in the mazzards, had
significantly softer mean fruit firmness than the majority of progeny homozygous for the firm diplotypes. Scientific Reports | (2019) 9:5008 | https://doi.org/10.1038/s41598-019-41484-8 Results
Ph Of these two genes, Pav_sc0000029.1_g070.1.mk, is a homolog of
the peach NAC gene ppa008301m that has been predicted to control maturity date21,22. The final candidate gene,
Pav_sc0000975.1_g210.1.mk, was predicted to be a Squamosa promoter-Binding Protein which has been found
to be associated with fruit ripening in tomato23. Discussion
i d In addition, three of the old cultivars
included in this study, ‘Moreau’, ‘Cristobalina’ and ‘Early Burlat’, have relatively soft fruit and their qP-FF4.1 geno-
types include one “soft” and one “firm” allele. In sour cherry, all of the germplasm are soft, as firmness comparable
to sweet cherry has not been a critical trait. This QTL region is the second region in sweet cherry that has been
shown to have been under selection, the first being a QTL region on LG 2 that contains a major QTL for fruit size24. Th
l
f
h ‘
’
‘ ’
l
d h
l
f
h
d
h
h “
ft” The results from the ‘Fercer’ × ‘X’ population and the RosBREED germplasm further indicate that the “soft”
alleles present in the mazzard accessions are dominant, or at least partially dominant over the “firm” alleles pres-
ent in bred cultivars. This is consistent with the findings in sour cherry where no individuals exhibited the firm-
ness of bred sweet cherry cultivars. No sour cherry individual was homozygous for the “firm” allele at qP-FF4.1, Scientific Reports | (2019) 9:5008 | https://doi.org/10.1038/s41598-019-41484-8 7 www.nature.com/scientificreports/ Figure 7. A circos plot depicting linkage group (LG) 4 in the ‘Lovell’ peach reference genome v.2.0 (orange)
and the corresponding LG 4 in the sweet cherry genome (red). The firmness QTL region is highlighted in
translucent blue and mapped SNP markers as orange circular glyphs. The detailed information for SNP markers
in the QTL region is presented in Table S6. The blue and red histograms are annotated genes, with direction of
transcription as blue (forward) or red (reverse). Candidate genes are labeled in the sweet cherry region. Figure 7. A circos plot depicting linkage group (LG) 4 in the ‘Lovell’ peach reference genome v.2.0 (orange)
and the corresponding LG 4 in the sweet cherry genome (red). The firmness QTL region is highlighted in
translucent blue and mapped SNP markers as orange circular glyphs. The detailed information for SNP markers
in the QTL region is presented in Table S6. The blue and red histograms are annotated genes, with direction of
transcription as blue (forward) or red (reverse). Candidate genes are labeled in the sweet cherry region. and therefore all the sour cherry individuals had at least one “soft” allele for qP-FF4.1. Discussion
i d These results are also
consistent with the results from progeny from a cross between a sweet cherry cultivar ‘Emperor Francis’ and
the mazzard accession NY 54. A major QTL for fruit size associated with domestication was identified in this F1
population25; however, QTL for fruit firmness could not be identified from this population because all the prog-
eny had soft fruit. Given that the qP-FF4.1 diplotypes for ‘Emperor Francis’ and NY 54 are H1H1 and H13H13,
respectively, and the conclusion that soft fruit is dominant over firm fruit, this result would be expected.iii
26h p
yti
p
In peach, a major QTL for fruit firmness was also identified on LG 4, first reported by Dettori et al.26. This
locus, termed F-M, controls both peach fruit firmness and flesh adhesion to the endocarp, with soft (melting)
fruit dominant to firm (non-melting) fruit. Two genes encoding endopolygalacturonase (endoPG) are considered
to be the causal genes at this locus. The peach physical map (v.2.0) positions of these two genes are as follows:
ppa006839m 19046344-19049605 bp, and ppa006857m 19081325-19083984 bp. Using the peach genome as a
proxy for cherry, this places the endoPGs and the F-M locus, ~ 8 Mbp distal to the qP-FF4.1. In contrast to peach,
no studies in cherry have associated endoPG with flesh firmness, nor were any endoPG genes been identified in
the qP-FF4.1 region. Peach and cherry also differ in their ethylene requirement for ripening. Peach is a climacteric
fruit meaning that it has a strong requirement for ethylene to ripen, while cherry is a non-climacteric fruit. Taken
together, these results suggest that the genetic control of fruit firmness in cherry evolved separately from that
of peach. This conclusion is consistent with qP-FF4.1 being associated with domesticated and bred germplasm. Discussion
i d Note: aThe cherry and peach genome sequences used were from Shirasawa et al.42 and Verde
et al.39, respectively. bNot available (NA) as no sequence match was found in the peach genome sequence. cThe
description is the Blast2GO ontology term. dThe tomato annotation was included in italic when it differed from
the cherry and/or peach annotation(s) and suggested a potential involvement with fruit firmness. Table 4. Summary of 25 candidate genes for fruit firmness located in the qP-FF4.1 interval on chromosome 4
in sweet cherry. Note: aThe cherry and peach genome sequences used were from Shirasawa et al.42 and Verde
et al.39, respectively. bNot available (NA) as no sequence match was found in the peach genome sequence. cThe
description is the Blast2GO ontology term. dThe tomato annotation was included in italic when it differed from
the cherry and/or peach annotation(s) and suggested a potential involvement with fruit firmness. QTL hotspot of qP-FF4.1 region. The scope of the work presented herein is limited to fruit firmness;
however, the qP-FF4.1 region is an important QTL “hotspot” for cherry breeders because major QTL for other
traits map to this region. LG 4 loci for two phenology traits, bloom and maturity date, have been conserved across
multiple Prunus species27. For bloom time, the major locus named Lb, was first reported in almond by Ballester
et al.28 and subsequently identified in multiple Prunus species29–32. In sour cherry, the peak peach genome v2.0
position for the bloom time QTL on chromosome 4 was ~10.8 Mbp33. For maturity date, a major QTL termed
qMD4.1, was identified first in peach34,35 and subsequently in cherry36,37. The most likely candidate gene for the
peach QTL qMD4.1 is ppa008301m, which is believed to be an NAC transcription factor. It maps to ~11.106 Mbp
on the peach genome sequence v1.038 which is equivalent to ~11.117 Mbp on the peach genome sequence v2.039. In a recent study, Isuzugawa et al.36 found that two sweet cherry candidate genes, homologous to the NAC tran-
scription factors identified in peach, also mapped within the maturity date QTL on LG 4. In addition, QTL for
fruit weight and soluble solids content have also been reported in this qP-FF4.1 region in peach34,35. An analysis of
maturity date for the ‘Fercer’ × ‘X’ population used in our study identified a QTL that explained on average 50%
of the phenotypic variance for maturity time37. Discussion
i d 8 Scientific Reports | (2019) 9:5008 | https://doi.org/10.1038/s41598-019-41484-8 www.nature.com/scientificreports/ Cherry candidate genes
Transcript start and stop (bp)
Descriptionc
Biological process
Cherry genomea
Peach genomea
Pav_sc0003492.1_g360.1.br
13090321~13090626
NAb
hypothetical protein PRUPE_7G080000/BHLH
transcription factord
Ethylene signaling pathway
Pav_sc0003492.1_g260.1.mk
13134328~13147715
10270962~10272561
UDP-glycosyltransferase 79B6-like
Plant cell wall metabolism
Pav_sc0003492.1_g250.1.mk
13147752~13148159
10276520~10277987
UDP-glycosyltransferase 79B6-like
Plant cell wall metabolism
Pav_sc0003492.1_g040.1.br
13251924~13253405
NA
Zinc finger MYM-type protein 1-like
Brassinosteroid signaling pathway
Pav_sc0000975.1_g110.1.mk
13329847~13330536
10435004~10436194
Ethylene-responsive transcription factor 4
Ethylene signaling pathway
Pav_sc0000975.1_g200.1.mk
13386372~13389094
10505281~10508243
Probable protein phosphatase 2C29/MYB
transcription factor
Auxin signaling pathway
Pav_sc0000975.1_g210.1.mk
13389781~13391094
10508967~10510314
Squamosal promoter-Binding Protein 1-like
Fruit ripening regulator
Pav_sc0002828.1_g670.1.mk
13612734~13616541
10718283~10722430
Beta-glucuronosyltransferase GlcAT14B
Plant cell wall metabolism
Pav_sc0002828.1_g650.1.mk
13627727~13637959
10738335~10747338
Probable serine/threonine-protein kinase
At1g54610/BZIP family transcription factor
Gibberellin signaling pathway
Pav_sc0002828.1_g640.1.mk
13648670~13654523
10755244~10761213
Inner membrane protein oxaA/Beta-glucosidase
Plant cell wall metabolism
Pav_sc0002828.1_g510.1.mk
13741324~13741986
10815979~10816956
Putative RING-H2 finger protein ATL19
Brassinosteroid signaling pathway
Pav_sc0002828.1_g470.1.mk
13776148~13776732
10818810~10819608
Putative RING-H2 finger protein ATL19
Brassinosteroid signaling pathway
Pav_sc0002828.1_g460.1.mk
13778460~13779378
10821182~10821971
Putative RING-H2 finger protein ATL19
Brassinosteroid signaling pathway
Pav_sc0002828.1_g410.1.mk
13809261~13815575
10861181~10863415
Expansin-A12
Plant cell wall metabolism
Pav_sc0002828.1_g380.1.mk
13831978~13833770
10882666~10885574
F-box/FBD/LRR-repeat protein At4g26340-like
Gibberellin signaling pathway
Pav_sc0002828.1_g310.1.mk
13868641~13869483
10929344~10930120
Glycine-rich cell wall structural protein 2
Plant cell wall metabolism
Pav_sc0002828.1_g280.1.mk
13892641~13893365
NA
Agamous-like MADS-box protein AGL29
Ethylene signaling pathway
Pav_sc0002828.1_g060.1.mk
14014881~14016967
11036801~11039019
Fiber protein Fb34/Zinc binding dehydrogenase
ABA signaling pathway
Pav_sc0000029.1_g020.1.mk
14069597~14074899
11087505~11091386
Beta-glucosidase 18-like isoform X1
Plant cell wall metabolism
Pav_sc0000029.1_g040.1.br
14079090~14079410
11092451~11093649
Beta-glucosidase 18-like isoform X1
Plant cell wall metabolism
Pav_sc0000029.1_g050.1.mk
14080593~14083893
11093783~11095088
Beta-glucosidase 18-like isoform X1
Plant cell wall metabolism
Pav_sc0000029.1_g070.1.mk
14097151~14098925
11116814~11118655
NAC domain-containing protein 72
Ethylene signaling pathway
Pav_sc0000029.1_g090.1.mk
14118138~14120442
11138518~11140641
NAC transcription factor 56
Ethylene signaling pathway
Pav_sc0002607.1_g160.1.mk
14294246~14301322
11300896~11310515
B3 domain-containing protein Os07g0679700-
like isoform X1
ABA signaling pathway
Pav_sc0000124.1_g110.1.mk
14575143~14616823
11627434~11632547
Cellulose synthase-like protein G2 isoform X2
Plant cell wall metabolism
Table 4. Summary of 25 candidate genes for fruit firmness located in the qP-FF4.1 interval on chromosome 4
in sweet cherry. Note: aThe cherry and peach genome sequences used were from Shirasawa et al.42 and Verde
et al.39, respectively. bNot available (NA) as no sequence match was found in the peach genome sequence. cThe
description is the Blast2GO ontology term. dThe tomato annotation was included in italic when it differed from
the cherry and/or peach annotation(s) and suggested a potential involvement with fruit firmness. Table 4. Summary of 25 candidate genes for fruit firmness located in the qP-FF4.1 interval on chromosome 4
in sweet cherry. www.nature.com/scientificreports/ www.nature.com/scientificreports/ having a firm fruit in a short period of time between blooming and maturity. This would be in particular the case
of cultivar ‘Early Burlat’, which has one of the shortest developmental periods between blooming and maturity. Hence, the recent method of developing sweet cherry cultivars which come to maturity at the same period as
‘Early Burlat’, but exhibit a significantly higher firmness, was to use genitors with an extra-early blooming time41. In sour cherry, none of the individuals evaluated had the genotype BBBB for ss490552928; therefore, the ‘A’ allele
was always present. This is consistent with “soft” fruit alleles for this locus being dominant to “firm” fruit alleles. In
sour cherry, bloom and maturity time are also correlated, however all individuals whether early or late maturing
have soft fruit compared to the firm fruit associated with bred sweet cherries.h t
pi
The multiple QTLs in the qP-FF4.1 region should be taken into consideration when performing breeding
selection in both sweet and sour cherry. Hence, it is of utmost importance to disentangle the genetic determinism
of the traits’ variation within this QTL; in particular, it would be helpful for breeders to know whether maturity
date and firmness are controlled by the same pleiotropic locus or by two closely linked genes. Fine mapping
initiatives might be conducted in order to search for recombinants within this narrow genetic interval. More
specifically, using cultivars such as ‘Early Burlat’ and ‘Fercer’ might be highly informative. Indeed, the predicted
diplotypes for both cultivars are H3H6 and H1H2, respectively; that is, each would have one “firm” and one “soft”
haplotype. However, ‘Fercer’ is known to be a significantly firmer cultivar as compared to ‘Early Burlat’. Multi-year
data from INRA indicate a mean firmness, as measured by Durofel, of 67 and 49 for ‘Fercer’ and ‘Early Burlat’,
respectively. This shows the complexity of the genetic determinism of fruit firmness, as already demonstrated
by Campoy et al.6 and might suggest as well the existence of epistatic interactions. To test the hypothesis that
maturity time and firmness are controlled by distinct genes in this LG 4 “hotspot”, breeders will need to produce
very large progeny populations when crossing ‘Early Burlat’ with other firmer but also later ripening cultivars in
order to obtain recombinants between the two hypothesized closely linked genes. www.nature.com/scientificreports/ For example, endoPG, the major enzyme associated with softening in peach,
was not identified in the qP-FF4.1 region.i Candidate genes controlling fruit firmness. The available sweet cherry genome sequence provided the
opportunity to identify agronomically important candidate genes for qP-FF4.142. In our study, the identification
of candidate genes was employed across species including sweet cherry, peach and tomato. Peach and sweet
cherry are closely related Prunus species and share a high level of synteny43. Therefore, prior to the publication of
the sweet cherry genome sequence, the peach genome was used as a proxy for candidate gene prediction in sweet
cherry6,27. Tomato was included as fruit firmness has been extensively studied in this species44,45, and like cherry
and peach, tomato is a fleshy carpel. Although fleshy fruits are physiologically classified as climacteric (tomato
and peach) and non-climacteric (cherry), these fruits share some common characteristics such as the role of plant
hormones and their interplay related to changes in firmness during fruit softening19,44,46. For example, all fruits i
Among the candidate genes identified, an expansin gene was considered as the most promising candidate for
several reasons. Firstly, expansin genes have been thought to contribute to fruit softening by weakening nonco-
valent interactions between cellulose microfibrils and hemicellulose components50. In tomato, expansin genes
have been shown to be associated with fruit ripening and firmness51,52. Secondly, the candidate expansin gene
in sweet cherry has sequence homology to the expansin gene PcEXP4 previously reported to be upregulated
during tissue softening in sour cherry16. Thirdly, the expansin gene in sweet cherry was predicted to contain
two functional domains; one of them was commonly found in pollen allergens which were proposed as cell
wall-loosening agents to induce extension of the plant cell wall53. Lastly, this gene is very close to the peak of the
qP-FF4.1 region. Among other candidate genes, transcription factors, such as NAC domain protein, MADS-box
protein and Squamosa promoter-Binding Protein could also play roles in regulating fruit firmness as they have
been shown to be involved in fruit ripening process21,23,54. However, future work is needed to fine map this region
and ultimately identify and characterize the genes and their alleles that underlie these QTL. www.nature.com/scientificreports/ Finally, the fact that among the
founders used in modern breeding, the haplotype H3 was only found in ‘Early Burlat’ agrees with the ‘originality’
of this cultivar in terms of developmental cycle; as already stated, ‘Early Burlat’ has a rather intermediate bloom
time but is one of the earliest maturing cultivars. Candidate genes controlling fruit firmness. The available sweet cherry genome sequence provided the
opportunity to identify agronomically important candidate genes for qP-FF4.142. In our study, the identification
of candidate genes was employed across species including sweet cherry, peach and tomato. Peach and sweet
cherry are closely related Prunus species and share a high level of synteny43. Therefore, prior to the publication of
the sweet cherry genome sequence, the peach genome was used as a proxy for candidate gene prediction in sweet
cherry6,27. Tomato was included as fruit firmness has been extensively studied in this species44,45, and like cherry
and peach, tomato is a fleshy carpel. Although fleshy fruits are physiologically classified as climacteric (tomato
and peach) and non-climacteric (cherry), these fruits share some common characteristics such as the role of plant
hormones and their interplay related to changes in firmness during fruit softening19,44,46. For example, all fruits
appear to respond to abscisic acid (ABA) and ethylene; but, in non-climacteric fruit, even if ABA has a more
dominant role, the fruit still exhibit characteristics of ethylene-dependent ripening19. Moreover, recent studies
seem to indicate that the classification of fruits as either climacteric or non-climacteric is an oversimplification44. Some fruits, like melons, can display both climacteric and non-climacteric behaviors47 while kiwi fruit can display
non-climacteric behaviors in the first stage of ripening and climacteric behaviors and in the second stage of ripen-
ing48. The fruit softening process involves the physiological modification of the cell wall and during the ripening
phase of the fruit, plant hormones play important roles in this process49. Therefore, genes related to plant cell wall
metabolism or various hormone signaling pathways were considered as candidates in this study. Much of tissue
firmness work in tomato also focused on characterizing the potential role of cell wall–modifying genes and the
transcription factors involved in hormone signaling pathways11,23. Although a cross-species strategy could help
identify more candidate genes for fruit firmness, it should be noted that mechanisms controlling firmness in these
three species are possibly different. Discussion
i d This QTL was detected in the same region as the one for firmness;
however, the peak detected with the multi-year analysis was at 36.1 cM, which is almost 2 cM downstream from
the peak for the firmness QTL.h pi
Q
The clustering of these QTLs is in agreement with the correlations observed in the populations studied among
the two fruit traits: firmness and maturity date. Indeed, within the ‘Fercer’ × ‘X’ population, early maturing
individuals bear smaller and softer cherries than the late maturing ones. In the RosBREED germplasm, a prior
study of this germplasm identified a QTL for maturity date that overlapped with qP-FF4.140. In this prior study,
ss490552928 was associated with maturity date with the ‘A’ and ‘B’ alleles associated with “early” and “late” matu-
rity, respectively; however, one of the haplotypes that contained the ‘A’ allele was associated with late maturity40. A similar result was obtained from the INRA sweet cherry germplasm collection population where the ‘A’ for
ss490552928, was also associated with early maturity and soft fruit. The phenomenon of early maturing cultivars
which tend to be softer than late maturing ones could probably result from a developmental impossibility of Scientific Reports | (2019) 9:5008 | https://doi.org/10.1038/s41598-019-41484-8 9 Methods The data for the sweet and sour cherry individuals were collected for two
years (sweet cherry, 2011 and 2012; sour cherry, 2011 and 2013). Since different methods were used to measure
firmness in the RosBREED and INRA populations, the data for these populations were analyzed separately.h i
p p
p p
y
p
y
The phenotypic data were analyzed using SAS version 9.13 (SAS Institute Inc.) and the model PROC MIXED
was used to obtain the variance components. PROC CORR was performed to calculate the correlation coefficients
of fruit firmness among different years. Broad-sense heritability (H2) was calculated using estimates for the indi-
viduals based on the following random linear model: =
+
+
+
µ
Y
y
g
e
ij
i
j
ij where Yij is the phenotypic value of the jth individual in ith year; µ is the mean value of fruit firmness; yi is the ran-
dom effect of the ith year on the phenotype; gj is the random genotypic effect of jth individual; and eij is the model
residual. H2 was calculated using the following equation: H2 = σ2
g/(σ2
g + σ2
e/n), where σ2
g is the genetic variance,
σ2
e is the residual error, and n is the number of years. where Yij is the phenotypic value of the jth individual in ith year; µ is the mean value of fruit firmness; yi is the ran-
dom effect of the ith year on the phenotype; gj is the random genotypic effect of jth individual; and eij is the model
residual. H2 was calculated using the following equation: H2 = σ2
g/(σ2
g + σ2
e/n), where σ2
g is the genetic variance,
σ2
e is the residual error, and n is the number of years. Genotyping and genetic map. All individuals from the three sweet cherry populations were genotyped
with the RosBREED Illumina Infinium cherry SNP array of 5,696 SNP markers58 and SNP genotypes were scored
using the Genotyping Module of GenomeStudio Data Analysis software59. For the ‘Fercer’ × ‘X’ F1 population, a
total of 724 SNP markers were polymorphic and segregating in this population. A linkage map was constructed
using JoinMap 4.060 and Kosambi’s mapping function was used to convert recombination frequency into map
distance. The two resulting parental maps consisted of 110 and 87 SNP markers, polymorphic for ‘Fercer’ and ‘X’,
respectively. Methods Plant materials. Three sweet cherry populations were used in this study. These were: (1) an INRA bi-pa-
rental F1 population, (2) the INRA sweet cherry germplasm collection, and (3) RosBREED (www.rosbreed.org)
pedigreed population. The INRA bi-parental F1 population consisted of a progeny of 67 individuals derived from
a cross between cultivar ‘Fercer’ and an unknown parent called ‘X’. The INRA sweet cherry germplasm collection,
maintained by INRA’s Prunus Genetic Resources Center at Bourran, France, included 193 sweet cherry accessions
collected from France and other 15 countries of America, Asia and Europe55. RosBREED pedigreed populations
consisted of a set of 65 elite sweet cherry and mazzard clones, and 463 unselected F1 seedlings from 86 crosses
from the Washington State University sweet cherry breeding program (Table S2). The germplasm of RosBREED
pedigreed population, spanning six generations, was considered representative of U.S. public breeding germplasm
for this crop24. For sour cherry, a total of 338 individuals including parents, ancestors and offspring from five
bi-parental F1 populations, were used. These individuals were grown at the Michigan State University Clarksville
Research Station, Clarksville, Michigan. A detailed description of these sour cherry plant materials is presented
in Cai et al.33. Phenotyping and phenotype modeling. Fruit were harvested from the field when ripe based on a sub-
jective assessment of skin color, texture and taste56,57, placed in coolers for transport back to the laboratory, and
evaluated the same day. Fruit firmness (g/mm2) was evaluated using different methods for INRA’s populations
and RosBREED’s sweet and sour cherry pedigreed populations. For the two INRA populations (F1 population and
the sweet cherry germplasm collection), fruit firmness was measured using a Durofel (Setop Giraud Technologie,
Cavaillon, France) texture analyzer on the day of harvest. A 3-mm probe was applied at two points on the fruit
equator, the movement of the probe was recorded and the average of the two measures on ten fruits was used. Data were collected from seven years (2009–2013, 2015 and 2016) for the F1 population and two years for the ger-
mplasm collection (2014 and 2015). For the RosBREED sweet cherry and sour cherry individuals, fruit firmness
was measured from 25 fruits that were at room temperature using the compression test of BioWorks FirmTech 2
(Wamego, KS, USA). Compression was performed from the fruit cheek and with the stems still on the fruit. The
mean value of 25 measures were used. www.nature.com/scientificreports/ The haplotypes and
their germplasm sources described in this study provide a genetic framework for this future discovery.ii In conclusion, we identified a major QTL for fruit firmness in three sweet cherry populations that is associated
with domesticated and bred germplasm. As all commercial sweet cherry cultivars must meet consumer demands
for firm fruit, knowledge of the desirable alleles at this QTL would be targets for marker-assisted breeding. For
example, it would be especially useful to select against “soft” alleles in cases where wild germplasm is being use as
breeding parents. Candidate genes for this fruit firmness QTL were proposed; however, future fine mapping and
transcriptomic analysis is needed to enable the identification of the underlying gene(s). Scientific Reports | (2019) 9:5008 | https://doi.org/10.1038/s41598-019-41484-8 10 www.nature.com/scientificreports/ Methods The genome-wide significance cutoff was set at 4 × 10−5 (0.05/1215).t gh
g
gif
(
)
QTL analysis in RosBREED sweet cherry pedigreed population was done using FlexQTL software that is
designed for use with multiple pedigree-connected families65. Markov Chain Monte Carlo (MCMC) simulation,
implemented in FlexQTL software, was applied to obtain samples from the joint posterior distribution of the
model. A total of 1,000 samples (500,000 iterations with thinning value of 500) were stored for each simulation
and then used for statistical inference. The inference on the number of QTLs was based on a pairwise compar-
ison of models differing from each other by one QTL. The Bayes factor parameters (2lnBF) were interpreted as
non-significant (0–2), positive (2–5), strong (5–10) or decisive (>10) evidence for the presence of QTL. The
inference on the QTL position was based on posterior intensity and the inference on the QTL contribution was
based on the posterior mean estimates of the QTL effect size. Both additive and dominant genetic models were
tested using a maximum number of 10 QTLs. Prior number of QTL was set to 1 or 3 and genome-wide analyses
were performed twice for each prior using different seed numbers to test the robustness of the analysis. Haplotype (diplotype) analysis. Haplotypes (i.e. alleles) for the fruit firmness QTL were identified for
the INRA F1 population and the RosBREED sweet and sour cherry pedigreed germplasm. Five SNPs covering
the consensus QTL region identified from the sets of plant materials were chosen for haplotype construction. Phased SNP marker information was obtained for each individual from the RosBREED sweet cherry germplasm
from the FlexQTL output. Haplotypes were assigned using the PediHaplotyper software66. The SNP phasing for
the sour cherry haplotypes was described in Cai et al.33. Unique haplotypes were named from H1 to H16 ran-
domly. Statistical analyses for the association between haplotype (diplotype) and fruit firmness were performed
using the software R version 3.1.367. QTL genotypes (QQ, Qq, qq) predicted by FlexQTL were used to deduce if
a haplotype was associated with soft or firm fruit. As ‘Q’ is assigned to the higher phenotypic value, in this case
increased firmness, QQ, Qq, and qq genotypes correspond to two “firm” alleles, one “firm” and one “soft” allele,
and two “soft” alleles, respectively. Haplotypes were also deduced to be associated with soft or firm fruit based on
comparison of diplotypes. In silico Candidate Genes. Methods For the INRA sweet cherry germplasm collection, marker data curation was described in Campoy
et al.55. A total of 1,215 SNP markers were retained after removing the following four SNP types: (1) SNPs failing
to generate clear genotype clusters; (2) SNPs with missing genotypes greater than 5%; (3) SNPs showing high
distortion for Hardy-Weinberg equilibrium (>0.0001); and (4) SNPs with minor allele frequencies lower than
5%. For RosBREED pedigreed population, marker data curation was described in Cai et al.24. A total of 1,617
SNPs were identified as robust markers and therefore used for QTL analysis. Genetic positions for these markers
were determined by aligning and integrating these physical positions (based on Peach Genome v2.0)39 with the
sweet cherry ‘Regina’ × ‘Lapins’ SNP linkage map61. The sour cherry plant materials were also genotyped using
the RosBREED Illumina Infinium cherry SNP array58. The generation of the sour cherry genetic data, including
haplotype reconstruction was described in Cai et al.33. QTL analysis. QTL analyses were performed for all three sweet cherry populations using different mapping
softwares. For ‘Fercer’ × ‘X’, QTL mapping was carried out using MultiQTL v2.6 software with the multiple inter-
val mapping (MIM) approach used (MultiQTL Ltd, Haifa, Israel, 2005, www.multiQTL.com). Different types
of analyses were performed for single year and multiple years, respectively. Each year was first analyzed inde-
pendently in order to examine the stability of the QTLs. An analysis combining all years was performed using
the multiple environment option with increase of the accuracy of the QTL detection. The detailed QTL mapping
methodology was as described in Castède et al.27. The graphical presentation of QTL locations on the linkage
groups was obtained using the MapChart software version 2.262.h g
p
g
pt
A genome-wide association analysis was done for the INRA sweet cherry germplasm collection. This analy-
sis tested the association between fruit firmness and the SNP markers on the chromosome where the firmness
QTL was located. A total of 1215 SNP markers across the sweet cherry genome were used in the analysis and Scientific Reports | (2019) 9:5008 | https://doi.org/10.1038/s41598-019-41484-8 11 www.nature.com/scientificreports/ the SNPs associated with fruit firmness were identified according to a mixed linear model (MLM) using the
software TASSEL version 5.2.6163. Corrections for population structure and kinship were adopted in the model. Population structure was described in the study of Campoy et al.55. The relative kinship matrix was calculated
using SPAGeDi64. Methods The list of genes within the QTL interval and their functional annotations in
sweet cherry were obtained from the sweet cherry genome42 available on the Genome Database of Rosaceae
website (GDR, https://www.rosaceae.org). The corresponding predicted cherry protein sequences obtained from
GDR were blasted against the National Center for Biotechnology Information (NCBI) database to obtain gene
ontology terms using BLASTP in the program Blast2GO68 with an E-value cutoff of 0.001. The sequences of the
genes within the QTL interval were also blasted to the peach genome v2.039 available at GDR and the tomato
genome ITAG 2.4069 available at Sol genomics Network (https://solgenomics.net), and their best gene matches
and annotations were extracted. Overall, three annotations (sweet cherry, peach and tomato) and gene ontology
results from Blast2GO were considered for the identification of candidate genes for fruit firmness. Candidate
genes considered were those genes predicted to be involved in plant cell wall metabolism or various hormone
(Ethylene, Brassinosteroid, Auxin, Gibberellin, and ABA) signaling pathways associated with fruit ripening. A
circular plot of the mapping and candidate gene data was prepared with the Circos plotting tool70. Alignment and
analysis of the expansin genes was performed with ClustalW v2.1 multiple sequences alignment tool. A dendro-
gram of the four genes was constructed using the Jukes-Cantor genetic distance model, and the neighbor-joining
tree building method within the GeneiousV.11.1.4 GUI software71,72. Data Availability
G
d
f
h Genotypic data for the INRA bi-parental population and RosBREED pedigreed populations is available at the
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This project was supported in part by the USDA-NIFA-Specialty Crop Research Initiative project, RosBREED:
Enabling marker-assisted breeding in Rosaceae (2009-51181-05808) and RosBREED 2: Combining disease
resistance with horticultural quality in new rosaceous cultivars (2014-51181-22378). This project was also
supported by the CEP Innovation, the private partner of INRA breeding program. We thank Dr. José Antonio
Campoy and Mr. Guillaume Lalanne-Tisné for the curation of SNP data of ‘Fercer’ × ‘X’ progeny and the
construction of the ‘Fercer’ and ‘X’ parental maps. We also thank the technical staff of A3C team and UEA Unit
for the management of trees as well as the phenotyping activities and the INRA’s ‘Prunus Genetic Resources
Center’ for preserving and managing the sweet cherry germplasm collections. Author Contributions A.I. designed the experiments and provided financial support; L.C. and J.Q. analyzed the data; T.B. provided data
for INRA sweet cherry germplasm collection; E.D. and C.S. provided candidate gene list; L.C., J.Q. and A.I. wrote
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66. Voorrips, R. E., Bink, M. C. A. M., Kruisselbrink, J. Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-41484-8. Competing Interests: The authors declare no competing interests. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional affiliations Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and
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Validation of Biomarkers in Gene Expression Datasets of Inflammatory Bowel Disease: IL13RA2, PTGS2 and WNT5A as Predictors of Responsiveness to Infliximab Therapy
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Journal of proteomics and bioinformatics
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cc-by
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Győrffy et al., J Proteomics Bioinform 2014, 7:9
http://dx.doi.org/10.4172/jpb.1000329 Proteomics & Bioinformatics Research Artic
Research Article pen Access
Open Access Validation of Biomarkers in Gene Expression Datasets of Inflammatory
Bowel Disease: IL13RA2, PTGS2 and WNT5A as Predictors of
Responsiveness to Infliximab Therapy Validation of Biomarkers in Gene Expression Datasets of Inflammatory
Bowel Disease: IL13RA2, PTGS2 and WNT5A as Predictors of
Responsiveness to Infliximab Therapy é Kormos1, Luca Bartha1, András Szabó2, Balázs Győrffy2,3, Jan Budczies4 and Barna Vásárhelyi1,5* András Győrffy1, Máté Kormos1, Luca Bartha1, András Szabó2, Balázs Győrffy2,3, Jan Budczies4 and Barna Vásárhelyi1,5* 1MTA-SE Pediatrics and Nephrology Research Group, Budapest, Hungary
22nd Department of Pediatrics, Semmelweis University, Budapest, Hungary
3MTA TTK Lendület Cancer Biomarker Research Group, Budapest, Hungary
4Institute of Pathology, Charité-Universitätsmedizin Berlin, 10117 Berlin, Germany
5Department of Laboratory Medicine, Semmelweis University Budapest, Hungary J Proteomics Bioinform
ISSN: 0974-276X JPB, an open access journal Introduction Infliximab therapy controls effectively the signs and symptoms
of inflammatory bowel disease (IBD) in the majority of patients who
fail to respond conventional therapy. About 40% of IBD patients,
however, are not responding and are exposed to risks of IFX therapy
without clinical benefits [1]. Identification of non-responding patients
before the therapy is, therefore, a big challenge for clinicians. Clinical
factors determining pharmacokinetics [2] such as baseline laboratory
data, medicinal history, smoking, dosing schedules are implicated
in therapeutic failure. However, patients with comparable clinical
characteristics still differ largely in term of their responsiveness to IFX
therapy. Therefore, biomarkers are needed to classify IBD patients
whether they are candidate or not to IFX treatment. The availability of recent gene expression microarray data obtained
in IBD groups later responding and non-responding to IFX therapy
provides an opportunity to re-analyze how the published genes would
perform in another IBD population. In this study we cross-validated published data with GEO datasets. We also analyzed whether biomarkers identified in blood or biopsy
samples reflect gene expression patterns in biopsies and peripheral
blood mononuclear cells, respectively. As a result of this work we *Corresponding author: Barna Vásárhelyi, Department of Laboratory
Medicine, Semmelweis University Nagyvárad tér 4., 1089 Budapest,
Hungary, Tel: 0036-20-6663246; Fax: 0036-1- 2100278 Ext. 56492; E-mail:
vasarhelyi.barna@med.semmelweis-univ.hu *Corresponding author: Barna Vásárhelyi, Department of Laboratory
Medicine, Semmelweis University Nagyvárad tér 4., 1089 Budapest,
Hungary, Tel: 0036-20-6663246; Fax: 0036-1- 2100278 Ext. 56492; E-mail:
vasarhelyi.barna@med.semmelweis-univ.hu Biomarkers may vary in term of their nature, physiologic function
and the sample of origin. While antibodies against IFX are associated
with loss of response [3], they are not suitable to predict response
in IFX-naive patients. Recently, in addition to routine laboratory
indicators and immune markers [4] much research activity focused on
the measurement of gene expression values in colon biopsy samples
and peripheral blood to predict the individual response to IFX in IFX-
naive IBD patients. However, study designs including methods used
to determine gene expression and patient populations to be enrolled
were quite heterogeneous. In addition, the majority of these studies
investigated some specific elements or pathways of disease pathogenesis
and did not analyze the gene expression patterns as a whole. Therefore, Received July 24, 2014; Accepted August 28, 2014; Published September 01,
2014 Citation: Győrffy A, Kormos M, Bartha L, Szabó A, Győrffy B, et al. *Corresponding author: Barna Vásárhelyi, Department of Laboratory
Medicine, Semmelweis University Nagyvárad tér 4., 1089 Budapest,
Hungary, Tel: 0036-20-6663246; Fax: 0036-1- 2100278 Ext. 56492; E-mail:
vasarhelyi.barna@med.semmelweis-univ.hu *Corresponding author: Barna Vásárhelyi, Department of Laboratory
Medicine, Semmelweis University Nagyvárad tér 4., 1089 Budapest,
Hungary, Tel: 0036-20-6663246; Fax: 0036-1- 2100278 Ext. 56492; E-mail:
vasarhelyi.barna@med.semmelweis-univ.hu Citation: Győrffy A, Kormos M, Bartha L, Szabó A, Győrffy B, et al. (2014)
Validation of Biomarkers in Gene Expression Datasets of Inflammatory Bowel
Disease: IL13RA2, PTGS2 and WNT5A as Predictors of Responsiveness to
Infliximab Therapy. J Proteomics Bioinform 7: 272-277. doi:10.4172/jpb.1000329 Abstract Background: Some patients with inflammatory bowel disease (IBD) do not respond to infliximab (IFX) therapy. Gene expression studies revealed genes that may help to predict non-responding IBD patients. Our purpose was to
validate the discriminating power of published genes. Methods: Microarray datasets of IBD patients responding or non-responding to IFX were downloaded from
GEO database (‘transcriptomic arm’). Published genes discriminating responding and non-responding patients were
identified in PubMed (‘biomarker arm’). Using ROC-analysis, the discriminating performance of genes in ‘biomarker
arm’ were re-tested in each datasets of ‘transcriptomic arm’. We also performed an independent analysis of colon
biopsy datasets to identify novel discriminating genes. Results: The transcriptomic arm of four GEO datasets (3 and 1 from colon biopsies and blood cells, respectively)
included 99 patients (of those, 59 and 40 were IFX responder and non-responder, respectively). Of the 65 candidate
genes reported in biopsy specimens 25 genes discriminated significantly (p<0.05) infliximab responders and non-
responders in the three biopsy datasets consistently. Of the 39 candidate genes reported in peripheral blood 9 genes
provided significant discrimination after re-testing. Independent analysis of three biopsy datasets identified the top
five genes. Three genes (IL13RA2, PTGS2 and WNT5A) were highly effective discriminator in each analysis. Conclusion: This analysis identified IL13RA2, PTGS2 and WNT5A, three genes in colonic tissues of IBD
patients as suitable to discrimination between patients responding and non-responding to IFX therapy. These genes
encode proteins implicated in intestinal pathology; the high expression in non-responding patients may indicate
important targets in IBD therapy. Keywords: Infliximab; Gene expression; Inflammatory bowel disease
I t
d
ti the information originated from these studies is unavoidably skewed to
these factors and may ignore important detail beyond the scope of that
research. These limitations make the comparison and validation of the
individual study results quite difficult. Literature search We have set up the present meta-analysis according to "Preferred
Reporting Items for Systematic Reviews and Meta-Analyses" guidelines
published in 2009 (PRISMA)[5]. We used a pipeline requiring for both
the markers to be validated and the data to be used for validation to be
identified by a search of available publications and datasets. The design
for cross-validation process (including identification of transcriptome
datasets and published biomarkers) is presented in Figure 1. Genetic biomarkers of IFX responsiveness: the ‘biomarker
arm’ Genetic biomarkers of IFX responsiveness: the ‘biomarker
arm’ identified genes that were consistently suitable to discriminate between
IBD patients responding and non-responding to IFX therapy in all
analyzed datasets of colon biopsy specimens. identified genes that were consistently suitable to discriminate between
IBD patients responding and non-responding to IFX therapy in all
analyzed datasets of colon biopsy specimens. A search was performed in Pubmed using combinations of the
keywords ”infliximab”, ”response”, ”marker” and ”gene expression”. Only publications in English language were considered. Repeated
publications were identified and excluded so that each gene was included
just once in the final database. In addition, only studies publishing
gene expression biomarkers for inflammatory bowel disease, Crohn’s
disease and ulcerative colitis were included. We also recorded whether
the biomarker relates to a gene determined in blood or a biopsy sample. The unique gene symbols and names were identified for each gene by
querying the online repository of the HUGO Gene Nomenclature
Committee (http://www.genenames.org). Construction of GEO-based microarray database: the
transcriptome arm Receiver Operating Characteristics (ROC) analysis was performed
in the R statistical environment (http://www.r-project.org) using
Biobase Bioconductor libraries (http://www.bioconductor.org/). In
a ROC analysis the true positive rate (=sensitivity of the marker) is
compared to the false positive rate (=100-specificity of the marker)
for each available cut-off point of a parameter. Thus, each value of the
ROC curve represents a sensitivity/specificity pair corresponding to a
particular decision threshold. Then, area under the ROC curve (AUC)
is computed to measure of how well this parameter can distinguish
between the two diagnostic groups (responder/nonresponder). The
AUC value of a perfect biomarker is 1.0. We have searched GEO (http://www.ncbi.nlm.nih.gov/gds) using
the keywords "infliximab" and "response" to identify transcriptomic
datasets publishing response data against infliximab treatment. Only
datasets including at least five patients for each included cohort
(responder and non-responder) were considered. Patients receiving
placebo were also excluded from the analysis. The series matrix file for
each eligible dataset was downloaded and subsequently each dataset
was processed separately. RECEIVER OPERATING
CHARACTERISTIC (ROC)
Ulcerative colitis,
patient n=79
Crohn’s disease,
patient n=20
Dataset
Responder
Nonresponder
GSE12251 (UC)
12
11
GSE16879 (UC)
8
16
GSE23597 (UC)
25
7
GSE42296 (CD)
14
6
Identification
Screening
Eligibility
Included
Analysis
GEO search: ”infliximab ” +
”response”
Available series,
dataset n=13
Inflammatory bowel
diseases, n=4
PubMed search: ”infliximab”+ ”response” +
”marker” + ”gene expression”
Re-publication of previous
marker, n=31
No marker published, n=98
Not IBD
(rheumatoid arthritis
n=48; psoriasis n=23;
ankylosing spondylitis
n=19, other n=45)
Inflammatory bowel
diseases, n=28
Genes in these
studies, n=24
All available publications,
n=292
Ulcerative colitis,
n=6
Crohn’s disease,
n=13
IBD,
n=9
Genes in these
studies, n=63
Genes in these
studies, n=52
BIOMARKER ARM
TRANSCRIPTOMIC ARM
Other disease, n=9
(RA, JIA, depression)
Measured in at least one transcriptomic study, n=104
Blood-based markers, n=39
Biopsy-based markers, n=65
Figure 1: A flow diagram depicting the exploration of infliximab therapy response biomarker candidates in the published scientific literature and the search
for transcriptomic datasets in GEO (www.ncbi.nlm.nih.gov/geo/) according to the PRISMA guidelines. Introduction (2014)
Validation of Biomarkers in Gene Expression Datasets of Inflammatory Bowel
Disease: IL13RA2, PTGS2 and WNT5A as Predictors of Responsiveness to
Infliximab Therapy. J Proteomics Bioinform 7: 272-277. doi:10.4172/jpb.1000329 Copyright: © 2014 Győrffy A, et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and
source are credited Volume 7(9) 272-277 (2014) - 272 Citation: Győrffy A, Kormos M, Bartha L, Szabó A, Győrffy B, et al. (2014) Validation of Biomarkers in Gene Expression Datasets of Inflammatory
Bowel Disease: IL13RA2, PTGS2 and WNT5A as Predictors of Responsiveness to Infliximab Therapy. J Proteomics Bioinform 7: 272-277. doi:10.4172/jpb.1000329 Biomarkers We identified 292 publications investigating candidate biomarkers
of non-responsiveness to IFX therapy. Thirty-one papers repeatedly
tested biomarkers of previous studies, while 98 studies investigated
biomarkers other than gene expression. Of the remaining 163
publications, 28 studies enrolled IBD patients. In these studies a total of
139 genes were tested as possible biomarkers of IFX responsiveness. Of
these, 104 were present in at least one of the 4 GEO datasets (Figure 1). Identification of transcriptomic databases The database search in GEO resulted in all together 13 datasets,
of which four datasets were related to IBD. Three and one GEO
dataset were originated from colon biopsies and blood mononuclear
cells (PBMC), respectively. These were related to 59 and 40 IFX
responder and non-responder patients, respectively. Table 1 presents
the characteristics of these 4 datasets; clinical characteristics for each
patient are listed in Supplemental Table 2. The entire database contains
20,168 genes–one should note, however, that some inconsistencies
regarding gene lists measured in individual datasets exist. Genes
originated from biomarker studies were verified by each of the four
independent datasets. Construction of GEO-based microarray database: the
transcriptome arm Then, 65 and 39 genetic biomarkers identified in biopsy samples and
blood were related to GEO datasets of biopsy samples and PBMC. The
complete results of analysis for each of the 104 candidate genes within
each of the four datasets and the link to the original publication in
PubMed are listed in Supplemental Table 3. set. Instead, we performed the entire analysis in each of the datasets. Then, 65 and 39 genetic biomarkers identified in biopsy samples and
blood were related to GEO datasets of biopsy samples and PBMC. The
complete results of analysis for each of the 104 candidate genes within
each of the four datasets and the link to the original publication in
PubMed are listed in Supplemental Table 3. Of the 65 candidate genes reported as possible biomarker in
biopsy specimens just 25 genes discriminated significantly (p<0.05)
infliximab responders and non-responders in the three biopsy datasets
consistently. The discriminative power of these biomarkers is listed in
Table 2A in term of ROC AUC (area under the curve) values. Of note,
just three biomarkers (S100A8, SELP and CD86) were significant also
in PBMC dataset. Of the 39 candidate genes reported as possible biomarker in
peripheral blood just 9 genes in PBMC datasets provided significant
discrimination (p<0.05) between infliximab responders and non-
responders. The discriminative power of these biomarkers is listed in
Table 2B in term of ROC AUC values; just three biomarker (WARS,
MAP1LC3B and ODC1) were significant also in biopsies. With this approach, we re-tested the discrimination performance
of each of the 104 published genes in the 4 GEO datasets. Statistical
significance was set at p<0.05 and the genes had to reach statistical
significance using the average p value across all datasets. Discussion The discrimination of IBD patients as possible responders and non-
responders before the initiation of IFX therapy is a still unmet clinical
need. Previous attempts to predict therapeutic response identified
several genes in colon biopsy samples and peripheral blood. Before one would start to investigate clinical usefulness of biomarker
cited as predictive for therapeutic response in a larger population, the
published data are worth to be re-validated in independent cohorts. In
our study the cross-validation of the discriminating biomarkers was
performed with ROC analysis. ROC AUC curve and associated p values
were generated using an R-script (available as supplemental material). With this approach we demonstrated that merely 17 and 23 per cent of
published biomarkers provided consistently significant discrimination
between responding and non-responding patients in transcriptomic
datasets of the same kind of sample (colon and PBMC, respectively). Of note, these biomarkers were not overlapping: biomarkers identified
in colon biopsy samples were not identical to those in peripheral blood. Therefore, datasets of PBMC and biopsy samples are not comparable
and their information content cannot be pooled. Analysis of datasets irrespectively of published results The complete results of analysis including AUC values and p values
for all available 20,168 genes in each of the four datasets is presented as
Supplemental Table 4. In order to identify the most robust markers, we
considered only those markers which were significant across all three
independent analyses of colon biopsy samples and with the average of
ROC values. The top five of these markers are listed in Table 3; of those,
three (IL13RA2, PTGS2 and WNT5A) were also identified in earlier
studies. An independent analysis of GEO datasets was also performed using
the same statistical approach. Citation: Győrffy A, Kormos M, Bartha L, Szabó A, Győrffy B, et al. (2014) Validation of Biomarkers in Gene Expression Datasets of Inflammatory
Bowel Disease: IL13RA2, PTGS2 and WNT5A as Predictors of Responsiveness to Infliximab Therapy. J Proteomics Bioinform 7: 272-277.
doi:10.4172/jpb.1000329 J Proteomics Bioinform
ISSN: 0974-276X JPB, an open access journal Construction of GEO-based microarray database: the
transcriptome arm (UC: Ulcerative Colitis, IBD: Inflammatory Bowel
Disease, CD: Crohn’s Disease, JIA: Juvenile Idiopathic Arthritis, HUGO: Human Genome Organization Gene Nomenclature Committee) GEO search: ”infliximab ” +
”response” PubMed search: ”infliximab”+ ”response” +
”marker” + ”gene expression” All available publications,
n=292 Not IBD
(rheumatoid arthritis
n=48; psoriasis n=23;
ankylosing spondylitis
n=19, other n=45) Re-publication of previous
marker, n=31 RECEIVER OPERATING
CHARACTERISTIC (ROC) Figure 1: A flow diagram depicting the exploration of infliximab therapy response biomarker candidates in the published scientific literatu
for transcriptomic datasets in GEO (www.ncbi.nlm.nih.gov/geo/) according to the PRISMA guidelines. (UC: Ulcerative Colitis, IBD: Inf
Disease, CD: Crohn’s Disease, JIA: Juvenile Idiopathic Arthritis, HUGO: Human Genome Organization Gene Nomenclature Committee) Figure 1: A flow diagram depicting the exploration of infliximab therapy response biomarker candidates in the published scientific literature and the search
for transcriptomic datasets in GEO (www.ncbi.nlm.nih.gov/geo/) according to the PRISMA guidelines. (UC: Ulcerative Colitis, IBD: Inflammatory Bowel
Disease, CD: Crohn’s Disease, JIA: Juvenile Idiopathic Arthritis, HUGO: Human Genome Organization Gene Nomenclature Committee) J Proteomics Bioinform
ISSN: 0974-276X JPB, an open access journal Volume 7(9) 272-277 (2014) - 273 ISSN: 0974-276X JPB, an open access journal We also set up an R function to enable instant reproduction of
all of the results published in our present study. An R script for ROC
analysis was also added to the supplemental material (Supplemental
R script 1.R) with an additional input file needed for designation of
the samples into responder and non-responder cohorts-these data
were originally published in the series matrix files in GEO, but could
not be downloaded in an automated way (Supplemental Table 1). The
ROC_stats function in the supplemented script calculates the p-values
that test the null hypothesis that the area AUC really equals 0.5. First,
the script calculates the standard error of our null hypothesis from
which the function derives the z-score. Finally, the p-value is calculated
by the transformation of the z-score using the normal distribution. In
addition, the function performs downloading and parsing for each of
the original series matrix files using the GEOquery package (http://
watson.nci.nih.gov/~sdavis/), therefore, there is no need to include
these in the supplemental material. This new ROC analysis script
not only enables the user to validate the results of present study, but
also provides an easy to use tool for independent validation of new
biomarker candidates. set. Instead, we performed the entire analysis in each of the datasets. Table 1: Summary of the GEO datasets included in the construction of the
transcriptomic database for the meta-analysis of predictive power for infliximab
therapy response. ISSN: 0974-276X JPB, an open access journal Published biomarkers of response to infliximab therapy Additionally, to confirm the possible relevance of some published
genes, we also performed a de novo analysis on biopsy datasets. Of
the top five genes we identified as very efficient discriminators in
microarray datasets, two genes were novel, while three genes (IL13RA2,
PTGS2 and WNT5A) published earlier were validated in our study. All
of these genes exhibited significantly increased expression in colon
biopsy samples of IBD patients not responding to IFX therapy. (We
should note, however, that the prediction would be further improved if
instead of single genes combinations of gene expression levels would be To avoid batch effects, the datasets were not combined into one
Dataset
n
Platform
Tissue
Reference
GSE16879
24
GPL570
colon biopsy
[23]
GSE12251
23
GPL570
colon biopsy
[24]
GSE23597
32
GPL570
colon biopsy
[25]
GSE42296
20
GPL6244
PBMC
[26]
Table 1: Summary of the GEO datasets included in the construction of the
transcriptomic database for the meta-analysis of predictive power for infliximab
therapy response. To avoid batch effects, the datasets were not combined into one Dataset
n
Platform
Tissue
Reference
GSE16879
24
GPL570
colon biopsy
[23]
GSE12251
23
GPL570
colon biopsy
[24]
GSE23597
32
GPL570
colon biopsy
[25]
GSE42296
20
GPL6244
PBMC
[26] Volume 7(9) 272-277 (2014) - 274 Citation: Győrffy A, Kormos M, Bartha L, Szabó A, Győrffy B, et al. (2014) Validation of Biomarkers in Gene Expression Datasets of Inflammatory
Bowel Disease: IL13RA2, PTGS2 and WNT5A as Predictors of Responsiveness to Infliximab Therapy. J Proteomics Bioinform 7: 272-277. doi:10.4172/jpb.1000329 Citation: Győrffy A, Kormos M, Bartha L, Szabó A, Győrffy B, et al. (2014) Validation of Biomarkers in Gene Expression Datasets of Inflammatory
Bowel Disease: IL13RA2, PTGS2 and WNT5A as Predictors of Responsiveness to Infliximab Therapy. J Proteomics Bioinform 7: 272-277. Published biomarkers of response to infliximab therapy doi:10.4172/jpb.1000329 Original study
GSE12251
COLON BIOPSY
GSE16879
COLON BIOPSY
GSE23597
COLON BIOPSY
GSE42296
PBMC
Symbol
Full name
Disease
n
Ref
AUC
p-value
AUC
p-value
AUC
p-value
AUC
p-value
WNT5A
wingless-type MMTV integration site
family, member 5A
CU
46
[24]
0.95*
1.00E-10*
0.92
4.70E-13
0.77
8.1E-03
0.59
0.17
CSF3
colony stimulating factor 3
IBD
16
[27]
0.89
8.30E-10
0.84
1.30E-05
0.81
2.77 E-03
0.58
0.19
PTGS2
prostaglandin-endoperoxide synthase
2
IBD
16
[27]
0.97
<1E-16
0.87
5.20E-07
0.83
1.11 E-04
0.61
0.12
TNF
tumor necrosis factor
IBD
16
[27]
0.78
2.70E-03
0.72
0.02
0.82
3.37 E-04
0.63
0.08
G0S2
G0/G1switch 2
CD
37
[28]
0.99
<1E-16
0.85
8.71E-05
0.80
1.97 E-04
0.59
0.17
S100A8
S100 calcium binding protein A8
CD
37
[28]
0.89
1.30E-09
0.84
7.60E-05
0.85
1.58 E-06
0.66
0.03
IL6
interleukin 6
IBD
16
[27]
0.92
1.49E-11
0.87
5.35E-07
0.81
1.62 E-04
0.57
0.22
IL8
interleukin 8
IBD
16
[27]
0.95
<1E-16
0.8
8.51E-04
0.75
4.80 E-03
0.51
0.45
CXCL6
chemokine (C-X-C motif) ligand 6
CU
46
[24]
0.73*
0.018*
0.88
4.10E-07
0.70
0.044
0.62
0.10
SELP
selectin P
IBD
16
[27]
0.9
2.70E-10
0.68
0.077
0.85
1.83 E-05
0.73
0.003
ACSL4
acyl-CoA synthetase long-chain family
member 4
CU
46
[24]
0.75*
0.041*
0.89
5.60E-09
0.84
1.30E-05
0.64
0.06
CD80
CD80 molecule
IBD
16
[27]
0.68
0.06
0.78
0.0020
0.69
0.07
0.61
0.13
CD86
CD86 molecule
IBD
16
[27]
0.76
0.024
0.75
0.012
0.87
7.80E-07
0.78
2.40E-04
CXCL10
chemokine (C-X-C motif) ligand 10
IBD
16
[27]
0.68
0.08
0.71
0.03
0.8
0.0004
0.58
0.21
CSF2
colony stimulating factor 2
(granulocyte-macrophage)
IBD
16
[27]
0.69
0.1
0.8
0.001
0.79
0.0007
0.54
0.32
IGFBP5
insulin-like growth factor binding
protein 5
CU
46
[24]
0.72*
0.07*
0.77
0.005
0.91
2.91E-13
0.57
0.22
IL10
interleukin 10
CU
37
[28]
0.72
0.038
0.74
0.011
0.87
4.20E-06
0.61
0.12
IL13RA2
interleukin 13 receptor, alpha 2
CU
46
[24]
0.78*
0.043*
0.9
1.30E-09
0.95
<1E-16
0.64
0.058
IL1A
interleukin 1, alpha
IBD
16
[27]
0.73
0.07
0.94
<1E-16
0.77
0.003869
0.62
0.09
IL1B
interleukin 1, beta
IBD
16
[27]
0.72
0.15
0.97
<1E-16
0.81
0.0003
0.5
0.5
LPHN2
latrophilin 2
CU
46
[24]
0.72*
0.06*
0.78
0.006
0.88
1.17E-08
0.58
0.20
PTPRC
protein tyrosine phosphatase, receptor
type, C
IBD
16
[27]
0.69
0.047
0.7
0.039
0.78
2.00E-03
0.63
0.07
RGS2
regulator of G-protein signaling 2,
24kDa
CU
46
[24]
0.71*
0.05*
0.9
2.79E-10
0.69
0.04
0.65
0.05
S100A9
S100 calcium binding protein A9
CD
37
[28]
0.77
0.042
0.95
<1E-16
0.88
4.40E-05
0.62
0.09
SELE
selectin E
IBD
16
[27]
0.73
0.09
0.91
4.74E-10
0.84
0.0003
0.56
0.28
Gene
Original study
GSE42296in PBMC
Sample biopsy datasets (n=3)
Symbol
Full name
Disease
Type of sample
n
Ref
AUC
p-value
Average AUC Average p value
C1R
complement component 1, r
subcomponent
IBD
serum
18
[29]
0.75
1.50E-03
0.72
0.06
MAP1LC3B
microtubule-associated protein 1 light
chain 3 beta
CD
peripheral
20
[26]
0.74
2.12E-04
0.69
0.02
WARS
tryptophanyl-tRNA synthetase
CD
peripheral
20
[26]
0.71
7.10E-03
0.78
0.01
APOA4
Apolipoprotein A-IV
IBD
serum
18
[29]
0.7
1.24E-03
0.58
0.26
CA2
carbonic anhydrase II
CD
peripheral
20
[26]
0.69
1.40E-03
0.65
0.11
ODC1
ornithine decarboxylase 1
CD
peripheral
20
[26]
0.69
1.76E-03
0.76
0.006
BMP6
bone morphogenetic protein 6
CD
peripheral
20
[26]
0.69
1.82E-03
0.53
0.41
PBX1
pre-B-cell leukemia homeobox 1
CD
peripheral
20
[26]
0.68
1.98E-03
0.64
0.12
CYP1B1
cytochrome P450, family 1, subfamily
B, polypeptide 1
CD
peripheral
20
[26]
0.65
4.64E-03
0.56
0.32
A
B
C
Source: biopsy
Source: blood
All published markers
65
39
Of these: validation in same tissue is OK
25
9
Of these: validation in different tissue is OK
3
3
Table 2: Detailed results for previously published biomarker candidates including characteristics of the original studies describing the marker as well as performance of the
gene to discriminate responder and non-responder patients after infliximab therapy in each available dataset. J Proteomics Bioinform
ISSN: 0974-276X JPB, an open access journal Table 2: Detailed results for previously published biomarker candidates including characteristics of the original studies describing the marker as well as performance of the
gene to discriminate responder and non-responder patients after infliximab therapy in each available dataset. Biomarkers identified in colon biopsy specimens (A) include
genes with a p value <0.05 as the average of the three biopsy-based dataset is listed. Biomarkers identified in blood samples (B) and number of genes with a consistent
discriminator performance in each dataset of the same type of sample (C). Ref #17 has more patients as in Table 1 because in the original study additional samples were
included in the discovery set. *Genes identified in GSE12251 were also described in the paper where GSE12251 was published–these are included because their average
p-value is significant in the two other datasets. Citation: Győrffy A, Kormos M, Bartha L, Szabó A, Győrffy B, et al. (2014) Validation of Biomarkers in Gene Expression Datasets of Inflammatory
Bowel Disease: IL13RA2, PTGS2 and WNT5A as Predictors of Responsiveness to Infliximab Therapy. J Proteomics Bioinform 7: 272-277.
doi:10.4172/jpb.1000329 Published biomarkers of response to infliximab therapy doi:10.4172/jpb.1000329 Citation: Győrffy A, Kormos M, Bartha L, Szabó A, Győrffy B, et al. (2014) Validation of Biomarkers in Gene Expression Datasets of Inflammatory
Bowel Disease: IL13RA2, PTGS2 and WNT5A as Predictors of Responsiveness to Infliximab Therapy. J Proteomics Bioinform 7: 272-277. doi:10.4172/jpb.1000329 Gene
GSE12251
GSE16879
GSE23597
Symbol
Full name
Average AUC
AUC
P-Value
AUC
P-Value
AUC
P-Value
IL13RA2
interleukin 13 receptor, alpha 2
0.91
0.90
1.31E-09
0.94
<1E-16
0.87
1.14E-07
PTGS2
prostaglandin-endoperoxide synthase 2
0.89
0.97
<1E-16
0.87
5.24E-07
0.83
0.0001
TFPI2
tissue factor pathway inhibitor 2
0.89
0.92
8.93E-14
0.91
9.83E-14
0.83
8.17E-05
STC1
stanniocalcin 1
0.89
0.95
<1E-16
0.88
8.49E-08
0.83
1.23E-05
WNT5A
wingless-type MMTV integration site family,
member 5A
0.88
0.95
<1E-16
0.92
4.71E-13
0.78
0.001
Table 3: Top five genes ranked by AUC with consistently significant predictive values in all the 3 GEO datasets of colon biopsy samples. Bold: genes overlapping
to Table 2. member 5A
Table 3: Top five genes ranked by AUC with consistently significant predictive values in all the 3 GEO datasets of colon biopsy samples. Bold: genes overlapping
to Table 2. used; however, the relatively small sample size would not allow to make
established conclusions.) studies, Wnt5a (a noncanonical Wnt ligand) potentiated colonic
crypt regeneration after tissue injury [17]. On the other hand, high
Wnt5a levels were associated with poor prognosis in colorectal cancer
patients and promoted human colon cancer cell migration in animals
[18]. The contribution of Wnt5a to polyp formation in colon was also
suggested [19]. Wnt5a is also involved in the induction of epithelial-
to-mesenchymal transition [20], a common phenomenon in chronic
inflammatory conditions. In addition to use as biomarkers to discriminate between IBD
patients responding and non-responding to IFX treatment these genes
may provide novel perspectives in IBD pharmacotherapy. While the
major limitation of our in silico study is the absence of protein levels,
one may assume that transcriptome is representative for the proteome. Therefore, one may speculate that proteins encoded by these genes as
PTGS2, IL13RA and Wnt5a may predict and, theoretically, contribute
to the responsiveness to IFX in IBD. Data suggest that the relative absence of an endogenous Wnt-
inhibitor in colonic myofibroblasts (and, consequently, the increase
of Wnt levels in crypts) is associated with an increased risk of cancer
in ulcerative colitis [21]. Published biomarkers of response to infliximab therapy Although there are no data available whether
IBD patients non-responding to IFX therapy are at an increased risk
for colon cancer, an animal study indicated that IBD animals treated
with IFX are protected from colon cancer [22-29]. Therefore, one may
also speculate that patients having high Wnt5a gene expression may
be subjected to IFX non-responsiveness and, also, are at a higher risk
of cancer. These data together are suggestive that the causative role of
Wnt5a in resistance to IFX therapy may be worth to be investigated
further. PTGS2 is the gene that encodes the enzyme prostaglandin-
endoperoxide synthase also called as cyclooxygenase-2 (COX-2) the
enzyme is responsible for the production of prostaglandin H2, an
inflammatory mediator from arachinodic acid. Previous observations
indicate higher than normal PTGS2 expression in colonic epithelial
cells in IBS [6] and that PTGS2 expression is associated with disease
activity [7]. In addition to these data our analysis indicates that patients
do not responding to IFX therapy may exhibit even higher PTGS
expression than those with appropriate response. This finding is in line
with the demonstrated contribution of inflammatory prostaglandins to
the pathogenesis of IBD. The major limitation of our in silico analysis is the verification of
our results in independent datasets. However, the lack of an available
biobank with IFX responding and non-responding patients’ biopsy
specimens prevented the prompt investigation of this issue; several
years are required to complete this task. Therefore, this analysis should
be regarded rather as a hypothesis-generating one. On the other hand, COX-2 derived prostaglandins also have a role in
mucosal defense in the small intestine and colon [8]. Hence, inhibition
of COX-2 is a double-edged sword as it is reflected by controversial
results of controlled trials and experimental colitis models with COX-2
inhibitors [9]. Administration of COX-2 inhibitors was associated with
the cited as a possible contributor to the integrity of intestinal mucosa,
in the healing of gastrointestinal ulcers and in the modulation of IBD
[10], but exacerbation of IBD was also reported in several studies with
COX-2 inhibition [11]. In conclusion, our analysis revealed that IL13RA2, PTGS2 and
WNT5A, genes expressed in colonic tissues of IBD patients are
suitable to discriminate patients responding and non-responding to
IFX therapy. As all three genes encode proteins that are implicated in
intestinal homeostasis and pathology; the difference in their expression
in responding and non-responding patients may indicate important
diagnostic targets in IBD therapy. J Proteomics Bioinform
ISSN: 0974-276X JPB, an open access journal Published biomarkers of response to infliximab therapy Interleukin (IL)-13 is an important regulator of epithelial apoptosis
in immune mediated disorders [12]. It plays a role in inflammation,
mucus production, tissue remodeling, and fibrosis [13]. Disturbed
IL-13-expression may play a role in immune dysregulation [14]. IL-13 signaling is mediated by the type-2 IL-4 receptor. The IL-13
receptor alpha 2 (IL13RA2) inhibits the activity of IL-13; IL-13Ralpha2
contributes to the down-regulation of a chronic and pathogenic Th2-
mediated immune response [15]. These literary data support the results
of our analysis indicating the association of an abnormal IL13RA2
expression with infliximab unresponsiveness. Acknowledgement Sources of support: OTKA grants K 108655 and 101661. 4. Zampeli E, Gizis M, Siakavellas SI, Bamias G (2014) Predictors of response
to anti-tumor necrosis factor therapy in ulcerative colitis. World J Gastrointest
Pathophysiol 5: 293-303. Published biomarkers of response to infliximab therapy Biomarkers identified in colon biopsy specimens (A) include
genes with a p value <0.05 as the average of the three biopsy-based dataset is listed. Biomarkers identified in blood samples (B) and number of genes with a consistent
discriminator performance in each dataset of the same type of sample (C). Ref #17 has more patients as in Table 1 because in the original study additional samples were Table 2: Detailed results for previously published biomarker candidates including characteristics of the original studies describing the marker as well as performance of the
gene to discriminate responder and non-responder patients after infliximab therapy in each available dataset. Biomarkers identified in colon biopsy specimens (A) include
genes with a p value <0.05 as the average of the three biopsy-based dataset is listed. Biomarkers identified in blood samples (B) and number of genes with a consistent
discriminator performance in each dataset of the same type of sample (C). Ref #17 has more patients as in Table 1 because in the original study additional samples were
included in the discovery set. *Genes identified in GSE12251 were also described in the paper where GSE12251 was published–these are included because their average
p-value is significant in the two other datasets. Table 2: Detailed results for previously published biomarker candidates including characteristics of the original studies describing the marker as well as performance of the
gene to discriminate responder and non-responder patients after infliximab therapy in each available dataset. Biomarkers identified in colon biopsy specimens (A) include
genes with a p value <0.05 as the average of the three biopsy-based dataset is listed. Biomarkers identified in blood samples (B) and number of genes with a consistent
discriminator performance in each dataset of the same type of sample (C). Ref #17 has more patients as in Table 1 because in the original study additional samples were
included in the discovery set. *Genes identified in GSE12251 were also described in the paper where GSE12251 was published–these are included because their average
p-value is significant in the two other datasets. J Proteomics Bioinform
ISSN: 0974-276X JPB, an open access journal Volume 7(9) 272-277 (2014) - 275 Citation: Győrffy A, Kormos M, Bartha L, Szabó A, Győrffy B, et al. (2014) Validation of Biomarkers in Gene Expression Datasets of Inflammatory
Bowel Disease: IL13RA2, PTGS2 and WNT5A as Predictors of Responsiveness to Infliximab Therapy. J Proteomics Bioinform 7: 272-277. References Expression profiling of Wnt family of
genes in normal and inflammatory bowel disease primary human intestinal
myofibroblasts and normal human colonic crypt epithelial cells. Inflamm Bowel
Dis. 2011;17:213-20. 8. Wallace JL (2001) Prostaglandin biology in inflammatory bowel disease. Gastroenterol Clin North Am 30: 971-980. 9. Paiotti AP, Marchi P, Miszputen SJ, Oshima CT, Franco M, et al. (2012) The
role of nonsteroidal antiinflammatory drugs and cyclooxygenase-2 inhibitors on
experimental colitis. In Vivo 26: 381-393. 22. Kim YJ, Hong KS, Chung JW, Kim JH, Hahm KB (2010) Prevention of colitis-
associated carcinogenesis with infliximab. Cancer Prev Res (Phila) 3: 1314-
1333. 10. Cipolla G, Crema F, Sacco S, Moro E, de Ponti F, et al. (2002) Nonsteroidal
anti-inflammatory drugs and inflammatory bowel disease: current perspectives. Pharmacol Res 46: 1-6. 23. Arijs I, De Hertogh G, Lemaire K, Quintens R, Van Lommel L, et al. (2009)
Mucosal gene expression of antimicrobial peptides in inflammatory bowel
disease before and after first infliximab treatment. PLoS One 4: e7984. 11. Kefalakes H, Stylianides TJ, Amanakis G, Kolios G (2009) Exacerbation of
inflammatory bowel diseases associated with the use of nonsteroidal anti-
inflammatory drugs: myth or reality? Eur J Clin Pharmacol 65: 963-970. 24. Arijs I, Li K, Toedter G, Quintens R, Van Lommel L, et al. (2009) Mucosal gene
signatures to predict response to infliximab in patients with ulcerative colitis. Gut 58: 1612-1619. 12. Schulzke JD, Bojarski C, Zeissig S, Heller F, Gitter AH, et al. (2006) Disrupted
barrier function through epithelial cell apoptosis. Ann N Y Acad Sci 1072: 288-
299. 25. Toedter G, Li K, Marano C, Ma K, Sague S, et al. (2011) Gene expression
profiling and response signatures associated with differential responses to
infliximab treatment in ulcerative colitis. Am J Gastroenterol 106: 1272-1280. 13. Mentink-Kane MM, Wynn TA (2004) Opposing roles for IL-13 and IL-13
receptor alpha 2 in health and disease. Immunol Rev 202: 191-202. 26. Mesko B, Poliska S, Váncsa A, Szekanecz Z, Palatka K, et al. (2013) Peripheral
blood derived gene panels predict response to infliximab in rheumatoid arthritis
and Crohn's disease. Genome Med 5: 59. 14. Mannon P, Reinisch W (2012) Interleukin 13 and its role in gut defence and
inflammation. Gut 61: 1765-1773. 27. Caprioli F, Bose F, Rossi RL, Petti L, Vigano C, et al. References 1. Ben-Horin S, Kopylov U, Chowers Y (2014) Optimizing anti-TNF treatments in
inflammatory bowel disease. Autoimmun Rev 13: 24-30. 1. Ben-Horin S, Kopylov U, Chowers Y (2014) Optimizing anti-TNF treatments in
inflammatory bowel disease. Autoimmun Rev 13: 24-30. 2. Ordás I, Mould DR, Feagan BG, Sandborn WJ (2012) Anti-TNF monoclonal
antibodies in inflammatory bowel disease: pharmacokinetics-based dosing
paradigms. Clin Pharmacol Ther 91: 635-646. 2. Ordás I, Mould DR, Feagan BG, Sandborn WJ (2012) Anti-TNF monoclonal
antibodies in inflammatory bowel disease: pharmacokinetics-based dosing
paradigms. Clin Pharmacol Ther 91: 635-646. Literary data on Wnt5a gene product are also promising. Members
of Wnt family are glycoproteins actively secreted by cells and along with
other pathways (e.g. FGF, Notch, BMP, and Hedgehog signalization)
they contribute to intestinal stem cell biology, the maintenance of
homeostasis, and, in case of malfunctioning, to pathological conditions
including inflammation and cancer [16]. Regarding Wnt5a, several
possible functions in gastrointestinal tract were reported. In animal 3. Nanda KS, Cheifetz AS, Moss AC (2013) Impact of antibodies to infliximab
on clinical outcomes and serum infliximab levels in patients with inflammatory
bowel disease (IBD): a meta-analysis. Am J Gastroenterol 108: 40-47. 3. Nanda KS, Cheifetz AS, Moss AC (2013) Impact of antibodies to infliximab
on clinical outcomes and serum infliximab levels in patients with inflammatory
bowel disease (IBD): a meta-analysis. Am J Gastroenterol 108: 40-47. 4. Zampeli E, Gizis M, Siakavellas SI, Bamias G (2014) Predictors of response
to anti-tumor necrosis factor therapy in ulcerative colitis. World J Gastrointest
Pathophysiol 5: 293-303. Volume 7(9) 272-277 (2014) - 276 5. Moher D, Liberati A, Tetzlaff J, Altman DG (2009) Preferred reporting items for
systematic reviews and meta-analyses: the PRISMA Statement. Open Med 3:
e123-e130. does not augment intestinal tumorigenesis in Apc1638N mice. Carcinogenesis
34: 2629-2638. 19. Lai C, Robinson J, Clark S, Stamp G, Poulsom R, et al. (2011) Elevation of
WNT5A expression in polyp formation in Lkb1+/- mice and Peutz-Jeghers
syndrome. J Pathol 223: 584-592. 6. Singer II, Kawka DW, Schloemann S, Tessner T, Riehl T, et al. (1998)
Cyclooxygenase 2 is induced in colonic epithelial cells in inflammatory bowel
disease. Gastroenterology 115: 297-306. 20. Sipos F, Galamb O (2012) Epithelial-to-mesenchymal and mesenchymal-to-
epithelial transitions in the colon. World J Gastroenterol 18: 601-608. 7. Hendel J, Nielsen OH (1997) Expression of cyclooxygenase-2 mRNA in active
inflammatory bowel disease. Am J Gastroenterol 92: 1170-1173. 21. Hughes KR, Sablitzky F, Mahida YR. Citation: Győrffy A, Kormos M, Bartha L, Szabó A, Győrffy B, et al. (2014) Validation of Biomarkers in Gene Expression Datasets of Inflammatory
Bowel Disease: IL13RA2, PTGS2 and WNT5A as Predictors of Responsiveness to Infliximab Therapy. J Proteomics Bioinform 7: 272-277.
doi:10.4172/jpb.1000329 J Proteomics Bioinform
ISSN: 0974-276X JPB, an open access journal References (2013) Reduction of
CD68+ macrophages and decreased IL-17 expression in intestinal mucosa
of patients with inflammatory bowel disease strongly correlate with endoscopic
response and mucosal healing following infliximab therapy. Inflamm Bowel Dis
19: 729-739. 15. Chiaramonte MG, Mentink-Kane M, Jacobson BA, Cheever AW, Whitters MJ, et
al. (2003) Regulation and function of the interleukin 13 receptor alpha 2 during
a T helper cell type 2-dominant immune response. J Exp Med 197: 687-701. 16. Katoh M (2007) Networking of WNT, FGF, Notch, BMP, and Hedgehog
signaling pathways during carcinogenesis. Stem Cell Rev 3: 30-38. 28. Arijs I, Quintens R, Van Lommel L, Van Steen K, De Hertogh G, et al. (2010)
Predictive value of epithelial gene expression profiles for response to infliximab
in Crohn's disease. Inflamm Bowel Dis 16: 2090-2098. 17. Miyoshi H, Ajima R, Luo CT, Yamaguchi TP, Stappenbeck TS (2012) Wnt5a
potentiates TGF-β signaling to promote colonic crypt regeneration after tissue
injury. Science 338: 108-113. 29. Gazouli M, Anagnostopoulos AK, Papadopoulou A, Vaiopoulou A, Papamichael
K, et al. (2013) Serum protein profile of Crohn's disease treated with infliximab. J Crohns Colitis 7: e461-e470. 18. Bakker ER, Das AM, Helvensteijn W, Franken PF, Swagemakers S, et al. References (2013) Wnt5a promotes human colon cancer cell migration and invasion but Submit your next manuscript and get advantages of OMICS
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The final countdown: Presence of an invasive mosquito extends time to predation for a native mosquito
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The final countdown: Presence of an invasive
mosquito extends time to predation for a native
mosquito Alexis J Beckermann
Washington University in St Louis
Kim A Medley
Washington University in St Louis
Solny A Adalsteinsson
Washington University in St Louis
Katie Westby
(
kwestby@wustl.edu
)
Washington University In St Louis: Washington
1403-5362 The final countdown: Presence of an invasive
mosquito extends time to predation for a native
mosquito
Alexis J Beckermann
Washington University in St Louis
Kim A Medley
Washington University in St Louis
Solny A Adalsteinsson
Washington University in St Louis
Katie Westby
(
kwestby@wustl.edu
)
Washington University In St Louis: Washington University in St Louis
https://orcid.org/0000-0003-
1403-5362
Research Article
Keywords: predation cues, apparent mutualism, habitat structure, mosquito, Aedes, Toxorhynchites
Posted Date: October 28th, 2022
DOI: https://doi.org/10.21203/rs.3.rs-2139483/v1
License:
This work is licensed under a Creative Commons Attribution 4.0 International
License.
Read Full License
Version of Record: A version of this preprint was published at Biological Invasions on March 30th, 2023
See the published version at https://doi.org/10.1007/s10530-023-03051-1. The final countdown: Presence of an invasive
mosquito extends time to predation for a native
mosquito
Alexis J Beckermann
Washington University in St Louis
Kim A Medley
Washington University in St Louis
Solny A Adalsteinsson
Washington University in St Louis
Katie Westby
(
kwestby@wustl.edu
)
Washington University In St Louis: Washington University in St Louis
https://orcid.org/0000-0003-
1403-5362
Research Article
Keywords: predation cues, apparent mutualism, habitat structure, mosquito, Aedes, Toxorhynchites
Posted Date: October 28th, 2022
DOI: https://doi.org/10.21203/rs.3.rs-2139483/v1
License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License
Version of Record: A version of this preprint was published at Biological Invasions on March 30th, 2023
See the published version at https://doi.org/10.1007/s10530-023-03051-1. Research Article License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Version of Record: A version of this preprint was published at Biological Invasions on March 30th, 2023. See the published version at https://doi.org/10.1007/s10530-023-03051-1. Page 1/16 Abstract We added one Tx. rutilus to feed in each microcosm for 24h (experiment 1 and 2) and until all prey were consumed
(experiment 2 only). When reared alone, Ae. triseriatus had higher survival compared to Ae. japonicus in
experiment 1 (71% vs. 52%) but there were no significant differences at 24 hours in experiment 2. When
we followed the cohort to total predation, Ae. triseriatus had a lower daily survival rate compared to Ae. japonicus (hazard ratio 1.165) when the species were kept separately. When the species were mixed,
however, Ae. japonicus was more vulnerable than Ae. triseriatus (hazard ratio 1.763), prolonging Ae. triseriatus time to total cohort predation. Both species were less likely to be consumed in the presence of
predation cues. We detected no effect of habitat structure. These results demonstrate vulnerability is
context dependent and the presence of an invasive congener can relax predation pressure on a native
prey species when they co-occur in the same habitat. Abstract Larvae of the predatory mosquito Toxorhynchites rutilus consume arthropods within container habitats,
including native Aedes triseriatus and invasive Aedes japonicus mosquitoes. Previous studies, which did
not account for common habitat attributes such as habitat structure and predation cues, conflict on
whether Ae. triseriatus and Ae. japonicus differ in their vulnerability to predation. We conducted two
laboratory experiments to assess how habitat attributes modulate Tx. rutilus predation on Ae. triseriatus
and Ae. japonicus. In experiment 1, we added fine particulate organic matter (FPOM) and assessed
vulnerability for each species separately. Experiment 2 contained the following treatments: Larvae of the predatory mosquito Toxorhynchites rutilus consume arthropods within container habitats,
including native Aedes triseriatus and invasive Aedes japonicus mosquitoes. Previous studies, which did
not account for common habitat attributes such as habitat structure and predation cues, conflict on
whether Ae. triseriatus and Ae. japonicus differ in their vulnerability to predation. We conducted two
laboratory experiments to assess how habitat attributes modulate Tx. rutilus predation on Ae. triseriatus
and Ae. japonicus. In experiment 1, we added fine particulate organic matter (FPOM) and assessed
vulnerability for each species separately. Experiment 2 contained the following treatments:
presence/absence of predation cues, presence/absence of habitat structure (FPOM and leaves) and three
species combinations: Ae. triseriatus or Ae. japonicus alone, and both species together. We added one Tx. rutilus to feed in each microcosm for 24h (experiment 1 and 2) and until all prey were consumed
(experiment 2 only). When reared alone, Ae. triseriatus had higher survival compared to Ae. japonicus in
experiment 1 (71% vs. 52%) but there were no significant differences at 24 hours in experiment 2. When
we followed the cohort to total predation, Ae. triseriatus had a lower daily survival rate compared to Ae. japonicus (hazard ratio 1.165) when the species were kept separately. When the species were mixed,
however, Ae. japonicus was more vulnerable than Ae. triseriatus (hazard ratio 1.763), prolonging Ae. triseriatus time to total cohort predation. Both species were less likely to be consumed in the presence of
predation cues. We detected no effect of habitat structure. These results demonstrate vulnerability is
context dependent and the presence of an invasive congener can relax predation pressure on a native
prey species when they co-occur in the same habitat. presence/absence of predation cues, presence/absence of habitat structure (FPOM and leaves) and three
species combinations: Ae. triseriatus or Ae. japonicus alone, and both species together. Introduction Non-native species may become invasive if they undergo rapid population growth in the absence of their
co-evolved predators and parasites (Colautti et al. 2004; Heger and Jeschke 2014). However, non-native
species often encounter native predators in their new range (Pintor and Beyers 2015), and outcomes
resulting from invasive prey-native predator encounters partially depend on how well the two are able to
detect or avoid each other (Mennen and Laskowski 2018; Narimanov et al. 2021). Many prey species that
have evolved with predators, whether in their native or introduced habitat, have developed strategies to
detect them, or when predation has occurred, and modify their behavior to minimize their risk. The act
and byproducts of predation produce chemical cues that prey respond to (Sih 1986; Lima & Dill 1990;
Chivers et al. 2001; Juliano & Gravel 2002). However, the strength of the behavioral response to predation
cues seems to be a product of an evolutionary relationship between native predators and native prey. As
a result, non-native species may be more vulnerable to predation by novel (native) predators (Grill &
Juliano 1996; Juliano & Gravel 2002; Costanzo et al. 201; Twining et al. 2020) and may indirectly benefit
native prey by relaxing predation pressure (Rodriguez 2006; Skein et al. 2018) or competition (Wagner et
al. 2010). Small, discrete, aquatic container ecosystems; either natural (bromeliads, pitcher plants, treeholes) or
associated with humans (tires, buckets, trashcans), are dominated by mosquito larvae. Within these Page 2/16 Page 2/16 Page 2/16 habitats, predation-risk cues are primarily solid wastes and partially eaten conspecifics (Kesavaraju and
Juliano 2010). In response to detection of predation-risk cues, larval mosquitos exhibit anti-predatory
behaviors such as reduced foraging and increased resting near the water surface (Juliano and Gravel
2002). These behaviors reduce predation risk, but not without fitness trade-offs such as smaller body-
size, reduced fecundity, and increased development time (Juliano and Reminger 1992; Chandrasegaran
and Juliano 2019; Costanzo et al. 2011). Rapid behavioral adaptation to changes in predation pressure
have been observed in the Eastern treehole mosquito Aedes triseriatus in just two generations of selection
under laboratory conditions (Juliano and Gravel 2002). Additionally, interpopulation differences in
response to predation risk by the predator Toxorhychites rutilus between two geographically isolated
populations of Ae. triseriatus with low and high levels of cohabitation with Tx. rutilus, have been
observed, highlighting the importance of recent evolutionary history (Juliano and Gravel 2002). Introduction Within natural container habitats (treeholes) across the Eastern United States, the native Elephant
Mosquito, Tx. rutilus is a common and dominant predator that can exert strong pressure on prey
communities (Juliano et al. 2019; Griswold and Lounibos 2006). Wild larval Tx. rutilus are generalist
predators, voraciously feeding on smaller mosquito larvae, fallen terrestrial arthropods, other dipterans
(Psychodidae & Chironomidae), and microcrustaceans (copepods & ostracods) (Campos and Lounibos
2000). Tx. rutilus is an ambush predator that relies on mechanoreceptors (setae) all over its body to
detect prey movement while hunting (Steffan and Evenhuis 1981). There are some cases, however, where
Tx. rutilus and related species will exhibit searching behavior (Linley and Darling 1993; Linley 1995;
Zuharah et al. 2015). Typically, established container habitats such as treeholes and buckets hold large
amounts of benthic sediment (fine particulate organic matter, FPOM) and plant/insect detritus (Macia
and Bradshaw 2000, Yee et al. 2007, Beckermann and Westby, personal observation). Resuspension of
the sediment by prey activity can disclose prey location and reduce visibility, making predators harder to
detect. Alternatively, sediment/detritus adds complexity to the larval habitat, potentially giving prey
increased refuge from predation. Previous studies evaluating the effects of habitat complexity on
vulnerability of native Ae. triseriatus and invasive Aedes albopictus to Tx. rutilus found the addition of
artificial leaves as habitat structure did not alter predation on either species (Alto et al. 2005). However,
natural leaf substrate can alter the feeding behavior of another non-native invasive mosquito, Aedes
japonicus. Increased browsing activity has been observed for Ae. japonicus in the presence of senescent
white oak leaves compared to a plastic strip in the larval environment (O’Donnell and Armbruster 2007). The increase in browsing activity due to the presence of phagostimulants from natural leaf substrate (vs. artificial substrates) could potentially leave prey larvae more vulnerable to predation by Tx. rutilus. Browsing activity is considered one of the more risky behaviors (Juliano and Gravel 2002; Kesavaraju and
Juliano 2004). To our knowledge, the effects of FPOM on predation risk for mosquitoes has not been
tested and the role of natural substrates, such as leaves, is under-explored. Tx. rutilus commonly cohabitates and preys upon Ae. triseriatus within their respective native range in the
United States (Bradshaw and Holzapfel 1983). The non-native, invasive mosquito Ae. japonicus, which
established and began expanding its US range in the late 1990’s, has substantial niche overlap with Ae. Prey larvae rearing Ae. triseriatus and Ae. japonicus eggs were collected from Tyson Research Center (TRC) in Eureka, MO in
June of 2016 and stimulated to hatch by being submerged in a .35 g/L nutrient broth (Difco Laboratories,
Detroit, MI) solution for 24 h. Once hatched, larvae were reared on .001 g of 1:1 lactalbumin and liver
powder and 6 mL of deionized water per larva. Once larvae reached 2nd instar, they were identified and
separated by species and larvae were maintained in an environmental chamber held at 25℃ and a 16:8 h
(L:D) photophase. Introduction rutilus for the two
species to be insignificant (Murrell & Juliano 2013), others suggest Ae. japonicus may be more vulnerable
(Freed et al. 2014, Juliano et al. 2019) with larger population declines for Ae. japonicus compared to Ae. triseriatus in a manipulative field experiment (Juliano et al. 2019). How predation cues and habitat
structure change the relative vulnerability for the two species remains to be tested. Our goals were to identify differences in prey vulnerability to Tx. rutilus between native Ae. triseriatus and
invasive Ae. japonicus, when alone or together, in the presence or absence of predation risk cues and
realistic habitat complexity. No previous prey vulnerability studies between these two species, to our
knowledge, have included multiple levels of natural habitat structure and predation risk cues that mimic
established container habitats in the wild. Specifically, we predicted that 1) predation cues and habitat
structure alter predation rates for both species; 2) the two species will differ in their vulnerability to
predation; and 3) that vulnerability may change when the species are exposed to predation singly or
together. Introduction triseriatus, becoming a potential competitor and a new potential prey source for Tx. rutilus (Kaufman and Page 3/16 Page 3/16 Fonseca 2014). Aedes triseriatus and Ae. japonicus display differences in foraging behavior which may
make Ae. japonicus more vulnerable to predation by Tx. rutilus, and Ae. japonicus has been shown to
actively forage more often than Ae. albopictus,. Aedes triseriatus’ larval feeding behavior does not differ
substantially from Ae. albopictus (O’Donnell and Armbruster 2007; Skiff and Yee 2014), thus we expect
Ae. japonicus to spend more time foraging than Ae. triseriatus. Although it has not been quantified to our
knowledge, our personal observations of these two species in detritus rich larval habitats indicate that Ae. japonicus spends more time foraging near the bottom of their container in FPOM and leaf litter compared
to Ae. triseriatus. Additionally, both species exhibit increased resting behavior when exposed to predation
risk cues produced by Tx. rutilus (Kesavaraju et al. 2011; Kesavaraju and Juliano 2004; Kesavaraju et al. 2007). While one laboratory study found the differences in prey vulnerability to Tx. rutilus for the two
species to be insignificant (Murrell & Juliano 2013), others suggest Ae. japonicus may be more vulnerable
(Freed et al. 2014, Juliano et al. 2019) with larger population declines for Ae. japonicus compared to Ae. triseriatus in a manipulative field experiment (Juliano et al. 2019). How predation cues and habitat
structure change the relative vulnerability for the two species remains to be tested. Fonseca 2014). Aedes triseriatus and Ae. japonicus display differences in foraging behavior which may
make Ae. japonicus more vulnerable to predation by Tx. rutilus, and Ae. japonicus has been shown to
actively forage more often than Ae. albopictus,. Aedes triseriatus’ larval feeding behavior does not differ
substantially from Ae. albopictus (O’Donnell and Armbruster 2007; Skiff and Yee 2014), thus we expect
Ae. japonicus to spend more time foraging than Ae. triseriatus. Although it has not been quantified to our
knowledge, our personal observations of these two species in detritus rich larval habitats indicate that Ae. japonicus spends more time foraging near the bottom of their container in FPOM and leaf litter compared
to Ae. triseriatus. Additionally, both species exhibit increased resting behavior when exposed to predation
risk cues produced by Tx. rutilus (Kesavaraju et al. 2011; Kesavaraju and Juliano 2004; Kesavaraju et al. 2007). While one laboratory study found the differences in prey vulnerability to Tx. Prey larvae rearing Ae. triseriatus and Ae. japonicus were hatched from field collected eggs obtained in June of 2017. The
larvae were then reared via the same methods used in our 2016 study. Predatory larvae rearing Page 4/16
Wild Tx. rutilus larvae were collected in July of 2016 from 140 L barrels established in the woods at TRC
in 2013 (Westby and Juliano 2017). Individuals were separated into 20 mL vials with 15 mL of deionized
water and fed a diet of larval Aedes spp. (Ae. triseriatus and Ae. japonicus) with some Culex restuans, Page 4/16 Culex territans, Anopheles barberi, and Orthopodomyia signifera collected from barrels at TRC. Each larva
received 15 prey daily until they reached 4th instar, wherein we reduced the prey to 10 larvae per day. Predatory larvae rearing Tx. rutilus larvae were field-collected or hatched from field-collected eggs from TRC. Tx. rutilus larvae
were reared individually in 20 mL vials with 15 mL of deionized water. Each predator larva was fed a diet
of 15 field-collected prey larvae per day with a species composition of Ae. japonicus, Ae. triseriatus, Cx. restuans, Cx. territans, An. barberi, and Or. signifera. Once larvae reached 4th instar, they were fed 10 4th
instar field-collected larva per day with the same species composition previously mentioned. Predator
larvae were maintained in an environmental chamber held at 25℃ with a photophase of 16:8 h (L:D). Experiment 2: Habitat Structure, Predation Cues, Single And Mixed
Species Experiment 2: Habitat Structure, Predation Cues, Single And Mixed
Species Experimental Design We established 40 laboratory microcosms with 350 mL of deionized water, half of which received our
treatment: 40 mL of fine particulate organic matter (FPOM) collected from 1 year old field mesocosms. The FPOM was boiled twice to remove microbes, and then homogenized before addition to the
experimental microcosms. Each microcosm received 20 prey larvae of a single species (Ae. japonicus or
Ae. triseriatus), and one 4th instar Tx. rutilus larva. Tx. rutilus individuals were starved for 24h before we
conducted the experiment to standardize their level of hunger. The experiment was run for 24 h after
which predator larvae were removed and the remaining live prey larvae were counted. We analyzed the proportion of larvae alive using a GLM (generalized linear model) with a quasi binomial
error distribution and two fixed effects: prey species (Ae. triseriatus or Ae. japonicus) and FPOM (present
or absent). The analysis was performed in SAS 9.4 using the GLIMMIX procedure. Habitat and cue water creation We held Ae. triseriatus and Ae. japonicus larvae in three different prey larval assemblages: 20 Ae. triseriatus larvae, 20 Ae. japonicus larvae, and 10:10 of both species in the presence or absence of
predation cues and habitat structure. We created 6 replicates of each combination of larval assemblages,
predation cues, and habitat structure for a total of 69 (72 − 3 that were spilled or dropped) container
microcosms (experimental units). Each microcosm received 340 mL of deionized water which was
replenished as needed; habitat structure treatments received an 1.25 mL of FPOM and 1.25 g of
senescent sycamore leaves collected from the forest floor at TRC. FPOM was collected from 3-year-old Page 5/16 Page 5/16 field mesocosms, homogenized, and boiled. To create our predation risk cues, we modified the methods
presented by Costanzo et al. (2011). Predation water treatments were prepared over the course of five
days by adding the prey larvae according to our assemblages (e.g. 20 Ae. triseriatus for the Ae. triseriatus
treatment group) with one predator larva, Tx. rutilus. Each day prey larvae were counted, identified, and
replenished to the original number. Pupated or dead Tx. rutilus were removed and replaced. Control water
treatments were also prepared over the course of five days by holding prey larvae according to our
assemblages but in the absence of predation; prey larvae were counted, identified, and replaced if
mortality occurred. This was done as a control to account for chemicals cues left by prey larvae. Prey
larvae were not fed during this time. After the five preparation days, all prey and predator larvae were
removed. All detritus, cues, and microbes produced from the predator water and control treatments were left in the
microcosm. Experimental Tx. rutilus were starved for 24 h before our trial began. Experimental cohorts of
prey larvae according to our assemblages and one 3rd or 4th instar Tx. rutilus larva were added to each
microcosm. Every 24 h, prey larvae were identified and mortality was recorded. Pupated or dead Tx. rutilus larvae were removed from the microcosm and replaced by another 3rd or 4th instar larva. The
experiment was allowed to run until all prey larvae were consumed by Tx. rutilus. Experimental
microcosms were held in an environmental chamber at 25 ℃ with a 16:8 h (L:D) photophase. Habitat and cue water creation To directly compare our results from experiment 1 to experiment 2, we analyzed the proportion of larvae
alive at the 24 hour mark using two GLMs (generalized linear model) with a quasi binomial error
distribution, one for the when the prey species were held singly, and the second for when the species were
held together. The models contained three fixed effects: prey species (Ae. triseriatus or Ae. japonicus)
predation cues (present or absent), and habitat structure (present or absent). To assess the time to total
cohort predation, we used two separate Cox Proportional Hazards models due to the different numbers of
prey larvae of each species if they were in single species (20 Ae. triseriatus or 20 Ae. japonicus) or mixed
species (10 Ae. triseriatus and 10 Ae. japonicus). Both analyses included the same three fixed effects as
the above analysis. All analyses were performed in SAS 9.4 using either the GLIMMIX or PHREG
procedures. Experiment 2: Habitat Structure, Predation Cues, Single And Mixed
Species After the first 24-hours, there was no significant effect of species (single species: F1,44 =2.88, P = 0.0966 and mixed species: F1,44 =2.91, P = 0.0951) habitat structure (single species: F1,44
=0.12, P = 0.7360 and mixed species: F1,44 =1.16, P = 0.2883), or predation cues (single species: F1,44
=2.80, P = 0.1015 and mixed species: F1,44 =0.81, P = 0.3718) whether the species were held singly or
together. When we followed each cohort until all prey larvae had been consumed, there was no significant effect of
habitat structure on prey vulnerability to Tx. rutilus for either species (single species: 𝝌2 = 0.0005, P =
0.9813, mixed: 𝝌2 = 0.6926, P = 0.4019). When prey larvae were held singly (20 Ae. triseriatus or 20 Ae. japonicus), Ae. triseriatus had a lower daily survival rate compared to Ae. japonicus (𝝌2 = 7.3793, P =
0.0066, hazard ratio 1.165, Fig. 2A) However, when held together (10:10 Ae. japonicus and Ae. triseriatus)
Ae. japonicus had a lower daily survival rate (𝝌2 = 30.4292, P = < .0001, hazard ratio 1.763) and Ae. triseriatus had an extended time to total cohort predation (Fig. 2B). The presence of predation risk cues
significantly extended time to predation for both Ae. japonicus and Ae. triseriatus compared to treatments
without cues (single species: 𝝌2 = 24.1758, P = < .0001, mixed species:𝝌2 = 8.5044, P = 0.0033) (Figs. 2A
and 2B). Experiment 1: Habitat structure, single species There was no significant effect of habitat structure on vulnerability to predation by Tx. rutilus (F1,36 =1.92,
P = 0.17). There was a significant effect of species (F1,36 =6.83, P = 0.013) with a larger proportion of Ae. triseriatus surviving compared to Ae. japonicus (mean and stder 0.71 ± 0.05 vs. 0.52 ± 0.06; Fig. 1). The
interaction between species and habitat structure was not significant (F1,36 =1.75, P = 0.1943) Page 6/16 Discussion eyesight is poorly developed in larvae, potentially owing to the dark and turbid treehole habitat where this
species evolved (Steffen and Evenhuis 1981). Thus, Toxorhynchites spp. rely less on visual cues and
more on tactile cues such as water movement (Steffen and Evenhuis 1981). Aquatic ambush predators
may be unaffected by the addition of habitat structure whereas active searchers are impeded (Saha et al. 2009; Deboom and Wahl 2013); however, whether structure increases, decreases, or has a neutral effect
on predation, as observed here, is determined by a suite of biological, behavioral, and environmental
factors (Michel and Adams 2009, Klecka and Boukal 2014). We observed significant increases in the time to total cohort predation for both Ae. japonicus and Ae. triseriatus when held in water containing predation cues. These results support the conclusions reached
in behavioral assays using video capture that invasive Ae. japonicus is sensitive to predation cues
produced by Tx. rutilus, despite its recent evolutionary history with this predator (Kesavaraju et al. 2011). Ae. japonicus encounters predators in its native range (Sota 1996; Sunahara et al. 2002) and apparently
maintained its ability to detect and respond to predation cues during its establishment and range
expansion in the US. It is also plausible that Ae. japonicus has had sufficient time to evolve an
antipredatory response to Tx. rutilus specifically since its introduction in our study area at least ten years
before we performed these experiments (Gallitano et al. 2006). Prey naivete is known to decrease since
time of introduction (Anton et al. 2020) and rapid evolution in response to predation cues has been
observed for Ae. triseriatus in the laboratory (Juliano and Gravel 2002). Whether the native or non-native invasive species is more vulnerable to predation is condition-dependent:
our experiments came to different conclusions depending on when the data was collected (24 hr vs. time
to total predation) and if the species were offered as prey separately or together. We found evidence that
Ae. triseriatus was less vulnerable after 24 hrs (experiment 1), no difference between the species at 24 hrs
when held separately (experiment 2), Ae. triseriatus was more vulnerable to total predation when reared
alone (experiment 2) and that Ae. japonicus was more vulnerable to total predation when offered with Ae. triseriatus at the same time (experiment 2), which extended the time to total predation for Ae. triseriatus. Discussion Aedes triseriatus and Ae. japonicus are mosquito species of public health importance as known or
potential vectors of viruses (Westby et al. 2015; DeCarlo et al. 2020). Both species are vulnerable to
predation by Tx. rutilus, but there has been ambiguity among prior studies about whether the native
species Ae. triseriatus, which coevolved with Tx. rutilus, is more or less vulnerable than the invasive Ae. japonicus (Murrell and Juliano 2013; Freed et al. 2014). In this paper, we further explored this question
and added the nuances of: 1) aquatic habitat structure, which has been shown to act as a refuge for prey
in other systems and is abundant in mosquito habitat, and 2) the presence of predation cues. Additionally, we explored whether relative vulnerability changed when the prey species were available
alone or together and on short and long time scales. Interestingly, we observed no significant effects of habitat structure from either of our experiments. We
predicted that the addition of structure that closely mimics conditions experienced in treeholes would
impact predation rates for one or both of the prey species tested. Specifically, we predicted FPOM would
have a relatively greater effect on Ae. japonicus as we have frequently observed this species spending
more time burying themselves in sediment compared to Ae. triseriatus (Westby and Beckermann,
personal observations). Our current study did not include an assessment of prey behavior, so we are
unable to determine if habitat structure impacted behavior, or if the two species behaved differently. It
appears from our results, however, that if our habitat structure did influence behavior it was not sufficient Page 7/16 to alter predation rates. Our results are consistent with the conclusions of Alto et al. (2005) wherein Ae. albopictus did not alter its behavior with the addition of plastic artificial leaves as habitat structure;
however, prey larvae are known to browse the bacteria on natural substrates (Kaufman et al. 2008) and to
increase browsing behavior when offered natural leaves compared to plastic artificial leaves (O'Donnell
and Armbruster 2007). Browsing has repeatedly been shown to be risky in behavioral studies of mosquito
larvae (Juliano and Reminger 1992; Grill and Juliano 1996), but results from the studies presented here
and behavioral studies are not directly comparable. The addition of FPOM and real leaves make visual
recording prohibitive. One hypothesis as to why habitat structure did not affect predation rates is that Toxorhynchites spp. Discussion Based on the conflicting results from our own studies it is not surprising that there was ambiguity among
the other three published studies (Murell et al. 2013; Freed et al. 2014; Juliano et al. 2019). We suggest
that differences in experimental design, such as the length of the trials and the number of prey offered, Page 8/16 Page 8/16 Page 8/16 can alter the outcome of these experiments and that context dependence is an important driver of the
differences in results (Juliano 2009). In our second experiment, we found that when Ae. japonicus was offered as the single prey species, it
experienced extended time to predation compared to native Ae. triseriatus. Since Ae. triseriatus exhibits
such a strong anti-predatory behavioral response and shares a longer evolutionary history with Tx. rutilus,
this result was not in line with our original predictions. Nor did this align with the results from our first
experiment where Ae. triseriatus was seemingly less vulnerable to predation than Ae. japonicus. Moreover,
Murrell and Juliano’s (2013) single species prey vulnerability assays between Ae. japonicus and Ae. triseriatus showed predation rates of the two species by Tx. rutilus over a period of 4 days were
statistically indistinguishable. However, our methods and objectives differed significantly from Murrell
and Juliano (2013) which focused on attack rates over a 24 h period and replenished prey species after
they were eaten. We did not replenish prey species, instead allowing the predator to eat until local
extinction of prey occurred. This could explain the differences in our results since Tx. rutilus is a Type II
functional predator and changes its foraging strategies based on the proportion of available prey (Russo
1983). Our results indicate it is still unclear whether Ae. triseriatus or Ae. japonicus are less vulnerable to
predation from Tx. rutilus comparatively when offered separately. In our mixed species treatments, we found Ae. triseriatus was significantly less vulnerable to predation
when held with Ae. japonicus in both cue and non-cue treatments, respectively. This shows a reversal in
the results of the single species treatments where Ae. japonicus was less vulnerable to predation, and
perhaps a more realistic picture of prey vulnerability in the wild since container habitats frequently
contain multi-species assemblages. Most interestingly, we show that the presence of an invasive
competitor may assist a native species through predator-mediated effects. Discussion When the presence of one
species reduces vulnerability to a shared predator for another prey species, it is known as apparent
mutualism (Holt and Lawton 1994). This phenomenon is important to consider when studying systems
with polyphagous predators, such as Tx. rutilus, due to the possibility of the predator’s prey preference
changing when presented with multiple species. Testing the interaction of multiple prey species and their
shared predator also gives a better picture of how predation of invasive species may occur. As we saw in
the results from our two studies, when prey were offered as single species, or at 24 hrs, there was still
ambiguity on whether Ae. japonicus was more vulnerable, but when held with Ae. triseriatus, Ae. japonicus were more vulnerable to predation. In our case, had we not offered the prey species together, or
followed them over time, our conclusions from this study would be different, highlighting the need for
more context-dependent studies of predator-prey interactions (Juliano 2009). Furthermore, the results
from the Juliano et al. (2019) experimental field predator removal study reported strong evidence that Ae. japonicus were more negatively affected by Tx. rutilus predation, whereas there were no significant
differences in pupal and larval abundances of Ae. triseriatus whether Tx. rutilus was present or removed
from their habitat. There is now both field and laboratory evidence that Tx. rutilus predation more
negatively affects Ae. japonicus than its native competitor, Ae. triseriatus when held together. Page 9/16 Our results show that Ae. japonicus’ presence in container habitats can relax predation pressure on Ae. triseriatus (Juliano et al. 2019). This, coupled with its presence reducing gregarine parasite prevalence
(Westby et al. 2019) suggests that Ae. japonicus may be more of an apparent mutualist for Ae. triseriatus
than an antagonist. Furthermore, there is limited evidence of asymmetric competition between the two
species (Alto 2011; Freed et al. 2014) or of displacement of Ae. triseriatus in containers in forested areas
(Westby et al. 2019; Westby et al. 2021). Our results show that Ae. japonicus’ presence in container habitats can relax predation pressure on Ae. triseriatus (Juliano et al. 2019). This, coupled with its presence reducing gregarine parasite prevalence
(Westby et al. 2019) suggests that Ae. japonicus may be more of an apparent mutualist for Ae. triseriatus
than an antagonist. Furthermore, there is limited evidence of asymmetric competition between the two
species (Alto 2011; Freed et al. Discussion 2014) or of displacement of Ae. triseriatus in containers in forested areas
(Westby et al. 2019; Westby et al. 2021). Declarations Data availability statement Upon acceptance of this manuscript, data will be made publically available
through Dryad. The authors declare no conflicts of interest. Acknowledgements We would like to thank Brenden Sweetman, Thomas Van Horn, Hanna Peterman,
Leslie Sterling, and Delilah Sayer for laboratory and field assistance, Katie Costanzo for advice on
experimental design, and Susan Flowers for student program support. This work was funded by Tyson
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splendens (Diptera: Culicidae) predator on the behavior of three mosquito species. J Insect Sci
15:128. https://doi.org/10.1093/jisesa/iev115 Figures Figures Page 14/16 Figure 1
Proportion of prey alive after 24 hrs from experiment 1. Ae. japonicus was significantly more vulnerable
to predation compared to Ae. triseriatus. Figure 1
Proportion of prey alive after 24 hrs from experiment 1. Ae. japonicus was significantly more vulnerable
to predation compared to Ae. triseriatus. Figure 1 Proportion of prey alive after 24 hrs from experiment 1. Ae. japonicus was significantly more vulnerable
to predation compared to Ae. triseriatus. Page 15/16 Figure 2
(A) Experiment 2. Single species time to predation. Ae. japonicus had an extended time to predation
compared to Ae. triseriatus. (B) Mixed species time to predation. Ae. triseriatus had an extended time to
predation over Ae. japonicus regardless of the presence of predation risk cues. Figure 2 (A) Experiment 2. Single species time to predation. Ae. japonicus had an extended time to predation
compared to Ae. triseriatus. (B) Mixed species time to predation. Ae. triseriatus had an extended time to
predation over Ae. japonicus regardless of the presence of predation risk cues. Page 16/16 Page 16/16 Page 16/16
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Combined metagenomic and phenomic approaches identify a novel salt tolerance gene from the human gut microbiome
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Combined metagenomic and phenomic approaches
identify a novel salt tolerance gene from the human gut
microbiome Eamonn P. Culligan 1,2, Julian R. Marchesi 1,3,4*, Colin Hill 1,2* and Roy D. Sleator 1,5*
1 Alimentary Pharmabiotic Centre, Biosciences Institute, University College Cork, Cork, Ireland
2 School of Microbiology, University College Cork, Cork, Ireland
3 Cardiff School of Biosciences, Cardiff University, Cardiff, UK
4 Department of Surgery and Cancer, Centre for Digestive and Gut Health, Imperial College London, London, UK
5 Department of Biological Sciences, Cork Institute of Technology, Cork, Ireland Eamonn P. Culligan 1,2, Julian R. Marchesi 1,3,4*, Colin Hill 1,2* and Roy D. Sleator 1,5*
1 Alimentary Pharmabiotic Centre, Biosciences Institute, University College Cork, Cork, Ireland
2 School of Microbiology, University College Cork, Cork, Ireland
3 Cardiff School of Biosciences, Cardiff University, Cardiff, UK
4 Department of Surgery and Cancer, Centre for Digestive and Gut Health, Imperial College London, London, UK
5 Department of Biological Sciences, Cork Institute of Technology, Cork, Ireland In the current study, a number of salt-tolerant clones previously isolated from a human
gut metagenomic library were screened using Phenotype MicroArray (PM) technology to
assess their functional capacity. PM’s can be used to study gene function, pathogenicity,
metabolic capacity and identify drug targets using a series of specialized microtitre plate
assays, where each well of the microtitre plate contains a different set of conditions
and tests a different phenotype. Cellular respiration is monitored colorimetrically by
the reduction of a tetrazolium dye. One clone, SMG 9, was found to be positive for
utilization/transport of L-carnitine (a well-characterized osmoprotectant) in the presence
of 6% w/v sodium chloride (NaCl). Subsequent experiments revealed a significant growth
advantage in minimal media containing NaCl and L-carnitine. Fosmid sequencing revealed
putative candidate genes responsible for the phenotype. Subsequent cloning of two
genes did not replicate the L-carnitine-associated phenotype, although one of the genes,
a
54
σ
-dependent transcriptional regulator, did confer salt tolerance to Escherichia coli
when expressed in isolation. The original clone, SMG 9, was subsequently found to
have lost the original observed phenotype upon further investigation. Nevertheless, this
study demonstrates the usefulness of a phenomic approach to assign a functional role to
metagenome-derived clones. ORIGINAL RESEARCH ARTICLE
published: 29 April 2014 published: 29 April 2014
doi: 10.3389/fmicb.2014.00189 Combined metagenomic and phenomic approaches
identify a novel salt tolerance gene from the human gut
microbiome Edited by: Anton G. Kutikhin, Kemerovo State
Medical Academy, Russia Anton G. Kutikhin, Kemerovo State
Medical Academy, Russia Anton G. Kutikhin, Kemerovo State
Medical Academy, Russia Reviewed by:
Anton G. Kutikhin, Kemerovo State
Medical Academy, Russia
Arseniy E. Yuzhalin, University of
Oxford, UK *Correspondence:
Julian R. Marchesi, School of
Biosciences, Cardiff University,
Museum Avenue, Cardiff CF10 3AX,
UK Colin Hill, Alimentary Pharmabiotic
Centre, School of Microbiology,
University College Cork, Cork,
Ireland Keywords: metagenomics, functional metagenomics, gut microbiome, microbiota, salt tolerance, BIOLOG,
phenotype microarray, transcriptional regulator Keywords: metagenomics, functional metagenomics, gut microbiome, microbiota, salt tolerance, BIOLOG,
phenotype microarray, transcriptional regulator Keywords: metagenomics, functional metagenomics, gut microbiome, microbiota, salt tolerance, BIOLOG,
phenotype microarray, transcriptional regulator and pathogenesis of infection (Sleator et al., 2001; Wemekamp-
Kamphuis et al., 2004). Carnitine is also found abundantly in
animal tissues and red meat and is an important compound in
the host environment; for the human pathogen Listeria mono-
cytogenes, carnitine and its uptake system OpuC are critical for
infection in mice (Sleator et al., 2003). In addition to its osmopro-
tective properties, carnitine may also be catabolised as a carbon or
nitrogen source to generate energy (Wargo and Hogan, 2009). www.frontiersin.org INTRODUCTION Briefly, a total of 23,040 clones from the library were screened
on LB agar supplemented with 6.5% (w/v) NaCl (a concen-
tration which inhibits the growth of the cloning host, E. coli
EPI300) and 12.5 μg/ml Cm using a Genetix QPix 2 XT colony
picking/gridding robotics platform to identify clones with an
increased salt tolerance phenotype compared to the cloning host
(E. coli EPI300) carrying an empty fosmid vector (pCC1FOS). Identification of any salt tolerant clones will therefore most likely
be due to a gene (or genes) present on the metagenomic insert
from the human gut microbiota. Clones were gridded onto Q-
trays (Genetix) using the robotics platform. Q-trays were incu-
bated at 37◦C for 3 days and checked twice daily for growth of
likely salt-tolerant clones. Salt-tolerant clones were subsequently
replica plated onto LB agar containing 12.5 μg/ml Cm and 6.5%
NaCl and onto LB containing 12.5 μg/ml Cm, but without 6.5%
NaCl (which represented a positive control plate). Each salt toler-
ant clone identified was streaked on LB agar + 12.5 μg/ml Cm to
ensure a pure culture and all clones were maintained as glycerol
stocks at −80◦C. genes will be annotated as hypothetical proteins or have no
known function or homology to existing proteins (Bork, 2000;
Qin et al., 2010). Phenomic approaches can be used to study
hundreds of phenotypic profiles of different bacterial strains
concurrently. Comparing phenomic profiles of wild-type and
mutant derivatives or host strains and clones identified through
metagenomic screening can reveal differences between strains
relating to gene function, pathogenicity and metabolism for
example (Bochner et al., 2001; Bochner, 2009). p
(
)
With this in mind, we have utilized combined metagenomic
and phenomic approaches in this study to characterize a salt
tolerant clone identified from a human gut microbiome metage-
nomic fosmid library. An overview of the study design using
this combined approach can be seen in Figure 1.The BIOLOG
phenotype microarray (PM) system was used to compare phe-
notypes between metagenomic clones and cloning host (carrying
empty fosmid vector). PM plates measure cellular respiration
colorimetrically via reduction of a tetrazolium dye with elec-
trons from NADH generated during the process of respiration. Strongly metabolized substrates generate a more intense purple
color which is recorded with a camera on the Omnilog instru-
ment. INTRODUCTION The ability to adapt to and tolerate increases in extracellular
osmolarity is an important characteristic that enables bacteria to
survive in stressful environments. Increased osmolarity, caused by
sodium chloride (NaCl) for example, initiates a phased response
in bacteria. Firstly during the primary response, potassium ions
are rapidly accumulated within the bacterial cell to offset the
detrimental effects of water loss and influx of toxic sodium and
chloride ions (Sleator and Hill, 2002; Epstein, 2003). Once the cell
has been stabilized, the secondary response begins and involves
the synthesis or, more often the more energetically favorable,
uptake of osmoprotectant compounds (also termed compatible
solutes or osmolytes). Osmoprotectants are compatible with cel-
lular functions and can accumulate to very high concentrations
within the cell and function to protect proteins and to restore
cell volume and thus, turgor pressure (Kempf and Bremer, 1998;
Sleator and Hill, 2002; Kunte, 2006). Osmoprotectants can be
grouped broadly as amino acids, polyols, sugars, trimethyl- and
quaternary-ammonium compounds and their derivatives (Kempf
and Bremer, 1998). The most widely utilized and best character-
ized osmoprotectants are glycine betaine, carnitine, proline, and
ectoine. Numerous studies have shown carnitine to be impor-
tant not only for salt tolerance, but also for survival in vivo g
g
gy
g
g
The emergence of “omics” technologies, an umbrella term
to include analyses such as genomics, metagenomics, transcrip-
tomics, proteomics, metabolomics, and phenomics to name a few,
have been used to gain valuable information about the functions
and interactions of various biological systems as a whole and can
provide more information than more traditional and reductive
approaches to biological problems. Sequence-based and func-
tional metagenomic approaches have led to the discovery of many
novel and diverse genes (Beja et al., 2000; Gillespie et al., 2002;
Banik and Brady, 2008; Culligan et al., 2013; Yoon et al., 2013). While identifying clones which display a specific phenotype
through functional metagenomic screening yields worthwhile
results, characterizing the functional mechanisms responsible
for the observed phenotype can be sometimes difficult owing
to the fact that a large proportion of metagenome derived April 2014 | Volume 5 | Article 189 | 1 www.frontiersin.org Metagenomic novel gene discovery Culligan et al. Caucasian male was used to screen for salt-tolerant clones. The
library was screened as outlined previously (Culligan et al., 2012). FOSMID SEQUENCING AND ANALYSIS FOSMID SEQUENCING AND ANALYSIS
Fosmid DNA was isolated from SMG 9 as described above
to a concentration >200 ng/μl (approximately 5 μg in total). Sequencing of the full fosmid insert of SMG 9 was per-
formed by GATC Biotech (Konstanz, Germany) using 454-
pyrosequencing on a titanium mini-run of the Roche GS-FLX
platform, achieving approximately 65-fold coverage. Sequencing INTRODUCTION If desired, thousands of phenotypes may be monitored
simultaneously using the different available PM plates which can
be grouped as those that measure carbon, nitrogen, phospho-
rous and sulfur metabolism, response to different pH conditions,
osmolytes and chemicals such as anitbiotics. The full range of PM
plates and their constituents, for investigation of bacterial phe-
notypes can be found at: http://www.biolog.com/products-static/
phenotype_microbial_cells_use.php BACTERIAL STRAINS AND GROWTH CONDITIONS Bacterial
strains,
plasmids,
and
oligonucleotide
primers
(Eurofins, MWG Operon, Germany) used in this study are
listed in Table 1. Escherichia coli EPI300::pCC1FOS (Epicentre
Biotechnologies, Madison, WI, USA) was grown in Luria-Bertani
(LB) medium containing 12.5 μg/ml chloramphenicol (Cm). E. coli MKH13 was grown in LB medium and in LB medium
supplemented with 20 μg/ml Cm for strains transformed with
the plasmid pCI372. LB media was supplemented with 1.5% w/v
agar when required. All overnight cultures were grown at 37◦C
with shaking. PHENOTYPE MICROARRAY (PM) ASSAY The PM9 osmolytes microplate was used to compare the cel-
lular phenotypes of SMG 9 and the cloning host, E. coli
EPI300::pCC1FOS (containing an empty fosmid vector) under 96
different conditions. Preparation of the different IF (Inoculating
Fluids; proprietary formulation supplied by BIOLOG) solutions
and inoculation of the PM plates was performed according to
the BIOLOG PM protocol for E. coli and other Gram negative
bacteria. Briefly, IF-0 solution was prepared by adding 25 ml of
sterile water to 125 ml of 1.2× IF-0. IF-0 + dye mix A solu-
tion was prepared by adding 1.8 ml of dye mix A and 23.2 ml
of sterile water to another bottle containing 125 ml of 1.2× IF-
0. IF-10 solution was prepared by adding 1.5 ml of dye mix A
and 23.5 ml of sterile water to a bottle containing 125 ml of 1.2×
IF-10. E. coli strains were grown overnight on LB agar at 37◦C
by streaking from a frozen stock. Cells were sub-cultured by
streaking again on LB agar and grown overnight again. Isolated
colonies were removed from the agar plate using a sterile swab
and added to a tube containing 16 ml of IF-0 solution until a
cell suspension of 42% T (transmittance) was achieved using
the BIOLOG Turbidimeter. 15 ml of this 42% T solution was
diluted in 75 ml of IF-0 + dye mix A to achieve 85% T. 600 μl
of the 85% T cell suspension solution was added to 120 ml of
IF-10 + dye mix A. Finally, 100 μl of the final cell suspen-
sion was inoculated to each well of the PM 9 microplate. Plates
were incubated at 37◦C for 24 h in the Omnilog plate reader
(BIOLOG). As our primary interest is salt tolerance we used the PM
osmolyte plate (PM9) for analysis. The PM screen indicated that
clone SMG 9 was positive for L-carnitine utilization in the pres-
ence of 6% w/v NaCl. Sequencing of the fosmid insert and cloning
of two genes identified a novel salt tolerance gene, but did not
replicate the carnitine-associated phenotype originally observed. Subsequent investigation revealed SMG 9 had lost this phenotype. Notwithstanding this phenomenon, this study demonstrates the
usefulness of a phenomic approach to assign a functional role to
metagenomic library-derived clones. METAGENOMIC LIBRARY CONSTRUCTION AND SCREENING A previously constructed fosmid clone library (Jones and
Marchesi, 2007; Jones et al., 2008), created from metagenomic
DNA isolated from a fecal sample from a healthy 26 year old April 2014 | Volume 5 | Article 189 | 2 Frontiers in Microbiology | Evolutionary and Genomic Microbiology Culligan et al. Metagenomic novel gene discovery FIGURE 1 | Overview of the study design using combined functional
metagenomic and phenomic approaches. An overview of the study design
and experimental process incorporating metagenomic library creation and
screening, identification of salt tolerant clones, comparison of clone to
control strain using Phenotypic Microarray (PM) assay and further analyses
that may be performed to identify novel genes. FIGURE 1 | Overview of the study design using combined functional
metagenomic and phenomic approaches. An overview of the study design
and experimental process incorporating metagenomic library creation and screening, identification of salt tolerant clones, comparison of clone to
control strain using Phenotypic Microarray (PM) assay and further analyses
that may be performed to identify novel genes. Table 1 | Bacterial strains, plasmids and oligonucleotide primers used in this study. Table 1 | Bacterial strains, plasmids and oligonucleotide primers used in this study. Strain or plasmid
Genotype or characteristic(s)
Source or references
STRAINS
E. coli EPI300
F−mcrA (mrr-hsdRMS-mcrBC) 80dlacZM15 lacX74 recA1
endA1 araD139 (ara, leu)7697 galU galK λ−rpsL nupG trfA dhfr;
high-transformation efficiency of large DNA
Epicentre Biotechnologies, Madison, WI, USA
E.coli MKH13
MC4100(putPA)101D(proP)2D(proU)
Haardt et al., 1995
E. coli MKH13::pCI372-mfsT
MKH13 containing pCI372 with mfsT gene from SMG 9 (similar to
Bacteroides sp. CAG:545)
This study
E. coli MKH13::pCI372-sdtR
MKH13 containing pCI372 with sdtR gene from SMG 9 (similar to
Bacteroides sp. CAG:545)
This study
E. coli EPI300::pCI372-mfsT
EPI300 containing pCI372 with mfsT gene from SMG 9 (similar to
Bacteroides sp. CAG:545)
This study
E. coli EPI300::pCI372-sdtR
EPI300 containing pCI372 with sdtR gene from SMG 9 (similar to
Bacteroides sp. CAG:545)
This study
PLASMIDS
pCI372
Shuttle vector between E. coli and L. lactis, CmR
Hayes et al., 1990
pCC1FOS
Fosmid cloning vector, CmR
Epicentre Biotechnologies, Madison, WI, USA
PRIMERS
pCI372 FP
CGGGAAGCTAGAGTAAGTAG
This study
pCI372 RP
CCTCTCGGTTATGAGTTAG
This study
mfsT FP
AAAACTGCAGCGGACTCGTGGTGGATGAC
This study
mfsT RP
GCTCTAGACAAACACCTTGGTGTCATTAGC
This study
sdtR FP
AAAAGTCGACCGGCTGTATGGAAGTTCCTG
This study
sdtR RP
GCTCTAGAGCCTCCAAATTTTGCACCAAG
This study
FP, forward primer; RP, reverse primer; CmR, chlorpamphenicol resistance; Restriction enzyme cut sites are underlined; PstI, CTGCAG; XbaI, TCTAGA; SalI, GTCGAC. RESULTS FIGURE 2 | Appearance of PM 9 plates after incubation for 24 hours at
37◦C. (A) Control EPI300::pCC1FOS and (B) SMG 9. PM plates measure
cellular respiration colorimetrically via reduction of a tetrazolium dye with
electrons from NADH generated during the process of respiration. Strongly
metabolized substrates generate a more intense purple color. Development
of a strong purple color can be seen in well B12 in Figure 2B (circled in
red), which was inoculated with SMG 9, while no color development is
visible in B12 of the control plate. This indicates SMG 9 has a greater ability
to transport and utilise L-carnitine compared to the EPI300::pCC1FOS host
strain. FIGURE 2 | Appearance of PM 9 plates after incubation for 24 hours at
37◦C. (A) Control EPI300::pCC1FOS and (B) SMG 9. PM plates measure CONFIRMATORY EXPERIMENTATION OF PM ANALYSIS In an attempt to confirm and replicate the result from the PM
analysis, SMG 9 and EPI300::pCC1FOS were grown in M9MM
containing various concentrations of NaCl (0–6% w/v) and sup-
plemented with 1 mM L-carnitine when appropriate. Figure 4A
shows growth of both clones in M9MM in the presence and
absence of 1 mM L-carnitine. In the presence of L-carnitine, SMG CONFIRMATION TESTS FOR OBSERVED PHENOTYPE Growth experiments were performed in defined M9 minimal
media (M9MM) (Fluka) to confirm the observed phenotype. Single isolated colonies of SMG 9 and EPI300::pCC1FOS were
grown overnight in M9MM (containing final concentrations
of; D-glucose (0.4%), Bacto™casamino acids (w/v 0.2%)
(Becton, Dickinson and Co, Sparks, MD, USA), magnesium sul-
fate (MgSO4) (2 mM), calcium chloride (CaCl2) (0.1 mM) and
12.5 μg/ml Cm). Reagents were purchased from Sigma Aldrich
(St. Louis, MO, USA) unless otherwise stated. Cells were har-
vested by centrifugation, washed in ¼ strength Ringers solu-
tion and resuspended in fresh M9MM. A 2% v/v inoculum
was sub-cultured in fresh M9MM containing various concen-
trations of sodium chloride (0–8% w/v NaCl) and 1 mM of
L-carnitine when required. Triplicate wells of a 96-well micro-
titre plate were inoculated with 200 μl of the appropriate cell
suspension. Plates were incubated at 37◦C for 24–48 h in an
automated spectrophotometer (Tecan Genios) which recorded
the optical density at 595 nm (OD595 nm) every hour. After
48 h the data was retrieved and analyzed using the Magellan
3 software program and graphs were created with Sigma
Plot 10.0 (Systat Software Inc, London, UK). Results are pre-
sented as the average of triplicate experiments, with error
bars being representative of the standard error of the mean
(SEM). FIGURE 2 | Appearance of PM 9 plates after incubation for 24 hours at
37◦C. (A) Control EPI300::pCC1FOS and (B) SMG 9. PM plates measure
cellular respiration colorimetrically via reduction of a tetrazolium dye with
electrons from NADH generated during the process of respiration. Strongly
metabolized substrates generate a more intense purple color. Development
of a strong purple color can be seen in well B12 in Figure 2B (circled in
red), which was inoculated with SMG 9, while no color development is
visible in B12 of the control plate. This indicates SMG 9 has a greater ability
to transport and utilise L-carnitine compared to the EPI300::pCC1FOS host
strain. DNA MANIPULATIONS AND CLONING OF mfsT AND sdtR GENES A number of clones initially identified from the metagenomic
library
were
chosen
at
random
and
phenotypically
screened
using
PM
9
osmolytes
plate
from
BIOLOG. The
layout
and
contents
of
PM9
can
be
viewed
at
http://www.biolog.com/pdf/pm_lit/PM1-PM10.pdf. One clone,
SMG 9, gave a positive result under one of the 96 different condi-
tions tested. It was found that SMG 9 had an increased metabolic
response (causing a reduction of the tetrazolium dye to generate a
purple color, Figure 2A) compared to the cloning host containing
an empty fosmid vector, E. coli EPI300::pCC1FOS (Figure 2B)
in 6% w/v NaCl supplemented with L-carnitine. The color
formation within each well was measured by BIOLOG’s Omnilog
machine, which produces a color-coded graph. A comparison of
the kinetic data output from SMG 9 and EPI300::pCC1FOS can
be seen in Figure 3. Extraction of fosmids containing metagenomic DNA: 5 ml of bac-
terial culture was grown overnight with 12.5 μg/ml Cm. One
millilitre of culture was used to inoculate 4 ml of fresh LB
broth. To this, 5 μl of 1000× Copy Control Induction solution
(Epicentre Biotechnologies) and 12.5 μg/ml Cm were added. The
mixture was incubated at 37◦C for 5 h with vigorous shaking
(200–250 rpm) to ensure maximum aeration. Cells were har-
vested from the whole 5 ml of induced culture by centrifuging
at 2100 × g for 12 min. Qiagen QIAprep Spin mini-prep kit was
used to extract fosmids as per manufacturer’s instructions. PCR
products were purified with a Qiagen PCR purification kit and
digested with XbaI and PstI (Roche Applied Science) for mfsT
and with SalI and XbaI for sdtR, followed by ligation using the
FastLink DNA ligase kit (Epicentre Biotechnologies) to similarly
digested plasmid pCI372. Electrocompetent E. coli MKH13 and E. coli EPI300 were transformed with the ligation mixture and plated
on LB agar plates containing 20 μg/ml Cm for selection. Colony
PCR was performed on all resistant transformants using primers
across the multiple cloning site (MCS) of pCI372 to confirm the
presence and size of the insert. METAGENOMIC LIBRARY CONSTRUCTION AND SCREENING ; CmR, chlorpamphenicol resistance; Restriction enzyme cut sites are underlined; PstI, CTGCAG; XbaI, TCTAGA; SalI, GTCGAC. FP, forward primer; RP, reverse primer; CmR, chlorpamphenicol resistance; Restriction enzyme cut sites are underlined; PstI, CTGCAG; XbaI, TCTAGA; SalI, GTCGAC. and identify homologous sequences from the National Centre
for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih. gov/blast/Blast.cgi). The full fosmid insert sequence of SMG 9
was submitted to GenBank and assigned the following accession
number; KJ524644. reads were assembled into a single contig by GATC Biotech. The
retrieved sequence was analyzed using the FGENESB software
program (Softberry) to identify putative open reading frames
and translated nucleotide sequences were subjected to BLASTP
analysis to assign putative functions to the encoded proteins April 2014 | Volume 5 | Article 189 | 3 www.frontiersin.org www.frontiersin.org Metagenomic novel gene discovery Culligan et al. SCREENING OF METAGENOMIC LIBRARY Approximately 23,000 clones from a metagenomic fosmid library
from the human gut microbiome were screened previously and
resulted in the identification of 53 salt tolerant clones which
could grow on LB agar supplemented with 6.5% NaCl (a con-
centration which inhibits growth of the cloning host, E. coli
EPI300::pCC1FOS) (Culligan et al., 2012). The salt tolerant clones
identified were annotated as SMG 1-53 (Salt MetaGenome 1–53). April 2014 | Volume 5 | Article 189 | 4 Frontiers in Microbiology | Evolutionary and Genomic Microbiology Metagenomic novel gene discovery Culligan et al. 9 displays a growth defect, while growth is similar under all other
conditions. The growth defect is alleviated when grown at 4% w/v
NaCl + 1 mM L-carnitine and there is no difference in growth
between clones either in the presence or absence of L-carnitine
(Figure 4B). At 5% w/v NaCl however, SMG 9 has a significant
growth advantage compared to EPI300::pCC1FOS both in the
presence and absence of 1 mM L-carnitine (Figure 4C). The pos-
itive effect of L-carnitine on the growth of SMG 9 is evident with
cells entering logarithmic phase growth sooner and reaching a much higher final optical density (OD595 nm). A similar, positive
effect for L-carnitine on growth of SMG 9 is also seen at 6% w/v
NaCl (Figure 4D). SEQUENCING OF SMG 9 FOSMID INSERT AND ANALYSIS Fosmid SMG 9 was fully sequenced (454-pyrosequencing) and
assembled by GATC Biotech. This generated a total of 2.3 × 106
base pairs of sequence data in 6939 sequencing reads. The aver-
age read length was 334 base pairs and coverage of 64.5× was
achieved. Following vector trimming the length of the insert
was approximately 36.5 kb and the %G+C content was 49.43%. Twenty-four putative open reading frames were predicted using
Softberry’s FGENESB, bacterial operon and gene prediction soft-
ware (www.softberry.com) (Mavromatis et al., 2007). Translated
nucleotide sequences were functionally annotated by homology
searches using the BLASTP program to identify homologous
sequences and determine their taxonomic origin. All but two of
the genes encoded proteins with high identity (98–100% at the
amino acid level) to Bacteroides sp. CAG:545. A list of the genes
on SMG 9, their encoded functions and putative domains are pre-
sented in Table 2. The full fosmid insert sequence of SMG 9 has
been submitted to GenBank and assigned the accession number,
KJ524644. FIGURE 3 | Kinetic data measured by BIOLOG Omnilog system. Color
formation within each well was measured by BIOLOG’s Omnilog machine,
which produces a color coded graph. Kinetic data from two clones can be
compared. EPI300::pCC1FOS is shown in red and that from SMG 9 is
shown in green. The green color indicates more rapid metabolism by SMG
9 under the conditions in the well (6% NaCl + L-carnitine). CLONING OF mfsT AND sdtR GENES FIGURE 3 | Kinetic data measured by BIOLOG Omnilog system. Color
formation within each well was measured by BIOLOG’s Omnilog machine,
which produces a color coded graph. Kinetic data from two clones can be
compared. EPI300::pCC1FOS is shown in red and that from SMG 9 is
shown in green. The green color indicates more rapid metabolism by SMG
9 under the conditions in the well (6% NaCl + L-carnitine). Following initial inspection of the encoded proteins on SMG 9,
the presence of an L-carnitine or general osmoprotectant trans-
porter, nor indeed any protein with a functional link to carnitine
metabolism was not immediately obvious. Transposon mutage-
nesis was attempted in order to create a knock-out mutant; this FIGURE 4 | Growth in M9 minimal media with NaCl +/−1mM L-carnitine. Growth of E. coli EPI300::pCC1FOS and SMG 9 in (A) M9 minimal media, (B)
M9 minimal media + 4% NaCl, (C) M9 minimal media + 5% NaCl and (D)
M9 minimal media + 6% NaCl. Legend: E. coli EPI300::pCC1FOS (• closed
circle); SMG 9 (▽open triangle); E. coli EPI300::pCC1FOS + 1mM L-carnitine
(■closed square); SMG 9 + 1 mM L-carnitine, ( open diamond). M9 minimal media + 6% NaCl. Legend: E. coli EPI300::pCC1FOS (• closed
circle); SMG 9 (▽open triangle); E. coli EPI300::pCC1FOS + 1mM L-carnitine
(■closed square); SMG 9 + 1 mM L-carnitine, ( open diamond). FIGURE 4 | Growth in M9 minimal media with NaCl +/−1mM L-carnitine. Growth of E. coli EPI300::pCC1FOS and SMG 9 in (A) M9 minimal media, (B)
M9 minimal media + 4% NaCl, (C) M9 minimal media + 5% NaCl and (D) (■c April 2014 | Volume 5 | Article 189 | 5 www.frontiersin.org Metagenomic novel gene discovery Culligan et al. Table 2 | Proteins predicted to be encoded on SMG 9 fosmid insert. Gene #
Putative encoded function
# a.a
Closest hit organism
% Coverage
e-value
% ID
Domains
1
ATP-dependent chaperone ClpB
554
Bacteroides sp. CAG:545
97
0.00E + 00
99
COG0714; AAA ATPase
2
Preprotein translocase SecG subunit
121
Bacteroides sp. CAG:545
100
3.00E-77
100
SecG
3
Putative uncharacterized protein
187
Bacteroides sp. CAG:545
100
3.00E-134
100
None detected
4
Putative uncharacterized protein
177
Bacteroides sp. CAG:545
86
3.00E-108
99
LptE
5
Transcriptional regulator
432
Bacteroides sp. CAG:545
100
0.00E + 00
99
AAA ATPase; σ54 interaction domain; HTH_8
bacterial reg protein, Fis family domain
6
Putative uncharacterized protein
545
Bacteroides sp. April 2014 | Volume 5 | Article 189 | 6 DISCUSSION however, proved unsuccessful. Two genes (gene 5 and gene 18),
which we felt may be likely to have a possible role in L-carnitine
utilization based on bioinformatic analysis were cloned in iso-
lation to further examine the phenotype. Genes 5 and 18 were
annotated sdtR for sigma-dependent transcriptional regulator
and mfsT for major facilitator superfamily transporter, respec-
tively. In the present study we have identified a novel salt tolerance
gene from a metagenomic library clone from the human gut
microbiome using a combined functional metagenomic and
PM approach. The clone, SMG 9, was identified from a pre-
vious library screen to identify salt tolerant clones (Culligan
et al., 2012) and was further characterized in this study using
PM osmolyte plates. From the PM screen, SMG 9 showed an
increased metabolic profile in the presence of 6% NaCl + 1 mM
L-carnitine compared to the control strain carrying an empty
fosmid vector (EPI300::pCC1FOS), indicating this clone could
utilize or transport L-carnitine. Experiments to confirm the find-
ings of the PM assay showed that SMG 9 displayed an increased
growth profile at 5% and 6% NaCl in the presence of 1 mM L-
carnitine compared to controls, similar to observations in the
PM assay. Transposon mutagenesis was attempted to create phe-
notypic knock out mutants, using the EZTn5 system (Epicentre
Biotechnologies; Goryshin and Reznikoff, 1998) but this proved
unsuccessful. This may be due to the presence of a gene encoding
a DNA repair protein MutS on the fosmid insert, which has been
associated with transposon excision (specifically Tn5 and Tn10)
(Lundblad and Kleckner, 1985). Both mfsT and sdtR were cloned in the vector pCI372 and
expressed in both E. coli EPI300 and the osmosensitive strain E. coli MKH13 (Haardt et al., 1995). The effect of each gene on the
growth of each strain under salt stress in the presence and absence
of L-carnitine was assessed. The mfsT gene had no effect on
growth under any of the conditions tested (data not shown). The
sdtR gene on the other hand conferred a significant salt tolerance
phenotype to E. coli MKH13 when grown in media supplemented
with both 3 and 4% w/v NaCl (Figures 5C,D, respectively), while
growth was similar in media lacking NaCl and in media supple-
mented with 2% w/v NaCl (Figures 5A,B, respectively). CLONING OF mfsT AND sdtR GENES CAG:545
100
0.00E + 00
99
TadD
7
Putative uncharacterized protein
1015
Bacteroides sp. CAG:545
100
0.00E + 00
99
SecD, SecF
8
OmpA/MotB domain protein
618
Bacteroides sp. CAG:545
71
0.00E + 00
99
PD40, similar to WD40 domain
9
Putative uncharacterized protein
155
Bacteroides sp. CAG:545
100
4.00E-106
100
NfeD
10
uPF0365 protein AL1_06760
317
Bacteroides sp. CAG:545
100
0.00E + 00
99
YdfA_immunity superfamily
11
Putative uncharacterized protein
142
Bacteroides sp. CAG:545
100
1.00E-97
98
Lipocalin_4
12
Subtilisin-like serine protease
678
Bacteroides sp. CAG:545
100
0.00E + 00
99
Peptidase_S8_S53 superfamily
13
Uncharacterized protein
812
Bacteroides sp. CAG:545
99
0.00E + 00
99
None detected
14
RagB/SusD family protein
547
Bacteroides sp. CAG:545
100
0.00E + 00
99
Two SusD superfamily
15
Outer membrane receptor for ferrien-
terochelin and colicins
1068
Bacteroides sp. CAG:545
100
0.00E + 00
100
Can_B2; Plug; OM channel; OMP_RagA_
SusC
16
Alpha-L-fucosidase-like
513
Bacteroides sp. CAG:545
100
0.00E + 00
98
COG3669; Alpha_L_fucos; F5_F8_Type_C
17
Putative uncharacterized protein
446
Bacteroides sp. CAG:545
100
0.00E + 00
99
DHQ_FE-ADH (Dehydroquinate iron alde-
hyde dehydrogenase)
18
Major facilitator superfamily MFS_1
487
Bacteroides sp. CAG:545
100
0.00E + 00
100
MFS; UhpC, sugar phosphate permease
19
ThiF family protein
242
Bacteroides sp. CAG:545
100
4.00E-174
98
YgdL_like
20
Putative uncharacterized protein
649
Bacteroides sp. CAG:545
100
0.00E + 00
98
Glyco_hydro_97
21
DNA mismatch repair protein MutS
894
Bacteroides sp. CAG:545
98
0.00E + 00
99
PRK05399;
MutS-I;
MutS_II;
MutS_III;
ABC_MutS_1
22
Glycosyl transferase group 1
378
Bacteroides sp. CAG:545
100
0.00E + 00
99
RfaG
23
No significant similarity found
68
N/A
N/A
N/A
N/A
N/A
24
Hypothetical protein Fjoh_3657
162
Flavobacterium johnsoniae UW101
80
4.00E-25
40
AdkA, archaeal adenylate kinase
Functional assignment is based on BLASTP of amino acid sequences predicted by Softberry’s FGENESB. Abbreviations: # a.a, number of predicted amino acids; %ID, % identity at the amino acid level; N/A, not
applicable. Frontiers in Microbiology | Evolutionary and Genomic Microbiology Table 2 | Proteins predicted to be encoded on SMG 9 fosmid insert. April 2014 | Volume 5 | Article 189 | 6 Metagenomic novel gene discovery Culligan et al. DISCUSSION Cloning
and expression of sdtR in EPI300 resulted in an increase in salt
tolerance compare to wild-type EPI300 carrying an empty copy
of the plasmid pCI372. Addition of 1 mM L-carnitine increased
the growth rate and final optical density of both strains, but
its effect on the sdtR+strain was not significant relative to the
EPI300::pCI372 control (Figures 6A,B). Next generation sequencing of the full fosmid insert of SMG 9
and functional assignment of the encoded proteins using BLASTP
revealed sequences shared highest genetic identity to Bacteroides
sp. CAG:545. Species of Bacteroides are commonly found in the
human gut, where the resident microbiota is largely composed of
species from two dominant phyla, the Bacteroidetes and Firmicutes
(Qin et al., 2010). The %G+C content of the SMG 9 insert was
49.44% which close to the reported range of 40–48% for genomes Although sdtR did confer a salt tolerance phenotype, neither
of the cloned genes replicated the original phenotype related to
L-carnitine. We re-examined clone SMG 9 and carried out fur-
ther studies, however these revealed that SMG 9 had lost the
carnitine-associated phenotype seen originally and unfortunately,
it was therefore not possible to identify the gene(s) responsible. FIGURE 5 | Growth in LB broth with NaCl. Growth in of E. coli MKH13::pCI372 (• black circle) and MKH13::pCI372-sdtR (▽open triangle) in (A) LB broth and
LB broth supplemented with (B) 2% NaCl, (C) 3% NaCl and (D) 4% NaCl. FIGURE 5 | Growth in LB broth with NaCl. Growth in of E. coli MKH13::pCI372 (• black circle) and MKH13::pCI372-sdtR (▽open triangle) in (A) LB broth and
LB broth supplemented with (B) 2% NaCl, (C) 3% NaCl and (D) 4% NaCl. April 2014 | Volume 5 | Article 189 | 7 www.frontiersin.org Metagenomic novel gene discovery Culligan et al. FIGURE 6 | Growth in M9 minimal media with NaCl +/−L-carnitine. Growth of EPI300::pCI372 and EPI300::pCI372-sdtR in (A) M9 minimal
media and (B) M9 minimal media + 6% NaCl. Legend: E. coli
EPI300::pCI372 (• black circle); EPI300::pCI372 + 1 mM L-carnitine (▽open
triangle); EPI300::pCI372-sdtR (■closed square); EPI300::pCI372-sdtR +
1mM L-carnitine, ( open diamond). EPI300::pCI372 (• black circle); EPI300::pCI372 + 1 mM L-carnitine (▽open
triangle); EPI300::pCI372-sdtR (■closed square); EPI300::pCI372-sdtR +
1mM L-carnitine, ( open diamond). EPI300::pCI372 (• black circle); EPI300::pCI372 + 1 mM L-carnitine (▽open
triangle); EPI300::pCI372-sdtR (■closed square); EPI300::pCI372-sdtR +
1mM L-carnitine, ( open diamond). DISCUSSION FIGURE 6 | Growth in M9 minimal media with NaCl +/−L-carnitine. Growth of EPI300::pCI372 and EPI300::pCI372-sdtR in (A) M9 minimal
media and (B) M9 minimal media + 6% NaCl. Legend: E. coli of species of Bacteroides (Shah, 1992). Sequencing and subse-
quent functional annotation did not reveal any obvious genes
related to known L-carnitine transport or utilization systems,
suggesting a novel mechanism may be involved. We conducted
further bioinformatic analysis of the encoded proteins in an
attempt to identify any link to salt tolerance or osmoprotectant
transport. Gene 5 (sdtR) is predicted to encode a σ54-dependent
transcriptional regulator, which contains a number of domains
(see Table 2), including a helix-turn-helix 8 (HTH_8), Fis-family
protein domain, while gene 18 (mfsT) is predicted to encode a
major facilitator superfamily (MFS) protein with an UhpC sugar
phosphate permease domain. Both genes were chosen for further
study and cloning as Fis is a regulatory protein involved the reg-
ulation of proline (another important osmoprotectant) uptake
(Xu and Johnson, 1995, 1997; Typas et al., 2007), while we fur-
ther reasoned that sdtR could be regulating host EPI300 genes,
contributing the L-carnitine-associated phenotype, while MFS
transporters also play a role in osmoprotectant uptake (Culham
et al., 1993; Haardt et al., 1995; Pao et al., 1998). Despite the
presence of a sugar phosphate permease domain, indicating sugar
transport, MFS transporters are known to have a diverse substrate
range (Pao et al., 1998; Saier, 2000; Law et al., 2008). evident from Figure 4A, where a growth defect for SMG 9 is
apparent when grown in M9MM + 1 mM L-carnitine. This indi-
cates L-carnitine may be increasing the metabolic load on the cell
and this metabolic stress is only relieved in the presence of NaCl,
when L-carnitine may be utilized efficiently in an osmoprotec-
tive capacity. If the gene is constitutively expressed, a mutation
may have occurred to counteract this phenomenon. The presence
of a gene encoding MutS may also be relevant as mutations to
MutS can result in a mutator phenotype in E. coli cells (Wu and
Marinus, 1994). Furthermore, it is possible the original SMG 9
clone acquired a mutation on the fosmid insert that conferred
the carnitine-associated phenotype and a subsequent suppressor
mutation occurred to silence this mutation, returning the clone
to its original phenotype. ACKNOWLEDGMENTS The Alimentary Pharmabiotic Centre is a research center funded
by Science Foundation Ireland (SFI grant number 07/CE/B1368). We acknowledge the continued financial assistance of the
Alimentary Pharmabiotic Centre, funded by Science Foundation
Ireland. Julian R. Marchesi acknowledges funding from The
Royal Society which supports the bioinformatic cluster (Hive)
at Cardiff University, School of Biosciences. Roy D. Sleator is an
ESCMID Research Fellow and Coordinator of ClouDx-i an EU
FP7-PEOPLE-2012-IAPP project DISCUSSION In conclusion, we have identified a novel salt tolerance gene
from the human gut microbiome using a combined functional
metagenomic and PM approach. The gene originates from a
species of Bacteroides and encodes a putative transcriptional reg-
ulator. Overall this study demonstrates the utility of functional
metagenomics and phenomics for novel gene discovery and func-
tional characterization of metagenome-derived clones. The mfsT gene did not confer a salt tolerance or the L-carnitine
associated phenotype to transformed cells (data not shown). The
sdtR gene also did not confer the L-carnitine associated pheno-
type to E. coli, but did however confer an increased salt tolerance
phenotype. sdtR therefore represents a novel salt tolerance gene
and most likely functions by influencing expression (either posi-
tively or negatively) of host E. coli genes, although further work,
comprising expression studies and microarray analysis, will be
required to elucidate the genes involved as transcriptional regu-
lators can influence a wide variety of genes. Transcriptional regu-
lators are commonly involved in the response different stresses in
bacteria (Hengge-Aronis et al., 1991; Cheville et al., 1996; Battesti
et al., 2011; Hoffmann et al., 2013), while σ54 (RpoN) has been
shown to play a role in osmotolerance in Listeria monocytogenes
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10.1146/annurev.micro.61.080706.093329 Copyright © 2014 Culligan, Marchesi, Hill and Sleator. 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) or licensor are credited and that the original publication in this
journal is cited, in accordance with accepted academic practice. No use, distribution or
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et al. (2007). Use of simulated data sets to evaluate the fidelity of metagenomic
processing methods. Nat. Methods 4, 495–500. doi: 10.1038/nmeth1043 April 2014 | Volume 5 | Article 189 | 9 www.frontiersin.org
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English
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Hydrogen Gas in Cancer Treatment
|
Frontiers in oncology
| 2,019
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cc-by
| 8,252
|
Edited by:
Nelson Shu-Sang Yee,
Penn State Milton S. Hershey Medical
Center, United States ,
Reviewed by:
Leo E. Otterbein,
Beth Israelv Deaconess Medical
Center and Harvard Medical School,
United States
Paolo Armando Gagliardi,
University of Bern, Switzerland
*Correspondence:
Jin Zhou
zhou-jin-2008@163.com
Kang Peng
kds978@163.com
†These authors share
co-first authorship Reviewed by:
Leo E. Otterbein,
Beth Israelv Deaconess Medical
Center and Harvard Medical School,
United States
Paolo Armando Gagliardi,
University of Bern, Switzerland Keywords: hydrogen gas, ROS, inflammation, combination, anti-cancer Hydrogen Gas in Cancer Treatment
Sai Li 1†, Rongrong Liao 2†, Xiaoyan Sheng 2†, Xiaojun Luo 3, Xin Zhang 1, Xiaomin Wen 3,
Jin Zhou 2* and Kang Peng 1,3* 1 Department of Pharmacy, Integrated Hospital of Traditional Chinese Medicine, Southern Medical University, Guangzhou,
China, 2 Nursing Department, Integrated Hospital of Traditional Chinese Medicine, Southern Medical University, Guangzhou,
China, 3 The Centre of Preventive Treatment of Disease, Integrated Hospital of Traditional Chinese Medicine, Southern
Medical University, Guangzhou, China Gas signaling molecules (GSMs), composed of oxygen, carbon monoxide, nitric oxide,
hydrogen sulfide, etc., play critical roles in regulating signal transduction and cellular
homeostasis. Interestingly, through various administrations, these molecules also exhibit
potential in cancer treatment. Recently, hydrogen gas (formula: H2) emerges as another
GSM which possesses multiple bioactivities, including anti-inflammation, anti-reactive
oxygen species, and anti-cancer. Growing evidence has shown that hydrogen gas can
either alleviate the side effects caused by conventional chemotherapeutics, or suppress
the growth of cancer cells and xenograft tumor, suggesting its broad potent application
in clinical therapy. In the current review, we summarize these studies and discuss the
underlying mechanisms. The application of hydrogen gas in cancer treatment is still in its
nascent stage, further mechanistic study and the development of portable instruments
are warranted. INTRODUCTION Gaseous signaling molecules (GSMs) refer to a group of gaseous molecules, such as oxygen (1),
nitric oxide (2), carbon monoxide (3), hydrogen sulfide (4), sulfur dioxide (5, 6), ethylene (7, 8),
etc. These gaseous molecules possess multiple critical functions in regulating cell biology in vivo
via signal transduction (9). More importantly, certain GSMs could serve as therapeutic agents
in primary cancer, as well as in multidrug-resistant cancer treatment when used by directly or
certain pharmaceutical formulations (9–13). In addition, some of these GSMs can be generated in
body via different bacteria or enzymes, such as nitric oxide, hydrogen sulfide, indicating that they
are more compatible molecules that may exhibit less adverse effects compared with conventional
chemotherapeutics (9, 14, 15). Recently, hydrogen gas has been recognized as another important
GSM in biology, exhibiting appealing potential in health care for its role in preventing cell injury
from various attacking (16–19). Specialty section:
This article was submitted to
Cancer Molecular Targets and
Therapeutics,
a section of the journal
Frontiers in Oncology
Received: 02 May 2019
Accepted: 15 July 2019
Published: 06 August 2019
Citation:
Li S, Liao R, Sheng X, Luo X, Zhang X,
Wen X, Zhou J and Peng K (2019)
Hydrogen Gas in Cancer Treatment. Front. Oncol. 9:696. doi: 10.3389/fonc.2019.00696 Specialty section:
This article was submitted to
Cancer Molecular Targets and
Therapeutics,
a section of the journal
Frontiers in Oncology Specialty section:
This article was submitted to
Cancer Molecular Targets and
Therapeutics,
a section of the journal
Frontiers in Oncology
Received: 02 May 2019
Accepted: 15 July 2019
Published: 06 August 2019 Received: 02 May 2019
Accepted: 15 July 2019
Published: 06 August 2019 With the formula of H2, hydrogen gas is the lightest molecule in the nature which only
accounts for about 0.5 parts per million (ppm) of all the gas. Naturally, hydrogen gas is a colorless,
odorless, tasteless, non-toxic, highly combustible gas which may form explosive mixtures with air in
concentrations from 4 to 74% that can be triggered by spark, heat, or sunlight. Hydrogen gas can be
generated in small amount by hydrogenase of certain members of the human gastrointestinal tract
microbiota from unabsorbed carbohydrates in the intestine through degradation and metabolism
(20, 21), which then is partially diffused into blood flow and released and detected in exhaled breath
(20), indicating its potential to serve as a biomarker. REVIEW
published: 06 August 2019
doi: 10.3389/fonc.2019.00696 published: 06 August 2019
doi: 10.3389/fonc.2019.00696 Citation: Li S, Liao R, Sheng X, Luo X, Zhang X,
Wen X, Zhou J and Peng K (2019)
Hydrogen Gas in Cancer Treatment. Front. Oncol. 9:696. doi: 10.3389/fonc.2019.00696 August 2019 | Volume 9 | Article 696 Frontiers in Oncology | www.frontiersin.org Hydrogen Gas in Cancer Treatment Li et al. As the lightest molecule in natural, hydrogen gas exhibits
appealing penetration property, as it can rapidly diffuse through
cell membranes (22, 23). Study in animal model showed that,
after orally administration of hydrogen super-rich water (HSRW)
and intra-peritoneal administration of hydrogen super-rich
saline (HSRS), the hydrogen concentration reached the peak at
5 min; while it took 1 min by intravenous administration of HSRS
(23). Another in vivo study tested the distribution of hydrogen
in brain, liver, kidney, mesentery fat, and thigh muscle in rat
upon inhalation of 3% hydrogen gas (24). The concentration
order of hydrogen gas, when reached saturated status, was liver,
brain, mesentery, muscle, kidney, indicating various distributions
among organs in rats. Except the thigh muscle required a longer
time to saturate, the other organs need 5–10 min to reach Cmax
(maximum hydrogen concentration). Meanwhile, the liver had
the highest Cmax (24). The information may direct the future
clinical application of hydrogen gas. a mechanism known as oxidative stress (39, 40). Normally,
under physical condition, cells including cancer cells maintain
a balance between generation and elimination of ROS, which is
of paramount importance for their survival (41, 42). The over-
produced ROS, resulted from imbalance regulatory system or
outer chemical attack (including chemotherapy/radiotherapy),
may initiate inner apoptosis cascade, causing severely toxic
effects (43–45). Hydrogen gas may act as a ROS modulator. First, as shown
in Ohsawa et al.’s study, hydrogen gas could selectively scavenge
the most cytotoxic ROS, •OH, as tested in an acute rat
model of cerebral ischemia and reperfusion (26). Another study
also confirmed that hydrogen gas might reduce the oxygen
toxicity resulted from hyperbaric oxygen via effectively reducing
•OH (46). Second, hydrogen may induce the expression of some
antioxidant enzymes that can eliminate ROS, and it plays
key roles in regulating redox homeostasis of cancer cells (42,
47). Studies have indicated that upon hydrogen gas treatment,
the expression of superoxide dismutase (SOD) (48), heme
oxyganase-1 (HO-1) (49), as well as nuclear factor erythroid 2-
related factor 2 (Nrf2) (50), increased significantly, strengthening
its potential in eliminating ROS. HYDROGEN GAS SUPPRESSES
INFLAMMATORY CYTOKINES Inflammatory cytokines are a series of signal molecules that
mediate the innate immune response, whose dys-regulation may
contribute in many diseases, including cancer (53–55). Typical
inflammatory cytokines include interleukins (ILs) excreted by
white blood cells, tumor necrosis factors (TNFs) excreted by
macrophages, both of which have shown close linkage to cancer
initiation and progression (56–59), and both of ILs and TNFs can
be suppressed by hydrogen gas (60, 61). In the current review, we take a spot on its application in
cancer treatment. Typically, hydrogen gas may exert its bio-
functions via regulating ROS, inflammation and apoptosis events. Citation: Although hydrogen gas was studied as a therapy in a skin
squamous carcinoma mouse model back in 1975 (25), its
potential in medical application has not been vastly explored
until 2007, when Oshawa et al. reported that hydrogen could
ameliorate cerebral ischemia-reperfusion injury by selectively
reducing cytotoxic reactive oxygen species (ROS), including
hydroxyl radical (•OH) and peroxynitrite (ONOO-) (26),
which then provoked a worldwide attention. Upon various
administrative formulations, hydrogen gas has been served as a
therapeutic agent for a variety of illnesses, such as Parkinson’s
disease (27, 28), rheumatoid arthritis (29), brain injury (30),
ischemic reperfusion injury (31, 32), and diabetes (33, 34), etc. By regulating ROS, hydrogen gas may act as an adjuvant
regimen to reduce the adverse effects in cancer treatment
while at the same time doesn’t abrogate the cytotoxicity of
other therapy, such as radiotherapy and chemotherapy (48, 51). Interestingly, due the over-produced ROS in cancer cells (38), the
administration of hydrogen gas may lower the ROS level at the
beginning, but it provokes much more ROS production as a result
of compensation effect, leading to the killing of cancer cells (52). More importantly, hydrogen has been shown to improve
clinical end-points and surrogate markers, from metabolic
diseases to chronic systemic inflammatory disorders to cancer
(17). A clinical study in 2016 showed that inhalation of hydrogen
gas was safe in patients with post-cardiac arrest syndrome (35),
its further therapeutic application in other diseases became even
more appealing. HYDROGEN GAS SELECTIVELY
SCAVENGES HYDROXYL RADICAL AND
PEROXYNITRITE, AND REGULATES
CERTAIN ANTIOXIDANT ENZYMES Inflammation induced by chemotherapy in cancer patients
not only causes severely adverse effects (62, 63), but also
leads to cancer metastasis, and treatment failure (64, 65). By regulating inflammation, hydrogen gas can prevent tumor
formation, progression, as well as reduce the side effects caused
by chemotherapy/radiotherapy (66). By far, many studies have indicated that hydrogen gas doesn’t
target specific proteins, but regulate several key players in cancer,
including ROS, and certain antioxidant enzymes (36). ROS refers to a series of unstable molecules that contain
oxygen, including singlet oxygen (O2•), hydrogen peroxide
(H2O2), hydroxyl radical (•OH), superoxide (•O−
2 ), nitric
oxide (NO•), and peroxynitrite (ONOO−), etc. (37, 38). Once
generated in vivo, due to their high reactivity, ROS may attack
proteins, DNA/RNA and lipids in cells, eliciting distinct damage
that may lead to apoptosis. The presence of ROS can produce
cellular stress and damage that may produce cell death, via Frontiers in Oncology | www.frontiersin.org HYDROGEN GAS EXHIBITS POTENTIAL IN
CANCER TREATMENT Another study showed that both inhaling hydrogen gas (1%
hydrogen in air) and drinking hydrogen-rich water (0.8 mM
hydrogen in water) could reverse the mortality, and body-
weight loss caused by cisplatin via its anti-oxidant property. Both treatments improved the metamorphosis, accompanied
with decreased apoptosis in the kidney, and nephrotoxicity as
assessed by serum creatinine and BUN levels. More importantly,
hydrogen didn’t impair the anti-tumor activity of cisplatin
against cancer cell lines in vitro and in tumor-bearing mice
(85). Similar results were also observed in Meng et al.’s study,
as they showed that hydrogen-rich saline could attenuate the
follicle-stimulating hormone release, elevate the level of estrogen,
improve the development of follicles, and reduce the damage
to the ovarian cortex induced by cisplatin. In the study,
cisplatin treatment induced higher level of oxidation products,
suppressed the antioxidant enzyme activity. The hydrogen-
rich saline administration could reverse these toxic effects by
reducing MDA and restoring the activity of superoxide dismutase
(SOD), catalase (CAT), two important anti-oxidant enzymes. Furthermore, hydrogen-rich saline stimulated the Nrf2 pathway
in rats with ovarian damage (86). Hydrogen Gas Relieves the Adverse Effects
Related to Chemotherapy/Radiotherapy Chemotherapy and radiotherapy remain the leading strategies
to treat cancer (73, 74). However, cancer patients receiving
these treatments often experience fatigue and impaired quality
of life (75–77). The skyrocketed generation of ROS during
the treatment is believed to contribute in the adverse effects,
resulting in remarkable oxidative stress, and inflammation (41,
42, 78). Therefore, benefited from its anti-oxidant and anti-
inflammatory and other cell protective properties, hydrogen gas
can be adopted as an adjuvant therapeutic regimen to suppress
these adverse effects. Under treatment of epidermal growth factor receptor
inhibitor gefitinib, patients with non-small cell lung cancer
often suffer with severe acute interstitial pneumonia (79). In
a mice model treated with oral administration of gefitinib
and intraperitoneal injection of naphthalene which induced
severely lung injury due to oxidative stress, hydrogen-rich water
treatment significantly reduced the inflammatory cytokines, such
as IL-6 and TNFα in the bronchoalveolar lavage fluid, leading to
a relieve of lung inflammation. HYDROGEN GAS INHIBITS/INDUCES
APOPTOSIS Apoptosis, also termed as programed cell death, can be triggered
by extrinsic or intrinsic signals and executed by different
molecular pathways, which serve as one efficient strategy for August 2019 | Volume 9 | Article 696 Frontiers in Oncology | www.frontiersin.org 2 Hydrogen Gas in Cancer Treatment Li et al. rats, such as the serum brain natriuretic peptide (BNP), aspartate
transaminase (AST), alanine transaminase (ALT), albumin and
malondialdehyde (MDA) levels. Mechanistically, hydrogen-rich
saline significantly lowered the ROS level, as well as inflammatory
cytokines TNF-α, IL-1β, and IL-6 in cardiac and hepatic tissue. Hydrogen-rich saline also induced less expression of apoptotic
Bax, cleaved caspase-3, and higher anti-apoptotic Bcl-2, resulting
in less apoptosis in both tissues (71). This study suggested that
hydrogen-rich saline treatment exerted its protective effects via
inhibiting the inflammatory TNF-α/IL-6 pathway, increasing the
cleaved C8 expression and Bcl-2/Bax ratio, and attenuating cell
apoptosis in both heart and liver tissue (71). cancer treatment (67, 68). Generally, apoptosis can be induced
by (1) provoking the death receptors of cell surface (such as
Fas, TNF receptors, or TNF-related apoptosis-inducing ligand),
(2) suppressing the survival signaling (such as epidermal
growth factor receptor, mitogen-activated protein kinase, or
phosphoinositide 3-kinases), and (3) activating the pro-apoptotic
B-cell lymphoma-2 (Bcl-2) family proteins, or down-regulating
anti-apoptosis proteins (such as X-linked inhibitor of apoptosis
protein, surviving, and the inhibitor of apoptosis) (69, 70). Hydrogen
gas
can
regulate
intracellular
apoptosis
by
impacting the expression of apoptosis-related enzymes. At
certain concentration, it can either serve as apoptosis-inhibiting
agent via inhibiting the pro-apoptotic B-cell lymphoma-2-
associated X protein (Bax), caspase-3, 8, 12, and enhancing the
anti-apoptotic B-cell lymphoma-2 (Bcl-2) (71), or as apoptosis-
inducing agent via the contrast mechanisms (72), suggesting its
potential in protecting normal cells from anti-cancer drugs or in
suppressing cancer cells. Hydrogen-rich water also showed renal protective effect
against cisplatin-induced nephrotoxicity in rats. In the studies,
blood oxygenation level-dependent (BOLD) contrast magnetic
resonance images (MRI) acquired in different treated group
showed that the creatinine and blood urea nitrogen (BUN) levels,
two parameters that related to nephrotoxicity, were significantly
higher in cisplatin treated group than those in the control group. Hydrogen-rich water treatment could significantly reverse the
toxic effects, and it showed much higher transverse relaxation
rate by eliminating oxygen radicals (83, 84). Frontiers in Oncology | www.frontiersin.org August 2019 | Volume 9 | Article 696 HYDROGEN GAS EXHIBITS POTENTIAL IN
CANCER TREATMENT More importantly, hydrogen-rich
water didn’t impair the overall anti-tumor effects of gefitinib both
in vitro and in vivo, while in contrast, it antagonized the weight
loss induced by gefitinib and naphthalene, and enhanced the
overall survival rate, suggesting hydrogen gas to be a promising
adjuvant agent that has potential to be applied in clinical practice
to improve quality of life of cancer patients (80). The mFOLFOX6 regimen, composed with folinic acid, 5-
fluorouracil, and oxaliplatin, is used as first-line treatment for
metastatic colorectal cancer, but it also confers toxic effects
to liver, leading to bad quality of life of patient (87, 88). A
clinical study was conducted in China by investing the protective
effect of hydrogen-rich water on hepatic function of colorectal
cancer patients (144 patients were enrolled and 136 of them
were include in the final analysis) treated with mFOLFOX6
chemotherapy. The results showed that the placebo group
exhibited damaging effects caused by mFOLFOX6 chemotherapy
as measured by the elevated levels of ALT, AST and indirect
bilirubin (IBIL), while the hydrogen-rich water combinational
treatment group exhibited no differences in liver function during
the treatment, probably due to its antioxidant activity, indicating Doxorubicin, an anthracycline antibiotic, is an effective
anticancer agent in the treatment of various cancers, but its
application is limited for the fatal dilated cardiomyopathy
and hepatotoxicity (81, 82). One in vivo study showed that
intraperitoneal injection of hydrogen-rich saline ameliorated the
mortality, and cardiac dysfunction caused by doxorubicin. This
treatment also attenuated histopathological changes in serum of August 2019 | Volume 9 | Article 696 3 Hydrogen Gas in Cancer Treatment Li et al. it a promising protective agent to alleviate the mFOLFOX6-
related liver injury (51). it a promising protective agent to alleviate the mFOLFOX6-
related liver injury (51). significantly enhanced the anticancer efficacies of thermal
therapy, achieving a synergetic anticancer effect. In vivo safety
evaluation showed that the injection dose of 10 mg kg−1 PdH0.2
nanocrystals caused no death, no changes of several blood
indicators, and no affected functions of liver and kidney. In
4T1 murine breast cancer tumor model and B16-F10 melanoma
tumor model, the combined PdH0.2 nanocrystals and NIR
irradiation therapy exhibited a synergetic anticancer effect,
leading to remarkable tumor inhibition when compared with
thermal therapy. Meanwhile, the combination group showed no
visible damage to heart, liver, spleen, lung, and kidney, indicating
suitable tissue safety and compatibility (52). Hydrogen Gas Suppresses Tumor
Formation In a mouse model,
hydrogen-rich
water
administration
lowered
the
hepatic
cholesterol,
peroxisome
proliferator-activated
receptor-α
(PPARα) expression, and increased the anti-oxidative effects in
the liver when compared with control and pioglitazone treated
group (96). Hydrogen-rich water exhibited strong inhibitory
effects to inflammatory cytokines TNF-α and IL-6, oxidative
stress and apoptosis biomarker. As shown in NASH-related
hepatocarcinogenesis model, in the group of hydrogen-rich
water treatment, tumor incidence was lower and the tumor
volumes were smaller than control and pioglitazone treated
group. The above findings indicated that hydrogen-rich water
had potential in liver protection and liver cancer treatment (96). HYDROGEN GAS EXHIBITS POTENTIAL IN
CANCER TREATMENT Most of the ionizing radiation-induced adverse effects to
normal cells are induced by hydroxyl radicals. The combination
of radiotherapy with certain forms of hydrogen gas may be
beneficial to alleviate these side effects (89). Indeed, several
studies found that hydrogen could protect cells and mice from
radiation (48, 90). As tested in a rat model of skin damage established by using a
44 Gy electronic beam, the treated group by hydrogen-rich water
exhibited higher lever of SOD activity and lower MDA and IL-6
in the wounded tissues than the control group and the distilled
water group. Furthermore, hydrogen-rich water shortened the
healing time and increased the healing rate of skin injury (48). Hydrogen Gas Suppresses Tumor
Formation Gastrointestinal toxicity is a common side effect induced by
radiotherapy, which impairs the life quality of cancer patients
(91). As shown in Xiao et al.’s study in mice model, hydrogen-
water administration via oral gavage increased the survival
rate and body weight of mice which were exposed to total
abdominal irradiation, accompanied with an improvement in
gastrointestinal tract function and the epithelial integrity of
the small intestine. Further microarray analysis revealed that
hydrogen-water treatment up-regulated miR-1968-5p, which
then up-regulated its target myeloid differentiation primary
response gene 88 (MyD88, a mediator in immunopathology, and
gut microbiota dynamics of certain intestinal diseases involving
toll-like receptors 9) expression in the small intestine after total
abdominal irradiation (92). Li et al. reported that the consumption of hydrogen-rich
water alleviated renal injury caused by Ferric nitrilotriacetate
(Fe-NTA) in rats, evidenced by decreased levels of serum
creatinine and BUN. Hydrogen-rich water suppressed the Fe-
NTA-induced oxidative stress by lowering lipid peroxidation,
ONOO−, and inhibiting the activities of NADPH oxidase and
xanthine oxidase, as well as by up-regulating antioxidant catalase,
and restoring mitochondrial function in kidneys. Consequently,
Fe-NTA-induced inflammatory cytokines, such as NF-κB, IL-
6, and monocyte chemoattractant protein-1 were significantly
alleviated by hydrogen treatment. More importantly, hydrogen-
rich water consumption inhibited several cancer-related proteins
expression, including vascular endothelial growth factor (VEGF),
signal transducer and activator of transcription 3 (STAT3)
phosphorylation, and proliferating cell nuclear antigen (PCNA)
in rats, resulting in lower incidence of renal cell carcinoma
and the suppression of tumor growth. This work suggested that
hydrogen-rich water was a promising regimen to attenuate Fe-
NTA-induced renal injury and suppress early tumor events (66). Another study conducted in clinical patients with malignant
liver tumors showed that the consumption of hydrogen-rich
water for 6 weeks reduced the level of reactive oxygen metabolite,
hydroperoxide, and maintained the biologic antioxidant activity
in the blood. Importantly, scores of quality of life during
radiotherapy were significantly improved in hydrogen-rich water
group compared to the placebo water group. Both groups
exhibited similar tumor response to radiotherapy, indicating
that the consumption of hydrogen-rich water reduced the
radiation-induced oxidative stress while at the same time didn’t
compromise anti-tumor effect of radiotherapy (93). Non-alcoholic steatohepatitis (NASH) due to oxidative
stress induced by various stimuli, is one of the reasons that
cause hepatocarcinogenesis (94, 95). Frontiers in Oncology | www.frontiersin.org Hydrogen Gas Acts Synergistically With
Thermal Therapy py
Recently, one study found that hydrogen might enhance the effect
of photothermal therapy. Zhao et al. designed the hydrogenated
Pd nanocrystals (named as PdH0.2) as multifunctional hydrogen
carrier to allow the tumor-targeted delivery (due to 30 nm
cubic Pd nanocrystal) and controlled release of bio-reductive
hydrogen (due to the hydrogen incorporated into the lattice of
Pd). As shown in this study, hydrogen release could be adjusted
by the power and duration of near-infrared (NIR) irradiation. Treatment of PdH0.2 nanocrystals plus NIR irradiation lead
to higher initial ROS loss in cancer cells, and the subsequent
ROS rebound was also much higher than that in normal
cells, resulting in more apoptosis, and severely mitochondrial
metabolism inhibition in cancer cells but not in normal cells. The combination of PdH0.2 nanocrystals with NIR irradiation Hydrogen Gas Suppresses Tumor Growth
Not only working as an adjuvant therapy, hydrogen gas can also
suppress tumor and tumor cells growth. As shown in Wang et al.’s study, on lung cancer cell
lines A549 and H1975 cells, hydrogen gas inhibited the cell
proliferation, migration, and invasion, and induced remarkable
apoptosis as tested by CCK-8, wound healing, transwell assays August 2019 | Volume 9 | Article 696 4 Li et al. Hydrogen Gas in Cancer Treatment TABLE 1 | The Summary of various formulation, application, mechanisms of H2 in cancer treatment. Hydrogen Gas Acts Synergistically With
Thermal Therapy As a hydroxyl radical and peroxynitrite scavenger, and
due to its anti-inflammatory effects, hydrogen gas may work to
prevent/relieve the adverse effects caused by chemotherapy and
radiotherapy without compromise their anti-cancer potential (as
summarized in Table 1 and Figure 1). Hydrogen gas may also
work alone or synergistically with other therapy to suppress
tumor growth via inducing apoptosis, inhibiting CSCs-related
and cell cycle-related factors, etc. (summarized in Table 1). More importantly, in most of the research, hydrogen gas has
demonstrated safety profile and certain selectivity property to
cancer cells over normal cells, which is quite pivotal to clinical
trials. One clinical trials (NCT03818347) is now undergoing to
study the hydrogen gas in cancer rehabilitation in China. Due to its physicochemical characteristics, the use of hydrogen
gas has been strictly limited in hospital and medical facilities and
laboratories. Li et al. designed a solidified hydrogen-occluding-
silica (H2-silica) that could stably release molecular hydrogen
into cell culture medium. H2-silica could concentration-
dependently inhibit the cell viability of human esophageal
squamous cell carcinoma (KYSE-70) cells, while it need higher
dose to suppress normal human esophageal epithelial cells
(HEEpiCs), indicating its selective profile. This effect was further
confirmed by apoptosis and cell migration assay in these two
cell lines. Mechanistic study revealed that H2-silica exerted its
anticancer via inducing H2O2 accumulation, cell cycle arrest,
and apoptosis induction mediated by mitochondrial apoptotic
pathways (72). By far, several delivery methods have proved to be available
and convenient, including inhalation, drinking hydrogen-
dissolved water, injection with hydrogen-saturated saline and
taking a hydrogen bath (101). Hydrogen-rich water is non-
toxic, inexpensive, easily administered, and can readily diffuse
into tissues and cells (102), cross the blood-brain barrier (103),
suggesting its potential in the treatment of brain tumor. Further
portable devices that are well-designed and safe enough will
be needed. By far, several delivery methods have proved to be available
and convenient, including inhalation, drinking hydrogen-
dissolved water, injection with hydrogen-saturated saline and
taking a hydrogen bath (101). Hydrogen-rich water is non-
toxic, inexpensive, easily administered, and can readily diffuse
into tissues and cells (102), cross the blood-brain barrier (103),
suggesting its potential in the treatment of brain tumor. Further
portable devices that are well-designed and safe enough will
be needed. However, regarding its medicinal properties, such as dosage
and administration, or possible adverse reactions and use
in specific populations, less information is available. Hydrogen Gas Acts Synergistically With
Thermal Therapy Formulation
Application
Mechanism
References
H2-rich water
Prevention of lung injury induced by gefitinib
Inflammatory cytokines and oxidative stress inhibition
(80)
Prevention of nephrotoxicity induced by cisplatin
Oxygen radicals elimination
(83, 84)
Reversal of mortality and body-weight loss caused by
cisplatin
ROS and apoptosis inhibition
(85)
Amelioration of liver toxicity induced by mFOLFOX6
regimen
Oxidative stress inhibition
(51)
Reversal of skin damage established by 44 Gy electronic
beam
Inflammatory cytokines and oxidative stress inhibition
(48)
Amelioration of gastrointestinal toxicity induced by
radiotherapy
miR-1968-5p up-regulation
(92)
Improving the quality of life
Antioxidant activity
(93)
Renal injury prevention and tumor growth suppression
Inflammatory cytokines and oxidative stress inhibition
(66)
Tumor incidence and growth suppression
Inflammatory cytokines and oxidative stress inhibition,
apoptosis induction
(96)
Cancer stem cells inhibition
CSCs properties and angiogenesis inhibition
(99)
H2-rich saline
Amelioration of cardiac dysfunction induced by
doxorubicin
Inflammatory cytokines, ROS and apoptosis inhibition
(71)
Damage of ovarian cortex induced by cisplatin
Nrf2 pathway stimulation
(86)
H2 inhalation
Reversal of toxicity to kidney caused by cisplatin
ROS and apoptosis inhibition
(85)
Tumor growth suppression
Cell cycle arrest and apoptosis induction
(98)
Glioblastoma growth inhibition and survival rate
enhancement
Inhibition of CSCs properties and induction of glioma
stem-like cell (GSC) differentiation
(100)
H2 Pd
nanocrystals
Synergistic effect with thermal therapy
ROS provoking
(52)
H2-silica
Cancer cell viability inhibition
H2O2 induction, cell cycle arrest, and apoptosis
induction
(72) H2 Pd
nanocrystals
H2-silica FIGURE 1 | Hydrogen in cancer treatment. August 2019 | Volume 9 | Article 696 5 Frontiers in Oncology | www.frontiersin.org Hydrogen Gas in Cancer Treatment Li et al. and flow cytometry. Hydrogen gas arrested the cell cycle at
G2/M stage on both cell lines via inhibiting the expression of
several cell cycle regulating proteins, including Cyclin D1, CDK4,
and CDK6. Chromosomes 3 (SMC3), a complex required for
chromosome cohesion during the cell cycle (97), was suppressed
by hydrogen gas via ubiquitinating effects. Importantly, in vivo
study showed that under hydrogen gas treatment, tumor growth
was significantly inhibited, as well as the expression of Ki-67,
VEGF and SMC3. These data suggested that hydrogen gas could
serve as a new method for the treatment of lung cancer (98). inflammatory disease, neurodegenerative disorders, and cancer
(17, 60). FUNDING This
work
was
supported
in
part
by
grants
from
the
Natural
Science
Foundation
of
Guangdong
Province
(2018A030313987)
and
Traditional
Chinese
Medicine
Bureau of Guangdong Province (20164015 and 20183009)
and Science and Technology Planning Project of Guangdong
Province (2016ZC0059). This
work
was
supported
in
part
by
grants
from
the
Natural
Science
Foundation
of
Guangdong
Province
(2018A030313987)
and
Traditional
Chinese
Medicine
Bureau of Guangdong Province (20164015 and 20183009)
and Science and Technology Planning Project of Guangdong
Province (2016ZC0059). Hydrogen Gas Acts Synergistically With
Thermal Therapy Its
mechanism, target, indications are also not clear, further study
are warranted. Recently, hydrogen gas was found to inhibit cancer stem
cells (CSCs). Hydrogen gas reduced the colony formation and
sphere formation of human ovarian cancer cell lines Hs38.T
and PA-1 cells via inhibiting the proliferation marker Ki67, stem
cell markers CD34, and angiogenesis. Hydrogen gas treatment
significantly inhibited the proliferation, invasion, migration of
both Hs38.T and PA-1 cells. More important, inhalation of
hydrogen gas inhibited the tumor volume significantly as shown
in the Hs38.T xenografted BALB/c nude mice model (99). DISCUSSION We thank Miss Ryma Iftikhar, Dhiviya Samuel, Mahnoor Shamsi
(St. John’s University), and Mr. Muaz Sadeia for editing and
revising the manuscript. Hydrogen gas has been recognized as one medical gas that
has potential in the treatment of cardiovascular disease, AUTHOR CONTRIBUTIONS SL, XW, JZ, and KP: conceptualization. SL, RL, XS, XL, XZ, JZ,
and KP: writing. SL, RL, and XS: revising. SL, XW, JZ, and KP: conceptualization. SL, RL, XS, XL, XZ, JZ,
and KP: writing. SL, RL, and XS: revising. Another recent study also confirmed the effects of hydrogen
gas in suppressing glioblastoma (GBM), the most common
malignant brain tumor. In vitro study indicated that hydrogen
gas inhibited several markers involved in stemness, resulting in
the suppression of sphere formation, cell migration, invasion, and
colony formation of glioma cells. By inhaling hydrogen gas (67%)
1 h, 2 times per day, the GBM growth was significantly inhibited,
and the survival rate was improved in a rat orthotopic glioma
model, suggesting that hydrogen might be a promising agent in
the treatment of GBM (100). 3. Vitek L, Gbelcova H, Muchova L, Vanova K, Zelenka J, Konickova
R, et al. Antiproliferative effects of carbon monoxide on pancreatic
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13. doi: 10.1097/MCG.0000000000001062 Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest. Copyright © 2019 Li, Liao, Sheng, Luo, Zhang, Wen, Zhou and Peng. This is an
open-access article distributed under the terms of the Creative Commons Attribution
License (CC BY). The use, distribution or reproduction in other forums is permitted,
provided the original author(s) and the copyright owner(s) are credited and that the
original publication in this journal is cited, in accordance with accepted academic
practice. No use, distribution or reproduction is permitted which does not comply
with these terms. 95. Ma C, Zhang Q, Greten TF. Non-alcoholic fatty liver disease promotes
hepatocellular carcinoma through direct and indirect effects on hepatocytes. Febs J. (2018) 285:752–62. doi: 10.1111/febs.14209 Copyright © 2019 Li, Liao, Sheng, Luo, Zhang, Wen, Zhou and Peng. This is an
open-access article distributed under the terms of the Creative Commons Attribution
License (CC BY). The use, distribution or reproduction in other forums is permitted,
provided the original author(s) and the copyright owner(s) are credited and that the
original publication in this journal is cited, in accordance with accepted academic
practice. No use, distribution or reproduction is permitted which does not comply
with these terms. 96. Kawai D, Takaki A, Nakatsuka A, Wada J, Tamaki N, Yasunaka T, et al. Hydrogen-rich water prevents progression of non-alcoholic steatohepatitis
and accompanying hepatocarcinogenesis in mice. Hepatology. (2012)
56:912–21. doi: 10.1002/hep.25782 August 2019 | Volume 9 | Article 696 Frontiers in Oncology | www.frontiersin.org 9
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A Functional Spiking Neural Network of Ultra Compact Neurons
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To cite this version: Pablo Stoliar, Olivier Schneegans, Marcelo Rozenberg. A Functional Spiking Neural Network of Ultra
Compact Neurons. Frontiers in Neuroscience, 2021, 15, 10.3389/fnins.2021.635098. hal-03323460 HAL Id: hal-03323460
https://enpc.hal.science/hal-03323460v1
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teaching and research institutions in France or
abroad, or from public or private research centers. ORIGINAL RESEARCH
published: 25 February 2021
doi: 10.3389/fnins.2021.635098 Edited by:
Paolo Milani,
University of Milan, Italy
Reviewed by:
Anup Das,
Drexel University, United States
Matteo Mirigliano,
University of Milan, Italy
*Correspondence:
Marcelo J. Rozenberg
marcelo.rozenberg@universite-paris-
saclay.fr
†These authors have contributed
equally to this work Edited by:
Paolo Milani,
University of Milan, Italy Edited by:
Paolo Milani,
University of Milan, Italy
Reviewed by:
Anup Das,
Drexel University, United States
Matteo Mirigliano,
University of Milan, Italy Reviewed by:
Anup Das,
Drexel University, United States
Matteo Mirigliano,
University of Milan, Italy *Correspondence:
Marcelo J. Rozenberg
marcelo.rozenberg@universite-paris-
saclay.fr †These authors have contributed
equally to this work †These authors have contributed
equally to this work Specialty section:
This article was submitted to
Neuromorphic Engineering,
a section of the journal
Frontiers in Neuroscience Specialty section:
This article was submitted to
Neuromorphic Engineering,
a section of the journal
Frontiers in Neuroscience
Received: 29 November 2020
Accepted: 22 January 2021
Published: 25 February 2021
Citation:
Stoliar P, Schneegans O and
Rozenberg MJ (2021) A Functional
Spiking Neural Network of Ultra
Compact Neurons. Front. Neurosci. 15:635098. doi: 10.3389/fnins.2021.635098 Keywords:
spiking
neural
networks,
neuron
models,
leaky-integrated-and-fire,
artificial
intelligence,
neuromorphic electronic circuits, neuromorphic computers, Jeffress model A Functional Spiking Neural Network
of Ultra Compact Neurons
Pablo Stoliar1†, Olivier Schneegans2† and Marcelo J. Rozenberg3*†
1 National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan, 2 Université Paris-Saclay,
Sorbonne Université, CentraleSupélec, CNRS, Laboratoire de Génie Électrique et Électronique de Paris, Gif-sur-Yvette,
France, 3 Université Paris-Saclay, CNRS, Laboratoire de Physique des Solides, Orsay, France We demonstrate that recently introduced ultra-compact neurons (UCN) with a minimal
number of components can be interconnected to implement a functional spiking
neural network. For concreteness we focus on the Jeffress model, which is a classic
neuro-computational model proposed in the 40’s to explain the sound directionality
detection by animals and humans. In addition, we introduce a long-axon neuron,
whose architecture is inspired by the Hodgkin-Huxley axon delay-line and where
the UCNs implement the nodes of Ranvier. We then interconnect two of those
neurons to an output layer of UCNs, which detect coincidences between spikes
propagating down the long-axons. This functional spiking neural neuron circuit with
biological relevance is built from identical UCN blocks, which are simple enough
to be made with off-the-shelf electronic components. Our work realizes a new,
accessible and affordable physical model platform, where neuroscientists can construct
arbitrary mid-size spiking neuronal networks in a lego-block like fashion that work in
continuous time. This should enable them to address in a novel experimental manner
fundamental questions about the nature of the neural code and to test predictions
from mathematical models and algorithms of basic neurobiology research. The present
work aims at opening a new experimental field of basic research in Spiking Neural
Networks to a potentially large community, which is at the crossroads of neurobiology,
dynamical systems, theoretical neuroscience, condensed matter physics, neuromorphic
engineering, artificial intelligence, and complex systems. INTRODUCTION Received: 29 November 2020
Accepted: 22 January 2021
Published: 25 February 2021 The basic understanding of the dynamical behavior of Spiking Neural Networks (SNN) in
Neuroscience is the focus of intense research. There are many relevant and pressing questions that
are coming into focus, for instance, breaking the neural code what is the nature of the neural code? how information is encoded and transmitted with spikes from one part of the brain to another? how does that depend on network topology? are brain networks close to a critical or a chaotic
state? what is the robustness of networks to chaos? how neurons may synchronize to form waves? how dynamical memories are realized and sustained? and many others (Korn and Faure, 2003;
Rabinovich et al., 2006). These issues are being studied either by in vivo and in vitro experiments in
neurobiology (Reyes, 2003; Yin et al., 2018), or theoretically by means of numerical simulations of Citation: Examples
of those digital neuron chips TrueNorth, Loihi, SpiNNaker, etc
(Furber, 2016; Thakur et al., 2018). These systems are multi-
core chips with neuromorphic architecture, that is, they have
vast numbers of relatively small memory and processing units,
which are densely interconnected. We may also include in this
category the recent implementations using Field Programmable
Gate Arrays (FPGA), which are making fast progress (Yang
et al., 2015, 2018, 2019, 2020). On the other hand, a qualitatively
different electronic engineering approach is aimed to design
CMOS VLSI circuits, called silicon neurons, which implement the
neuron models directly in hardware (Indiveri et al., 2011). Those
neurons can then be interconnected off-chip to form networks
by means, for instance, of the address event representation
(AER) (Liu et al., 2015). In this approach, no actual spikes
are transmitted between neurons, but it is the information of
a firing event which is sent using the AER protocol between
neuron addresses. One prominent example of a CMOS VLSI
system is the BrainScaleS chip, which implement AdEx neurons
(Schemmel et al., 2010) and further examples are discussed in
Furber (2016); Thakur et al. (2018). In those approaches, the
goal is to implement extremely large numbers of neurons (and
synapses) to achieve the computing capacity of a brain. Some
of their most significant challenges are to achieve low power
dissipation and miniaturization. To reduce the power, one may
work with transistors in the subthreshold regime (Mahowald
and Douglas, 1991; Benjamin et al., 2014), however, additional
issues arise in that case, such as device variability (Indiveri et al.,
2011). Regarding the neuron circuit miniaturization, reducing
its physical dimensions remains a challenge due to the relative
large size of the capacitor that is required to represent the voltage
of the membrane. y
Our methodology is built around a recently introduced
electronic neuron circuit, the ultra-compact neuron (UCN)
(Rozenberg et al., 2019). This UCN, like Mott neurons exploits a
memristive behavior, however, in contrast to those it is not based
on a quantum material but on a conventional electronics device,
the thyristor. So in this regard, the UCN may be considered a
silicon neuron model. The silicon neurons vary in their degree
of circuit complexity, for instance realizations of the AdEx model
may require tens of transistors (Indiveri et al., 2011). Citation: February 2021 | Volume 15 | Article 635098 1 Frontiers in Neuroscience | www.frontiersin.org Functional SNN of Ultra-Compact Neurons Stoliar et al. mathematical models of neural networks (Izhikevich and
Edelman, 2008; Stimberg et al., 2019). In the first case, the
neurobiological experiments are technically challenging and,
evidently, one cannot systematically modify the neural networks. In the second, the results on mathematical modeling may always
be questioned, as a peculiar result that may depend on the
assumptions made. For instance, it may be hard to assess if
the relative ubiquity of chaotic behavior (Korn and Faure, 2003;
Rudolph and Destexhe, 2007; Nobukawa et al., 2018) is of
biological relevance. Moreover, in a well-known study, Izhikevich
and Edelman (2008) observed the surprising result that the
suppression of a single neuron out of an ensemble of a thousand
spiking neurons may change the state of the entire network. electric spike when they are driven across the insulator to metal
transition (Janod et al., 2016). Despite intense activity, this field
of research is emerging and currently proposed devices are still
individual single neurons (Stoliar et al., 2017; Yi et al., 2018). The main challenges ahead are to achieve a reliable control
of the materials and the understanding of their fundamental
physical behavior (del Valle et al., 2019), before actual circuits
may be implemented. In the context of the approaches that deal with SNN systems
that we described above, our present methodology shares features
of many but is qualitatively different to all of them. The main
goal of the present work is to bring to the research community a
novel way to build and study SNNs of unprecedented simplicity,
which opens a new way to do experimental work in basic
neuroscience. We propose a methodology to build general neural
circuits of a priori arbitrary topology, where spiking neurons
are in direct interaction and are interconnected as lego-like
blocks. To illustrate this point, we shall adopt a classic model of
neuroscience: the Jeffress model of binaural detection of sound
directionality (Jeffress, 1948). In order to better position our current work with respect to
other approaches, we should also briefly describe the current
efforts on applied research in Neuromorphic Computational
Electronic Engineering for artificial intelligence (AI). That field
can be roughly divided in two big areas. One aims to implement
dedicated computer processors, which are optimized to run
algorithms based on mathematical models of neurons. Frontiers in Neuroscience | www.frontiersin.org Citation: The UCN,
in contrast, counts with a small number of components so
it can be considered a compact neuron model, following the
terminology introduced by Indiveri et al. (2011). Moreover, the
UCN can be termed ultra-compact as it requires a minimal
number of components. In fact, to realize a basic circuit of a
leaky-integrate-and-fire (LIF) neuron model, the UCN requires,
like most other silicon neurons, a capacitor to integrate charge,
and a resistor to mimic the leaky feature. However, the novelty
of the UCN is that the fire functionality can be realized by a
single silicon controlled rectifier (SCR or thyristor), which is
a conventional electronic device introduced in 1956. The key
insight is to realize that the I-V characteristics of the SCR display
memristive features analogous to that of Mott materials, which
enable the neuromorphic electric spiking behavior. However, the
SCR present the advantage that they are available off-the-shelf to
implement artificial neurons (Rozenberg et al., 2019), avoiding
the need to deal with the complexities of Mott quantum material
fabrication and control. We should also mention that the UCN
is not restricted to the LIF model as it can be easily extended
to realize a large variety of biologically relevant spiking neuron
models (Stoliar et al., 2020). We should make clear that the present UCN based electronic
circuit methodology is not aimed at competing with engineering
implementations for AI, therefore, speed, dissipation, and
physical size is not our immediate concern. Here, we explicitly
demonstrate that one can make a crucial step up and go
from the level of a single UCN spiking, to a functional
and biologically relevant circuit of more than ten neurons. The limitations of silicon neurons may be potentially
overcome by exploiting the neuromorphic functionalities of
quantum materials (del Valle et al., 2018). For instance, phase
change materials (Tuma et al., 2016), which accumulate phase
instead of charge. Another notable example are the Mott
insulators, whose memristive properties permit the firing of an February 2021 | Volume 15 | Article 635098 2 Functional SNN of Ultra-Compact Neurons Stoliar et al. part of the circuit, which generates the action potentials, only
requires three basic elements (see orange region in Figure 1A). Similar to most silicon neuron implementations (Indiveri et al.,
2011), it has a membrane capacitor (C) for the integrate function,
a resistor (R1 + R2) for the leaky function. Citation: However, in contrast
to conventional CMOS implementations, the fire function is
realized by a SCR. The fire function is most easily understood by
noting that the SCR, which is a pnpn device, can be considered as
a diode with a threshold (see Figure 1B). Thus, it is normally off
with a large resistance RHI, bigger than leak resistors (R1 + R2)
so that when current inputs the neuron, the capacitor gets
(leaky) charged, with time constant τ = (R1 + R2)C. The fire
occurs when the SCR suddenly commutes to the low resistance
value RLO. This happens when the SCR gate voltage reaches the
threshold value Vth, which is a parameter of the SCR. Thus, the
fire event can be easily tuned with the resistive voltage divider,
such that the condition Vth = Vc R2/(R1 + R2) is met. Or in
other words, this condition sets the critical value that Vc needs
to reach so that a spike is generated. At this point the resistance
of the SCR collapses, as its pn diode-like junctions become
forward polarized. Since the SCR resistance becomes much
smaller than the leak pair (R1 + R2), the membrane capacitor
rapidly discharges through it, producing a spike of voltage, or
action potential, on the small resistor R3. The time-scale for the
duration of the spike is ∼(R3 + RLO)C. The action potential
terminates when the current spike decreases beneath a value
Ihold, which is another parameter of the SCR. Thus, we see that
this hysteresis in the resistance, or memristive property, of the
SCR is the key feature behind the simplicity of the UCN. One qualitative difference with respect to neurocomputing
engineering implementations is that the UCN network operates
in real continuous time, with time scales in principle compatible
with those of biological systems. The aim of our work is a
proof-of-concept for a novel, flexible, and affordable platform
to construct spiking neural networks. This methodology can
be further scaled up to hundreds or possibly thousands of
neurons, which can be implemented with low-cost printed
circuits boards (PCB). This order of magnitude in the number
of neurons is sufficient to study questions of basic neuroscience,
such as those that we mentioned in the beginning. The Ultra Compact Neuron Circuit The Ultra Compact Neuron Circuit
The UCN circuit can display electric spiking behavior analogous
to a biological neuron and constitutes the basic building block of
our methodology. In (Rozenberg et al., 2019) we demonstrated
a key feature of UCN, namely, that one (upstream) UCN
block can be directly connected to a second (downstream)
UCN block, and that the spiking behavior in the former can
elicit spiking behavior in the latter. In the present work we
go beyond, and show that arbitrary functional circuits can be
implemented, rather straightforwardly, using the UCNs as a
lego-like constructive blocks without any need to use of AER
protocols. Our approach should be viewed as a novel, simple and
flexible experimental platform for neuroscience research in SNN,
alternative and complementary to numerical simulations, in vivo
and in vitro biological studies, and electronic engineering for
artificial intelligence applications. We shall next briefly describe
the basic behavior of the UCN, for the sake of completeness. Further details can be found in Rozenberg et al. (2019), Stoliar
et al. (2020). Citation: In other
words, we aim at opening a new experimental field of
basic research in Spiking Neural Networks to a potentially
large community, which is at the crossroads of neurobiology,
dynamical systems, theoretical neuroscience, condensed matter
physics, neuromorphic engineering, artificial intelligence, and
complex systems. The paper is organized as follows: We shall first describe
the basic features of the UCN circuit and of the Jeffress model
SNN. We shall then show how UCN blocks can be used to
implement the network, proceeding in two steps: Firstly, we shall
construct a novel long-axon neuron, which will serve as the delay-
line of the Jeffress model. Secondly, we shall combine two such
long-axon neurons with a layer of output neurons that detect
the coincidence of spikes propagating along the delay lines. We
shall then demonstrate the neuro-computational functionality of
sound directionality detection. Finally, we discuss the prospects
of adopting the UCN as a general and easy to implement spiking
neuron lego-like block for experimental electronic neuroscience
and neuro-engineering. Finally the action potential spike needs to be strengthened so
it can drive a downstream UCN connected to its output. This is
simply achieved by a transistor and a voltage source. The green
region in Figure 1A depicts the synaptic connections or dendritic
inputs of the neuron. For the Jeffress model implementation
we shall only need either one or two inputs (see Figure 1C). In the Supplementary Material we also provide further details
on the circuit implementation of the UCN units, along with
the list of off-the-shelf electronic components. Importantly for
the sake of adopting the UCN units as a physical platform for
arbitrary SNNs, we also present simulations on their fan-in/fan-
out performance. Frontiers in Neuroscience | www.frontiersin.org The Jeffress Model for Detection of
Sound Directionality We shall consider the Jeffress model (Jeffress, 1948) as a
prototypical SNN with a biological functional to show how such
a system can be implemented with the UCN blocks that we just
described. It is interesting to mention that this model was also
the focus of the pioneering work in neuromorphic engineering
by Lazzaro and Mead (1989), though more than 30 years
ago and following a qualitatively different implementation
based on CMOS. Subsequent work along those lines was
done by Bhadkamkar and Fowler (1993) who developed a
CMOS chip with about 50 thousand transistors working in
the subthreshold regime (Bhadkamkar, 1994). These lines of
work are and illustration of the electronic engineering approach In Figure 1, we show the electronic circuit of the UCN
(Rozenberg et al., 2019). The UCN is a minimal physical
implementation
of
a
leaky-integrate-and-fire
model
that
generates action potentials. This model is minimal since the key February 2021 | Volume 15 | Article 635098 Frontiers in Neuroscience | www.frontiersin.org Frontiers in Neuroscience | www.frontiersin.org 3 Functional SNN of Ultra-Compact Neurons Stoliar et al. FIGURE 1 | (A) Electronic circuit of the UCN unit. The input (green) is shown for the case of two dendrites. In orange we depict the section of the circuit where the
ction potentials are generated (see text for description). (B) Schematic I-V characteristics of the SCR and electronic symbol of the component. Note the hysteretic
ehavior similar to that observed in VO2 Mott neurons (del Valle et al., 2018). (C) Symbol of the artificial neuron for the case of a one-input (top) and two-input
bottom) UCN, which are used as lego-like building blocks (the list of the electronic components is provided in the Supplementary Material). FIGURE 2 | (A) The sound is incident with an angle α on the azimuthal plane that is transduced into an interaural time difference (ITD) for the excitation of the left and
ght ears auditory system. (B) The system generates spikes at the L and R input neurons that propagate down their respective long axons (delay lines). The ITD is
mapped onto the position of output neurons that detect the coincidence of spikes propagating down the long axons. Two spikes arriving in coincidence to the green
euron when α = 0 and ITD = 0. FIGURE 1 | (A) Electronic circuit of the UCN unit. The input (green) is shown for the case of two dendrites. The Jeffress Model for Detection of
Sound Directionality In orange we depict the section of the circuit where the
action potentials are generated (see text for description). (B) Schematic I-V characteristics of the SCR and electronic symbol of the component. Note the hysteretic
behavior similar to that observed in VO2 Mott neurons (del Valle et al., 2018). (C) Symbol of the artificial neuron for the case of a one-input (top) and two-input
(bottom) UCN, which are used as lego-like building blocks (the list of the electronic components is provided in the Supplementary Material). FIGURE 1 | (A) Electronic circuit of the UCN unit. The input (green) is shown for the case of two dendrites. In orange we depict the section of the circuit where the
action potentials are generated (see text for description). (B) Schematic I-V characteristics of the SCR and electronic symbol of the component. Note the hysteretic
behavior similar to that observed in VO2 Mott neurons (del Valle et al., 2018). (C) Symbol of the artificial neuron for the case of a one-input (top) and two-input
(bottom) UCN, which are used as lego-like building blocks (the list of the electronic components is provided in the Supplementary Material). FIGURE 2 | (A) The sound is incident with an angle α on the azimuthal plane that is transduced into an interaural time difference (ITD) for the excitation of the left and
right ears auditory system. (B) The system generates spikes at the L and R input neurons that propagate down their respective long axons (delay lines). The ITD is
mapped onto the position of output neurons that detect the coincidence of spikes propagating down the long axons. Two spikes arriving in coincidence to the green
neuron when α = 0 and ITD = 0. FIGURE 2 | (A) The sound is incident with an angle α on the azimuthal plane that is transduced into an interaural time difference (ITD) for the excitation of the left and
right ears auditory system. (B) The system generates spikes at the L and R input neurons that propagate down their respective long axons (delay lines). The ITD is
mapped onto the position of output neurons that detect the coincidence of spikes propagating down the long axons. Two spikes arriving in coincidence to the green
neuron when α = 0 and ITD = 0. and the challenges that subthreshold systems may present, as
described before. The Jeffress Model for Detection of
Sound Directionality front arrival at the left (L) and right (R) auditory systems
provokes the excitation of the respective input neurons. These
neurons send spikes that propagate down their long-axons,
similarly as a signal down a delay-line (Figure 2B). An layer of
output neurons detects the (time) coincidence of the propagating
spikes. Thus they provide a neurocomputation or transduction
of the incidence angle into an electric signal encoded by the
excitation of the output neuron array. The Jeffress model was introduced in 1948 to describe the
brain’s neural system for detection of sound directionality on
the azimuthal plane (Jeffress, 1948). It makes a mapping of
sound angle to neuronal space location. This is achieved by
exploiting the time difference of the arrival of sound to the
ear, and translating that difference into neuronal location by
means of long-axon neurons that act as homogeneously graded
delay lines. Neurons, which receive inputs from these graded
axonal lines, act as coincidence detectors which fire when
spikes simultaneously arrive from the two sides (Konishi, 1993). Remarkably, the relevance of the Jeffress model has survived
the ongoing revolution in experimental neuroscience (Joris
et al., 1998; Burger et al., 2011; Cariani, 2011). Moreover, it
has received some striking validations of its most basic aspects
through the neuronal mapping of the auditory system of birds
(Young and Rubel, 1983). As one can see from the diagram of Figure 2B, the
Jeffress model has two type of neurons: the input neurons are
characterized by a long axon and the output neurons have two
dendritic inputs to detect simultaneous spikes (i.e., coincidences)
occurring a two given points on the L and R axons. Frontiers in Neuroscience | www.frontiersin.org The Long-Axon Neuron These two “soma + axon-hillock” neurons may, in principle,
be setup to fire upon any desired excitation threshold, such as
depending on the intensity level, frequency, etc. Here, again for
the sake of simplicity, we set them to fire upon arrival of a single
above-threshold voltage pulse. potential leads to the local action potential generation in a given
segment of the axon. The propagation of the signal follows from
the fact that one action potential spike at one axon segment
induces a successive spike in the neighboring segment. The
propagation is initiated in the axon hillock of the cell soma,
which is the location where the soma connects to the axon. In this manner, the spike signal travels down the axon at
a constant speed. Finally, we may also mention that from a SNN architecture
point of view, the Jeffress model is neither a simple feed-forward
nor a recurrent NN. This can be appreciated in Figure 4C, where
we show the network architecture as layers. We can observe that
unlike feed-forward networks there is communication within a
given layer (the middle one), and also unlike recurrent networks,
there are no connections going backward. Since in each segment an action potential is generated, we may
use one UCN to describe each segment, and then build the axon
simply by means of a chain of UCNs. The key feature is that the
parameters of each UCN have to be chosen such that a single
spike of a given segment is sufficient to reliably elicit a spike in
the subsequent neighboring segment (Rozenberg et al., 2019). g
g
g
g
From the previous discussion, we may now introduce a
long-axon neuron model that emulates the mechanism of spike
propagation described before. The model is schematically shown
in Figure 3. It has an initial UCN unit that represents the soma-
plus-axon-hillock (S0), followed by a succession of UCN units
that represent the segments of the long axon (S1, S2, S3,. . .). As
in the HH model of a neuron axon, each one of those blocks
generates an action potential and induce a subsequent one on the
neighboring downstream segment. Thus, the long-axon neuron
is built as a one dimensional chain of spiking UCN units. For
simplicity, we adopt all the UCN blocks to be identical and set
to fire with just one single incoming pulse. The Long-Axon Neuron To implement the long-axon neuron circuit it is useful to recall
that the celebrated Hodgkin-Huxley (HH) model was introduced
to precisely describe the propagation of an action potential
along the axon of a neuron (Hodgkin and Huxley, 1952). It
was formulated as a set of differential equations of classic cable
theory. The parameters of the equation are the voltage-gated
conductance and capacitances per unit length. The non-linear
dependence of the ion channels conductance with the membrane In Figure 2 we show a schematic diagram of the Jeffress model
of binaural detection of sound direction. The sound is assumed
to originate along the azimuthal direction, thus depending on
the incidence angle the sound-wave front will arrive with an
interaural time difference (ITD) to the ears (Figure 2A). The February 2021 | Volume 15 | Article 635098 Frontiers in Neuroscience | www.frontiersin.org 4 Functional SNN of Ultra-Compact Neurons Stoliar et al. FIGURE 3 | Top: Schematic long-axon neuron. Bottom: Implementation of a
long-axon neuron by a uni-dimensional network of UCN units. S0 is the
soma + axon-hillock, S1, S2, . . . are axon segments. The list of components
of the UCN blocks are provided in the Supplementary Material. at their two dendritic inputs. The three output neurons depicted
in the diagram of Figure 4 are labeled “left,” “center,” and
“right” as they encode for sound signals incoming from those
respective sectors. The scheme of the circuit can be easily scaled up to increase
the system’s resolving power of the azimuthal angle. In the
Supplementary Material we provide an explicit realization
scaling up the number of UCNs in a long-axon neuron by means
of a realistic numerical simulation. However, in the following we
shall restrict ourselves to implement a small network where the
behavior of each neuron can be individually traced. FIGURE 3 | Top: Schematic long-axon neuron. Bottom: Implementation of a
long-axon neuron by a uni-dimensional network of UCN units. S0 is the
soma + axon-hillock, S1, S2, . . . are axon segments. The list of components
of the UCN blocks are provided in the Supplementary Material. In principle, a transducer, such as the cochlea in humans,
converts the incoming sound-wave front into input excitations
to the network. The interaural time difference of the wave front
results in a time delay between the spikes generated by L0 and
R0. RESULTS We now turn to describe the behavior of our neuromorphic
model. We begin with the long-axon neuron. In Figure 5 we show
the experimental data of the spike propagation along the axon. We observe that a spike travels down the axon as each pulse at a
given segment unit induces a subsequent pulse at its downstream
neighbor. For the chosen values of the circuit components (see
Supplementary Material) the pulse moves along the axon at a
constant rate of approximately 0.045 segment/µs. This rate may
be further increased by decreasing the capacitance of the UCN. We may now present the experimental data for the full Jeffress
model implementation. This is shown in Figure 6, where we
show two examples of the system’s behavior upon arrival of an
input to the left and to right ears, with a given ITD. We shall
begin with the case of no delay between arrivals, i.e., ITD = 0. This is shown in the Figure 6A. We can observe that the action
potentials propagate through each long-axon neuron in opposite
directions and at the same speed (first and second panels from
the top). Indeed, they arrive to the last segment of each long-axon
at about the same time 80 µs. The small differences between the
timings and propagations between the two long-axon neurons is
due to the small variability among electronic components, which
is specified by the maker. An important point to make here is that
the representation of time in SNNs is a non-trivial task (Liu et al.,
2015). It usually requires off-chip communication and an AER
protocol. In contrast, our circuit implementation is operating The Long-Axon Neuron However, it would be
easy to adapt the first “soma” block S0 to fire a spike upon any
other input signal of our choice. Frontiers in Neuroscience | www.frontiersin.org The Jeffress Model SNN Circuit We can now use the long-axon neurons to implement the
neural network of the Jeffress model that we show in Figure 4. We adopt one long-axon neuron to represent the left ear
input and another one for the right ear input. In the former
the spikes propagate from left to right and in the latter in
opposite direction. In addition, we need to interconnect the
long axons of these input neurons with an array of output
neurons that detect the coincidences. We implement the output
array also using UCN units, as shown in Figure 4. In this
case the UCN blocks are tuned to fire upon arrival of two
overlapping input signals, i.e., to detect coincidences of pulses February 2021 | Volume 15 | Article 635098 Frontiers in Neuroscience | www.frontiersin.org 5 Functional SNN of Ultra-Compact Neurons Stoliar et al. FIGURE 4 | (A) Schematic neuron network of the Jeffress model. (B) Corresponding spiking neural network implemented with UCN blocks. (C) SNN architecture. The Jeffress model is neither a simple feed-forward nor a recurrent neural network due to the intra-layer connections. Also notice if the hidden layers were redefined,
there would be connections toward the output layer that need to skip layers. FIGURE 4 | (A) Schematic neuron network of the Jeffress model. (B) Corresponding spiking neural network implemented with UCN blocks. (C) SNN architecture. The Jeffress model is neither a simple feed-forward nor a recurrent neural network due to the intra-layer connections. Also notice if the hidden layers were redefined,
there would be connections toward the output layer that need to skip layers. FIGURE 5 | Left panels: Measured action potential spike signal propagating
along a 7-segment long-axon neuron (top right panel). The top left panel
VC0 has the voltage on the membrane capacitor C (see Figure 4). The signals
S0 to S7 correspond to the action potential spike voltage measured on the
resistor R3 at the exit of the SCR (see Figure 4). The corresponding panels on
the right have the output of the respective neurons. The logic “0” corresponds
to a physical voltage value of 5 V, and the “1” to 0 V. that the corresponding “center” neuron senses the arrival of the
pulses in coincidence and consequently increases its potential
that overcomes the threshold of ∼2 V and fires. The Jeffress Model SNN Circuit The fire event of
the output neuron OC that signals the detection of sound from
the central quadrant can be seen in the bottom panels of the
Figure 6A. We see a strong spike in the trace of OC, however,
another smaller one can also be observed for OR. These latter one
can be considered a residual effect that are rather natural to this
system’s bio-mimetic neural architecture. We will come back to
this point later on. In the panels of Figure 6B we show the case of a finite delay
arriving to the L and R ear input neurons. As can be seen in the
top panels, the approximate delay is of 40 µs, arriving earlier at
the “left” ear L0, which indicates that the sound would have come
from the left sector. From the propagation of the spikes depicted in these panels,
we observe that coincidence is occurring between UCN pair L3-
R1, which is detected by the output neuron OL. In this case, the
third panel shows the membrane potential of “center” and “right,”
and we see that they detect the passing of spikes through the
long-axons but not in coincidence. Hence their voltage initially
increases and then starts to leak. By the time the second pulse
is detected, although the VC has not yet fully relaxed, the new
increase is not enough to reach the threshold. The VC fall just
short of overcoming the 2 V value. Thus, the sole coincidence is
detected by the “left” output neuron OL, as can be seen in from
its firing event in the bottom panel of the Figure 6B. FIGURE 5 | Left panels: Measured action potential spike signal propagating
along a 7-segment long-axon neuron (top right panel). The top left panel
VC0 has the voltage on the membrane capacitor C (see Figure 4). The signals
S0 to S7 correspond to the action potential spike voltage measured on the
resistor R3 at the exit of the SCR (see Figure 4). The corresponding panels on
the right have the output of the respective neurons. The logic “0” corresponds
to a physical voltage value of 5 V, and the “1” to 0 V. Left panels: Measured action potential spike signal propagating FIGURE 5 | Left panels: Measured action potential spike signal propagating
along a 7-segment long-axon neuron (top right panel). Frontiers in Neuroscience | www.frontiersin.org The Jeffress Model SNN Circuit The top left panel VC0 has the voltage on the membrane capacitor C (see Figure 4). The signals
S0 to S7 correspond to the action potential spike voltage measured on the
resistor R3 at the exit of the SCR (see Figure 4). The corresponding panels on
the right have the output of the respective neurons. The logic “0” corresponds
to a physical voltage value of 5 V, and the “1” to 0 V. Comparing the input spikes and the output fire, we may notice
the existence of a small delay, which is indicated in the traces of
the output neurons OC in Figure 6A and of OL in Figure 6B
with small arrows. The delay is of a few microseconds and is
simply due to the time it takes for the output neuron to integrate
the coincident input signals at its dendrites and fire, namely, to
perform its neurocomputation. directly in real continuous-time. Moreover, the time-scales of our
model are compatible with biological neuron networks ones. As shown in the diagram of Figure 4, we see that the detection
of coincidences is conducted between the UCN pairs L3-R1, L2-
R2, and L1-R3. From the data in Figure 6A we can see that
the “crossing” of the propagating spikes is, as expected, between
the second segment of each long axon, namely, the pair L2-R2. The traces of the membrane potentials of the output neurons
is shown in the third panel from the top of Figure 6A. We see In Figure 7 we finally show the systematic behavior of the
output as a function of the time difference between the arrival
of pulses to the left and right “ears” (L0 and R0) or ITD. We
observe and approximately left-right symmetric behavior of the February 2021 | Volume 15 | Article 635098 Frontiers in Neuroscience | www.frontiersin.org 6 Functional SNN of Ultra-Compact Neurons Stoliar et al. FIGURE 6 | Neurocomputation of ITD. Left panels (A) ITD = 0 µs and right panels (B) ITD = 40 µs. Top four rows show the traces of the propagation of the spikes
along the right-ear delay line, from R0 to R3, measured at the respective unit outputs. The next four lines show the respective signals for the spikes propagating in
the left-ear delay line, in opposite direction. The mid panel show the membrane voltage VC for the three output neurons. The Jeffress Model SNN Circuit As expected for the symmetric case of
ITD = 0 µs (α = 0), we observe the build-up of the potential of the central neuron OC (blue). When this membrane potential overcomes the threshold it sets off an
output spike as can be seen in the trace of the output neurons in the last three rows, where we indicated an additional delay by the small arrows. The right hand
panels have the respective traces for the case of a finite ITD = 40 µs, where we observe that the coincidence was detected by the OL neuron (green). FIGURE 6 | Neurocomputation of ITD. Left panels (A) ITD = 0 µs and right panels (B) ITD = 40 µs. Top four rows show the traces of the propagation of the spikes
along the right-ear delay line, from R0 to R3, measured at the respective unit outputs. The next four lines show the respective signals for the spikes propagating in
the left-ear delay line, in opposite direction. The mid panel show the membrane voltage VC for the three output neurons. As expected for the symmetric case of
ITD = 0 µs (α = 0), we observe the build-up of the potential of the central neuron OC (blue). When this membrane potential overcomes the threshold it sets off an
output spike as can be seen in the trace of the output neurons in the last three rows, where we indicated an additional delay by the small arrows. The right hand
panels have the respective traces for the case of a finite ITD = 40 µs, where we observe that the coincidence was detected by the OL neuron (green). FIGURE 7 | Systematic variation of the signals of the three output neurons of
the model as a function of the incoming time delay of ITD. The result of the
system’s neurocomputation is coded in terms of pulse width, which is also the
intensity of the spike generated by the output neurons. Note (Figure 6A) that
the pulse width indicates the degree of overlap or coincidence of the
propagating spikes. The delay 1t is defined as the time difference between
the pulses at L0 and R0. A positive 1t corresponds to a pulse generated on
the right side. For simplicity, the pulses were generated with a pulse generator. central sound incidence, or ITD = 0, shown in Figure 6A. The Jeffress Model SNN Circuit From this type of coding we observe that similarly as in
biological systems, there is a continuum of detection and we
adopt the strongest signal (winner takes all) as the result of the
neurocomputation. As it can also be seen in Figure 7, the resolution of the time
difference of our system is about 40 µs, which corresponds an
azimuthal angular of about 4◦, assuming an ear-to-ear distance
of 0.2 m and 343 m/s for the speed of sound (Glackin et al.,
2010; see Supplementary Material). In our present simple
implementation, the angular resolution depends on the time it
takes for a spike to propagate along two subsequent Ranvier
nodes of the long-axon neuron (see Figure 5 and Supplementary
Material). This timescale depends on the specific values of the
UCN parameters. For instance, a bigger capacitor would require
more time to get charged up to the fire threshold value, thus
would increase the time it takes the spike to travel down the long
axon. Hence this would decrease the angular resolution of the
system (see Supplementary Material). Conversely, the resolution
can be systematically improved by performing faster integration,
which can be achieved using smaller capacitors for the membrane
potential. Therefore the resolution depends on the specific values
of the circuit components of the UCN blocks of the long-axon
neuron (Rozenberg et al., 2019). Note, however, that given a delay
resolution, the maximal angle that the model may detect depends
on the number of Ranvier nodes implemented in the long-axon. FIGURE 7 | Systematic variation of the signals of the three output neurons of
the model as a function of the incoming time delay of ITD. The result of the
system’s neurocomputation is coded in terms of pulse width, which is also the
intensity of the spike generated by the output neurons. Note (Figure 6A) that
the pulse width indicates the degree of overlap or coincidence of the
propagating spikes. The delay 1t is defined as the time difference between
the pulses at L0 and R0. A positive 1t corresponds to a pulse generated on
the right side. For simplicity, the pulses were generated with a pulse generator. three output neurons with respect to the arrival delay. Frontiers in Neuroscience | www.frontiersin.org SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found
online
at:
https://www.frontiersin.org/articles/10.3389/fnins. 2021.635098/full#supplementary-material DISCUSSION We have shown how a functional spiking neural network
of biological relevance and be constructed using the recently
introduced ultra-compact neurons as building lego-like blocks. The specific neuronal network is the Jeffress model of the
auditory system of animals and humans, which remains a central
paradigm in the field more than 70 years after its original
proposal. This model was also adopted by Lazzaro and Mead
(1989) in their pioneering work to demonstrate the concept
of neurocomputing. DATA AVAILABILITY STATEMENT The original contributions presented in the study are included
in the article/Supplementary Material, further inquiries can be
directed to the corresponding author. The Jeffress Model SNN Circuit The
output is computed and coded in terms of the pulse width of
the output signal, as was indicated before in the case of the February 2021 | Volume 15 | Article 635098 Frontiers in Neuroscience | www.frontiersin.org Frontiers in Neuroscience | www.frontiersin.org 7 Functional SNN of Ultra-Compact Neurons Stoliar et al. In the Supplementary Material we provide details on how to
implement a large sound detection angle. In the Supplementary Material we provide details on how to
implement a large sound detection angle. the integrate and fire mathematical models and are a source of
concern for the stability of the dynamics (Korn and Faure, 2003;
Nobukawa et al., 2018). Finally, from the data of Figure 7 we may also notice that the
symmetry of the response is affected by a small off-set shift of
about 10 µs. This is simply due to the fact that the SCRs, as all
electronic components, have a small dispersion, or tolerance, in
their production. This feature can be easily corrected by a feed-
back loop of the output, which may tune one of the resistors at the
gate of the SCR that sets the firing threshold. This correction can
be cast in terms of a supervised learning of the auditory system,
where a correction pulse generated by the “wrong” output may
tune the value of a memristor at the gate of the SCR. Details of
that procedure are described in the Supplementary Material. One may envision that neuroscientists may exploit the
simplicity and convenience of the present scheme in multiple
manners. For instance, they may begin to build physical SNN of
UCNs to address under a new light open issues, such as the nature
of the neural code in feed-forward and recurrent neural networks. So far, the study of these important questions has essentially been
restricted to numerical simulations of schematic mathematical
models and algorithms. The present work introduces a novel experimental platform
for the systematic construction and study of the dynamical
behavior of SNN. This platform is simple, versatile and affordable,
so it may open a new avenue of research in experimental
neurobiology at a large and exciting interdisciplinary crossroad. FUNDING MR was supported by the French ANR project MoMA ANR-19-
CE30-0020. This work was also supported in part by the JSPS
KAKENHI Grant Number JP18H05911. MR was supported by the French ANR project MoMA ANR-19-
CE30-0020. This work was also supported in part by the JSPS
KAKENHI Grant Number JP18H05911. ACKNOWLEDGMENTS We
thank
R. Brette,
M. Stimberg,
and
P. Yger
for
valuable discussions. We also thank F. Simon for his help
concerning LTspice. The present work is a concrete demonstration of how the
UCN is a simple and versatile (Hodgkin and Huxley, 1952)
building block that allows to construct bio-inspired spiking
neural networks. This simplicity allows the UCN networks to
be readily made from off-the-shelf electronic components. We
should emphasize the important feature that the UCN is a
physical model of a spiking artificial neuron that does not include
unphysical features such a hard voltage reset nor an abstract
spike signal. Those artificial characteristics are indeed present in AUTHOR CONTRIBUTIONS To implement the Jeffress model, we have first introduced
a novel long-axon neuron, which we motivated by analogy
with the Hodgkin-Huxley model for axon propagation of
action potentials. Our long axon-neurons were built as a
chain of UCN units, where the initial one plays the role
of a soma + axon-hillock. The spikes then propagate along
a chain of UCN segments forming a transmission line of
action potentials, analogous to the nodes of Ranvier of
biological axons. Two of such long-axon units constitute the
input neurons that run antiparallel to each other. They are
interconnected via an output layer of two-dendritic inputs
neurons, which are also implemented by UCN blocks. The result
of the neuro-computation is coded by the spike intensity of
the output neurons. All authors listed have made a substantial, direct and intellectual
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distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the
original author(s) and the copyright owner(s) are credited and that the original
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 © 2021 Stoliar, Schneegans and Rozenberg. This is an open-access article
distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the
original author(s) and the copyright owner(s) are credited and that the original
publication in this journal is cited, in accordance with accepted academic practice. No
use, distribution or reproduction is permitted which does not comply with these terms. Copyright © 2021 Stoliar, Schneegans and Rozenberg. This is an open-access article
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1056 The use, distribution or reproduction in other forums is permitted, provided the
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publication in this journal is cited, in accordance with accepted academic practice. No
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integrate-and-fire model for building spiking neural networks. Sci. Rep. 9:11123. February 2021 | Volume 15 | Article 635098 Frontiers in Neuroscience | www.frontiersin.org 9
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https://europepmc.org/articles/pmc8583204?pdf=render
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English
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Gender Differences in The Factors associated with Hypertension in Non-Diabetic Saudi Adults—A Cross-Sectional Study
|
International journal of environmental research and public health/International journal of environmental research and public health
| 2,021
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cc-by
| 11,823
|
Citation: Al-Raddadi, R.;
Al-Ahmadi, J.; Bahijri, S.; Ajabnoor,
G.M.; Jambi, H.; Enani, S.;
Eldakhakhny, B.M.; Alsheikh, L.;
Borai, A.; Tuomilehto, J. Gender
Differences in The Factors associated
with Hypertension in Non-Diabetic
Saudi Adults—A Cross-Sectional
Study. Int. J. Environ. Res. Public
Health 2021, 18, 11371. https://
doi.org/10.3390/ijerph182111371
Academic Editor: Paul B. Tchounwou
Received: 26 August 2021
Accepted: 26 October 2021
Published: 29 October 2021 6
Department of Food and Nutrition, Faculty of Human Sciences and Design, King Abdulaziz University,
Jeddah 3270, Saudi Arabia Citation: Al-Raddadi, R.;
Al-Ahmadi, J.; Bahijri, S.; Ajabnoor,
G.M.; Jambi, H.; Enani, S.;
Eldakhakhny, B.M.; Alsheikh, L.;
Borai, A.; Tuomilehto, J. Gender
Differences in The Factors associated
with Hypertension in Non-Diabetic
Saudi Adults—A Cross-Sectional
Study. Int. J. Environ. Res. Public
Health 2021, 18, 11371. https://
doi.org/10.3390/ijerph182111371 Citation: Al-Raddadi, R.;
Al-Ahmadi, J.; Bahijri, S.; Ajabnoor,
G.M.; Jambi, H.; Enani, S.;
Eldakhakhny, B.M.; Alsheikh, L.;
Borai, A.; Tuomilehto, J. Gender
Differences in The Factors associated
with Hypertension in Non-Diabetic
Saudi Adults—A Cross-Sectional
Study. Int. J. Environ. Res. Public
Health 2021, 18, 11371. https://
doi.org/10.3390/ijerph182111371 7
King Abdullah International Medical Research Center (KAIMRC), King Saud bin Abdulaziz University for
Health Sciences (KSAU-HS), College of Medicine, King Abdulaziz Medical City, Jeddah 22384, Saudi Arabia
8
Department of Public Health, University of Helsinki, 00014 Helsinki, Finland
9
Public Health Promotion Unit, Finnish Institute for Health and Welfare, 00271 Helsinki, Finland 7
King Abdullah International Medical Research Center (KAIMRC), King Saud bin Abdulaziz University for
Health Sciences (KSAU-HS), College of Medicine, King Abdulaziz Medical City, Jeddah 22384, Saudi Arabia
8
Department of Public Health, University of Helsinki, 00014 Helsinki, Finland 8
Department of Public Health, University of Helsinki, 00014 Helsinki, Finland
9
Public Health Promotion Unit, Finnish Institute for Health and Welfare, 00271 Helsinki, Finland
*
Correspondence: sb@kau.edu.sa; Tel.: +966-564-370-571 p
y
9
Public Health Promotion Unit, Finnish Institute for Health and Welfare, 00271 Helsinki, Finland
*
Correspondence: sb@kau.edu.sa; Tel.: +966-564-370-571 p
y
9
Public Health Promotion Unit, Finnish Institute for Health and Welfare, 00271 Helsinki, Finlan Abstract: The association between lifestyle practices, obesity and increased BP are under-investigated. We aimed to investigate this association to identify the factors associated with hypertension and
prehypertension in Saudis. Non-diabetic adults were recruited from public healthcare centers using a
cross-sectional design. Recruits were interviewed using a predesigned questionnaire. Weight, height,
waist circumference (WC), hip circumference (HC), neck circumference (NC) and BP were measured. Article
Gender Differences in The Factors associated with
Hypertension in Non-Diabetic Saudi Adults—A
Cross-Sectional Study Rajaa Al-Raddadi 1,2,3
, Jawaher Al-Ahmadi 1,2,4, Suhad Bahijri 1,2,5,*
, Ghada M. Ajabnoor 1,2,5
,
Hanan Jambi 1,2,6, Sumia Enani 1,2,6
, Basmah Medhat Eldakhakhny 1,2,5
, Lubna Alsheikh 1,2, Anwar Borai 1,7
and Jaakko Tuomilehto 1,8,9 1
Saudi Diabetes Research Group, King Fahd Medical Research Center, King Abdulaziz University,
Jeddah 3270, Saudi Arabia; rmsalharbi@kau.edu.sa (R.A.-R.); jalahamade@kau.edu.sa (J.A.-A.);
gajabnour@kau.edu.sa (G.M.A.); hjambi@kau.edu.sa (H.J.); senani@kau.edu.sa (S.E.);
beldakhakhny@kau.edu.sa (B.M.E.); lloo86@hotmail.com (L.A.); Boraiaa@ngha.med.sa (A.B.);
jotuomilehto@gmail.com (J.T.) 1
Saudi Diabetes Research Group, King Fahd Medical Research Center, King Abdulaziz University,
Jeddah 3270, Saudi Arabia; rmsalharbi@kau.edu.sa (R.A.-R.); jalahamade@kau.edu.sa (J.A.-A.);
gajabnour@kau.edu.sa (G.M.A.); hjambi@kau.edu.sa (H.J.); senani@kau.edu.sa (S.E.);
beldakhakhny@kau.edu.sa (B.M.E.); lloo86@hotmail.com (L.A.); Boraiaa@ngha.med.sa (A.B.);
jotuomilehto@gmail.com (J.T.) 2
Food, Nutrition and Lifestyle Research Unit, King Fahd for Medical Research Centre, 2
Food, Nutrition and Lifestyle Research Unit, King Fahd for Medical Research Centre,
King Abdulaziz University, Jeddah 22252, Saudi Arabia 3
Department of Community Medicine, Faculty of Medicine, King Abdulaziz University,
Jeddah 22252, Saudi Arabia 4
Department of Family Medicine, Faculty of Medicine, King Abdulaziz University, Jeddah 22252, Saudi Arabia
5 5
Department of Clinical Biochemistry, Faculty of Medicine, King Abdulaziz University,
Jeddah 22252, Saudi Arabia
The variables were analyzed by comparing the prehypertensive and hypertensive groups with the
normotensive group. A total of 1334 adults were included. The study found that 47.2% of men and
24.7% of women were prehypertensive, and 15.1% of men and 14.4% of women were hypertensive. High BMI, WC, NC, and WC: HC ratios were associated with an increased risk of prehypertension
and hypertension in men and women. Low physical activity was associated with an increased
risk of elevated BP in men, while sleep duration of ≤6 h and sitting for ≥4 h were associated with
increased risk in women. Women from central Asia, southeast Asia, and those of mixed origin
had a higher prevalence of hypertension compared to those from Arabian tribes. In conclusion,
prehypertension and hypertension increase with age and obesity. Gender differences were apparent
in the association between several lifestyle practices and prehypertension or hypertension among
various ethnic/racial groups. Received: 26 August 2021
Accepted: 26 October 2021
Published: 29 October 2021 Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. Keywords: hypertension; prehypertension; lifestyle; obesity Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article
distributed
under
the
terms
and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/). International Journal of
Environmental Research
and Public Health 1. Introduction Hypertension is a strong risk factor for cardiovascular disorders and has been found
to be the main predictor of cardiovascular morbidity and mortality [1,2]. Indeed, a study Int. J. Environ. Res. Public Health 2021, 18, 11371. https://doi.org/10.3390/ijerph182111371 https://www.mdpi.com/journal/ijerph Int. J. Environ. Res. Public Health 2021, 18, 11371 2 of 14 published in 2008 reported that hypertension alone was the cause of 13.5% of the total
global premature deaths (7.6 million), as well as 54% of strokes, and 47% of incidences of
ischemic heart disease [3]. Moreover, various epidemiological studies on different populations and ethnicities
have shown that higher levels of BP, including levels in the prehypertension state, are
widely associated with increased risk of fatal and nonfatal cardiovascular events [4–6]. The
associated medical cost and human capital loss increases the burden to the economy [7,8]. In Saudi Arabia, the reported prevalence of hypertension has been increasing over
the years [9–14], with the most recent study reporting it to be 31.4% [15]. Furthermore,
hypertension was classified as the leading risk factor for death among Saudis in 2010 [16]. Unfortunately, as noted in other studies [17], hypertension was undetected in a large
percentage of the Saudi population during a 2013 national survey, and 57.8% of studied
people with hypertension were undiagnosed [14]. In view of these reports, steps should be
taken to increase awareness among the Saudi population to control the trend of increasing
hypertension-related mortality. In addition, healthcare professionals should be aware of
the risk factor associated with high blood pressure for this population. Increased blood pressure (BP) is often a consequence of obesity [18]. However, other
disease states, as well as some lifestyle practices, may lead to hypertension [19,20]. The
noted increase in the prevalence of hypertension in Saudi Arabia might be attributed to
lifestyle changes related to urbanization and the adoption of dietary eating habits that are
likely to result in hypertension, as well as the increasing prevalence of obesity [21]. The
relationship between lifestyle practices, different measures of obesity, and hypertension
has not been fully investigated in the Kingdom of Saudi Arabia. Furthermore, no studies of
the effect of ethnic origin on the prevalence of hypertension have been conducted in Saudi
Arabia, even though the Saudi people descend from different races, and racial disparity in
hypertension and hypertension-related outcomes has long been acknowledged [22]. 1. Introduction There-
fore, we aimed to investigate the factors associated with prehypertension and hypertension
among persons of various racial/ethnic groups in Saudi Arabia. 2. Materials and Methods A cross-sectional design was employed to conduct this study, which is part of a more
extensive study intended to validate a “dysglycemia risk score” for Saudi people that
has been outlined fully previously [23]. A total of 1477 adults were recruited. However,
after excluding those found to be diabetic and those with missing data, 1334 adults were
included in the study. Data were collected by trained medical students. Anthropometric
measurements (weight, height, waist circumference, hip circumference, and neck circum-
ference) and BP were taken using standardized techniques [24]. Recruits were interviewed
for medical history and demographic and lifestyle characteristics using a predesigned
questionnaire. Blood samples were obtained while fasting for an estimation of a glucose
and lipid profile. Another sample was taken one hour after the ingestion of a 50 g of oral
glucose load for the estimation of glucose and glycated hemoglobin (HbA1c) [25]. The definitions of hypertension and prehypertension followed those outlined in the
Joint National Committee on Prevention, Detection, Evaluation and Treatment of High
BP (JNC) VII report [24]. Prehypertension was defined as systolic blood pressure: 120
to 139 mm Hg and/or diastolic blood pressure 85 to 89 mm Hg, and hypertension was
defined as systolic ≥140, and/or diastolic ≥90 mm Hg or taking blood pressure-lowering
drug treatment. 3. Results A total 1334 adults were included in the current study, the mean age and age groups
according to BP status of men and women are presented in Table 1. Table 1. Mean and age groups of participating men and women according to their BP status. Variable
Men
Women
Total
Non-
Hypertensive
Prehypertensive
Hypertensive
p-Value
Total
Non-
Hypertensive
Prehypertensive
Hypertensive
p-Value
(N = 742)
(N = 280)
(N = 350)
(N = 112)
(N = 592)
(N = 361)
(N = 146)
(N = 85)
n; %
% (95% CI)
% (95% CI)
% (95% CI)
n; %
% (95% CI)
% (95% CI)
% (95% CI)
Age group
(years)
(mean ±
SD)
-
29.2 ± 9.0
29.9 ± 8.8
37.2 ± 14.3
b ***; c ***
-
31.4 ± 10.5
33.5 ± 12.3
40.9 ± 14.3
a *; b ***;
c ***
<35
549; 73.9
77.5
(72.2–82.3)
76.9 (72.1–81.2)
56.3
(46.6–65.6)
b ***; c ***
354; 59.8
65.9
(60.8–70.8)
59.6 (51.2–67.6)
34.1
(24.2–45.2)
a *; b ***;
c **
35–44
119; 16.0
17.1
(12.9–22.1)
14.0 (10.5–18.1)
19.5
(12.7–28.2)
125; 21.1
19.4
(15.4–23.9)
23.3 (16.7–31.0)
24.7
(16.0–35.3)
45–54
42; 5.7
3.6 (1.7–6.4)
7.1 (4.7–10.4)
6.3 (2.6–12.5)
76; 12.8
12.5
(9.2–16.3)
8.9 (4.8–14.8)
21.2
(13.1–31.4)
55–64
21; 2.8
1.1 (0.2–3.1)
1.7 (0.6–3.7)
10.7
(5.7–18.0)
30; 5.07
1.9 (0.7–4.0)
6.9 (3.3–12.2)
15.3
(8.4–24.7)
>64
11; 1.5
0.7 (0.1–2.6)
1; 0.3 (0.01–1.6)
7.1 (3.1–13.6)
7; 1.2
0.3 (0.01–1.5)
1.4 (0.2–4.9)
4.7 (1.3–11.6)
Proportion
of people
unaware of
their
condition
417; 56.2
NA
96.0 (93.4–97.8)
72.3
(63.1–80.4)
c ***
30.6
NA
93.2 (87.8–96.7)
52.9
(41.8–63.9)
c ***
N: total number of subjects in all subgroups; n: number of people in subgroup; Prehypertension was defined as systolic blood pressure
(SBP): 120 to 139 mm Hg, and/or diastolic blood pressure (DBP) 85 to 89 mm Hg; Hypertension was defined as SBP ≥140, and/or
DBP ≥90 mm Hg or taking blood pressure lowering treatment; a Non-hypertensive vs. Pre-hypertensive; b Non-hypertensive vs. hyper-
tensive; c Prehypertensive vs. hypertensive; *** p-value > 0.001; ** p-value > 0.01; * p-value > 0.05; NA, not applicable. Table 1. Mean and age groups of participating men and women according to their BP status. Mean and age groups of participating men and women according to their BP status. Statistical Methods Statistical analysis was carried out using SPSS, version 21. Descriptive statistics were
calculated for all measured and estimated parameters and are presented as mean ± standard
deviation (SD) for continuous variables and as frequency (percentages) for categorical
variables. Demographic, lifestyle and clinical factors of hypertension were analyzed
by comparing those of prehypertensive and hypertensive participants and those of nor- Int. J. Environ. Res. Public Health 2021, 18, 11371 3 of 14 motensive group. Factors with continuous variables were analyzed using ANOVA and
independent t-test, while those with categorical variables were analyzed using Chi-square
test or Fisher’s exact test, as appropriate. motensive group. Factors with continuous variables were analyzed using ANOVA and
independent t-test, while those with categorical variables were analyzed using Chi-square
test or Fisher’s exact test, as appropriate. pp
p
Multiple logistic regression analysis was used to adjust for age. Unadjusted and
adjusted Odds Ratio (OR) with its 95% Confidence Interval (CI) for the factors associated
with prehypertension and hypertension are presented. Statistical significance was assigned
at p < 0.05. 3. Results N: total number of subjects in all subgroups; n: number of people in subgroup; Prehypertension was defined as systolic blood pressure
(SBP): 120 to 139 mm Hg, and/or diastolic blood pressure (DBP) 85 to 89 mm Hg; Hypertension was defined as SBP ≥140, and/or
DBP ≥90 mm Hg or taking blood pressure lowering treatment; a Non-hypertensive vs. Pre-hypertensive; b Non-hypertensive vs. hyper-
tensive; c Prehypertensive vs. hypertensive; *** p-value > 0.001; ** p-value > 0.01; * p-value > 0.05; NA, not applicable. A high percentage of the participants were either prehypertensive (47.2% of men and
24.7% of women) or hypertensive (15.1% of men and 14.4% of women). Only 43.7% of men,
and 56.3% of women were found to have normal blood pressure. A higher percentage
of people were prehypertensive, or hypertensive due to elevated systolic blood pressure. More than 93% of Prehypertensive people, and even a considerable percentage of people
with hypertension (about 64%) were unaware of their condition. The mean age of hypertensive men and women was significantly higher than the mean
age of prehypertensive and normotensive corresponding groups. However, the mean age
of prehypertensive women was significantly higher than the mean age of the normotensive
group. Only 56.3% (95% CI: 46.6–65.6) of hypertensive men, and 34.1% (95% CI: 24.2–45.2)
of women were <35 years old, as shown in Table 1. y
Lifestyle, demographic and anthropometric characteristics in the hypertensive, prehy-
pertensive and non-hypertensive study groups are presented in Tables 2 and 3. 4 of 14 Int. J. Environ. Res. Public Health 2021, 18, 11371 Table 2. Association between demographic, lifestyle, vital signs, and anthropometric characteristics with prehypertension
and hypertension in the male participants. 3. Results Variable
Total
Non-Hypertensive
Prehypertensive
Hypertensive
p-Value
(N = 742)
(N = 280)
(N = 350)
(N = 112)
n; %
% (95% CI)
% (95% CI)
% (95% CI)
Body Mass Index (kg/m2)
(mean ± SD)
-
25.6 ± 5.2
28.0 ± 5.7
30.4 ± 6.7
a ***; b ***; c ***
<25
262; 35.3
47.1 (41.2–53–2)
30.6 (25.8–35.7)
20.5 (13.5–29.2)
a ***; b ***; c **
25–<30
275; 37.0
36.8 (31.1–42.7)
38.9 (33.7–44.2)
32.1 (23.6–41.6)
≥30
205; 27.6
16.1 (12.0–21.0)
30.6 (25.8–35.7)
47.3 (37.8–57.0)
Waist Circumference (cm)
(mean ± SD)
-
91.5 ± 13.8
97.7 ± 14.9
104.4± 16.6
a ***; b ***; c ***
Normal < 94 cm
350; 47.2
60.0 (54.0–65.8)
43.7 (38.5–49.1)
25.9 (18.1–35.0)
a ***; b ***; c **
Abdominal obesity Level 1 ≥94–102 cm
172; 19.1
21.8 (17.1–27.1)
25.1 (20.7–30.0)
20.5 (13.5–29.2)
Abdominal obesity Level 2 > 102 cm
211; 28.4
17.9 (13.6–22.9)
30.0 (25.2–35.1)
50.0 (40.4–59.6)
Waist to Height ratio
(mean ± SD)
-
0.53 ± 0.08
0.57± 0.09
0.61 ± 0.09
a ***; b ***; c ***
Normal ≤0.5
192; 25.9
36.1 (30.4–42.0)
22.0 (17.8–26.7)
12.5 (7.0–20.1)
a ***; b ***; c *
High > 0.5
541; 72.9
63.6 (57.6–69.2)
76.9 (72.1–81.1)
83.9 (75.8–90.2)
Waist to Hip ratio
(mean ± SD)
-
0.88 ± 0.07
0.91 ±0.08
0.93 ± 0.07
a ***; b ***; c *
Normal ≤0.95
581; 78.3
85.0 (80.3–89.0)
76.9 (72.1–81.1)
66.1 (56.5–74.8)
a *; b ***
High > 0.95
152; 20.5
14.6 (10.7–19.3)
22.0 (17.8–26.7)
30.4 (22.0–39.8)
Neck Circumference
(mean ± SD)
-
37.9 ± 3.7
40.0 ± 4.3
41.2 ± 4.3
a ***; b ***
Normal < 37
160; 21.6
32.9 (27.4–38.7)
16.0 (12.3–20.3)
10.7 (5.7–18.0)
a ***; b ***
High ≥37
572; 77.1
66.4 (60.6–71.9)
82.9 (78.5–86.7)
85.7 (77.8–91.6)
SBP (mmHg)
(mean ± SD)
-
110.1 ± 6.4
124.7 ± 5.6
136.9± 15.3
a ***; b ***; c ***
Individuals with Normal values
(SBP < 120 mmHg)
295; 39.8
100.0 (98.7–100.0)
2.0 (0.8–4.1)
7.1 (3.1–13.6)
a ***; b ***; c **
Individuals with high values
(SBP ≥120 mmHg)
447; 60.2
0
98.0 (95.9–99.2)
92.9 (86.4–96.9)
DBP (mmHg)
(mean ± SD)
-
68.2 ± 9.1
75.8 ± 8.0
88.1 ± 11.7
a ***; b ***; c ***
Individuals with normal values
(DBP ≥85 mmHg)
615; 82.9
100.0 (98.7–100.0)
85.7 (81.6–89.2)
31.3 (22.8–40.7)
a ***; b ***; c ***
Individuals with high values
(DBP ≥85 mmHg)
127; 17.1
0
14.3 (10.8–18.4)
68.8 (59.3–77.2)
Ethnic origin
Arabian tribes
596; 80.3
81.4 (76.4–85.8)
81.4 (77.0–85.4)
74.1 (65.0–81.9)
African tribes
28; 3.8
2.5 (1.01–5.1)
4.3 (2.4–7.0)
5.4 (2.0–11.3)
NS
Mediterranean countries
25; 3.4
2.1 (0.8–4.6)
4.3 (2.4–7.0)
3.6 (1.0–8.9)
Indian continent
56; 7.5
7.9 (5.0–11.7)
6.3 (4.0–9.4)
10.7 (5.7–18.0)
Central Asia
9; 1.2
1.1 (0.2–3.1)
1.4 (0.5–3.3)
0.9 (0.02–4.9)
South east Asia
13; 1.8
1.8 (0.6–4.1)
1.1 (0.3–2.9)
3.6 (1.0–8.9)
Mixed
15; 2.0
3.2 (1.5–6.0)
1.1 (0.3–2.9)
1.8 (0.2–6.3)
Physical activity
(30 min/day–5 days/week)
No
403; 54.3
48.6 (42.6–54.6)
56.9 (51.5–62.1)
60.7 (51.0–69.8)
a *; b *
Yes
339; 45.7
51.4 (45.4–57.4)
43.1 (37.9–48.5)
39.3 (30.2–49.0)
Sleep duration (h)
≤6
318; 42.9
42.5 (36.6–48.5)
42.9 (37.6–48.2)
43.8 (34.4–53.4)
>6–8
381; 51.3
51.4 (45.4–57.4)
52.9 (47.5–58.2)
46.4 (37.0–56.1)
NS
>8
43; 5.8
0.6 (3.6–9.5)
42.9 (24.2–69.7)
9.8 (5.0–16.9)
Sitting hours/day
<4
97; 13.1
15.4 (11.3–20.1)
12.3 (9.0–16.2)
9.8 (5.0–16.9)
4–5
225; 30.3
29.3 (24–35.0)
30.0 (25.2–35.1)
33.9 (25.3–43.5)
NS
6–8
258; 34.8
34.3 (28.7–40.2)
36.0 (31.0–41.3)
32.1 (23.6–41.6)
>8
162; 21.8
21.1 (16.5–26.3)
21.7 (17.5–26.4)
24.1 (16.5–33.1)
Smoking Habits
Non smoker
463; 62.4
61.4 (55.5–67.2)
65.1 (59.9–70.1)
56.3 (46.6–65.6)
NS
Smoker
279; 37.6
38.6 (32.8–44.6)
34.9 (29.9–40.1)
43.8 (34.4–53.4)
Daily fruit or vegetable intake (at least
one portion)
No
295; 39.8
36.8 (31.1–42.7)
42.9 (37.6–48.2)
37.5 (28.5–47.2)
NS
Yes
447; 60.2
63.2 (57.3–68.9)
57.1 (51.8–62.4)
62.5 (52.9–71.5)
N: total number of subjects in all subgroups; n: number of people in subgroup; Prehypertension was defined as systolic blood pressure
(SBP): 120 to 139 mm Hg, and/or diastolic blood pressure (DBP) 85 to 89 mm Hg; Hypertension was defined as SBP ≥140, and/or
DBP ≥90 mm Hg or taking blood pressure lowering treatment; a Non-hypertensive Vs. 3. Results Pre-hypertensive; b Non-hypertensive Vs. hypertensive; c Prehypertensive Vs. hypertensive; *** p-value > 0.001; ** p-value > 0.01; * p-value > 0.05; NS non-significant. Table 2. Association between demographic, lifestyle, vital signs, and anthropometric characteristics with prehypertension
and hypertension in the male participants. Table 2. Association between demographic, lifestyle, vital signs, and anthropometric characteristics with prehypertension
and hypertension in the male participants. N: total number of subjects in all subgroups; n: number of people in subgroup; Prehypertension was defined as systolic blood pressure
(SBP): 120 to 139 mm Hg, and/or diastolic blood pressure (DBP) 85 to 89 mm Hg; Hypertension was defined as SBP ≥140, and/or
DBP ≥90 mm Hg or taking blood pressure lowering treatment; a Non-hypertensive Vs. Pre-hypertensive; b Non-hypertensive Vs. hypertensive; c Prehypertensive Vs. hypertensive; *** p-value > 0.001; ** p-value > 0.01; * p-value > 0.05; NS non-significant. 5 of 14 Int. J. Environ. Res. Public Health 2021, 18, 11371 Table 3. Association between demographic, lifestyle, vital signs, and anthropometric characteristics with prehypertension
and hypertension in the female participants. Table 3. Association between demographic, lifestyle, vital signs, and anthropometric characteristics with prehypertension
and hypertension in the female participants. 3. Results Variable
Total
Non-Hypertensive
Prehypertensive
Hypertensive
p-Value
(N = 592)
(N = 361)
(N = 146)
(N = 85)
n; %
% (95% CI)
% (95% CI)
% (95% CI)
Body Mass Index (kg/m2)
(mean ± SD)
-
26.0 ± 5.5
28.7 ± 6.4
30.99 ± 7.15
a ***; b ***; c *
<25
223; 37.7
44.0 (38.9–49.3)
32.2 (24.7–40.4)
20.0 (12.1–30.1)
a ***; b ***; c *
25–<30
195; 32.9
35.5 (30.5–40.6)
30.8 (23.5–39.0)
25.9 (17.0–36.5)
≥30
174; 29.4
20.5 (16.5–25.0)
37.0 (29.2–45.4)
54.1 (43.0–65.0)
Waist Circumference (cm)
(mean ± SD)
-
84.3 ± 14.3
91.4 ±16.3
98.0 ± 16.2
a ***; b ***; c **
Normal < 80 cm
211; 35.6
44.0 (38.9–49.3)
28.1 (21.0–36.1)
25.9 (18.1–35.0)
a ***; b ***; c **
Abdominal obesity Level 1 ≥80–88 cm
95; 16.1
17.7 (13.9–22.1)
13.7 (8.6–20.4)
12.9 (6.6–22.0)
Abdominal obesity Level 2 > 88 cm
270; 45.6
35.2 (30.3–40.4)
54.8 (46.4–63.0)
74.1 (63.5–83.0)
Waist to Height ratio
(mean ± SD)
-
0.53 ± 0.09
0.58 ± 0.11
0.62 ± 0.1
a ***; b ***; c **
Normal ≤0.5
194; 32.8
40.2 (35.1–45.4)
26.0 (19.1–34.0)
12.9 (6.6–22.0)
a **; b ***
High > 0.5
382; 64.5
56.8 (51.5–62.0)
70.6 (62.5–77.8)
87.1 (78.0–93.4)
Waist to Hip ratio
(mean ± SD)
-
0.82 ± 0.09
0.85 ± 0.09
0.88 ± 0.09
a **; b ***; c *
Normal ≤0.8
216; 36.5
43.5 (38.3–48.8)
30.8 (23.5–39.0)
16.5 (9.3–26.1)
a **; b ***
High > 0.8
360; 60.8
53.5 (48.2–58.7)
65.8 (57.5–73.4)
83.5 (73.9–90.7)
Neck Circumference
(mean ± SD)
-
32.7 ± 3.4
34.5 ± 5.2
35.8 ± 3.9
a ***; b ***; c *
Normal < 34
314; 53.0
62.1 (56.8–67.1)
43.8 (35.6–52.3)
30.6 (21.1–41.5)
a ***; b ***; c *
High ≥34
261; 44.1
34.6 (29.7–39.8)
52.7 (44.3–61.1)
69.4 (58.5–79.0)
SBP (mmHg)
(mean ± SD)
-
104.2 ± 8.2
121.9 ± 4.9
133.4 ± 18.7
a ***; b ***; c ***
Individuals with Normal values
(SBP < 120 mmHg)
385; 65.0
100.0 (99.0–100.0)
7.5 (3.8–13.1)
15.3 (8.4–24.7)
a ***; b ***
Individuals with high values
(SBP ≥120 mmHg)
207; 35.0
0
92.5 (86.9–96.2)
84.7 (75.3–91.6)
DBP (mmHg)
(mean ± SD)
-
65.2 ± 7.9
74.8 ± 8.8
86.5 ± 11.7
a ***; b ***; c ***
Individuals with normal values
(DBP ≥85 mmHg)
513; 86.7
100.0 (99.0–100.0)
84.9 (78.1–90.3)
32.9 (23.1–44.0)
a ***; b ***; c ***
Individuals with high values
(DBP ≥85 mmHg)
79; 13.3
0
15.1 (9.7–21.9)
67.1 (56.0–76.9)
Ethnic origin
Arabian tribes
429; 72.5
74.5 (69.7–78.9)
77.4 (69.8–83.9)
55.3 (44.1–66.1)
b **
African tribes
43; 7.3
6.7 (4.3–9.7)
8.2 (4.3–13.9)
8.2 (3.4–16.2)
Mediterranean countries
37; 6.3
6.4 (4.1–9.4)
4.1 (1.5–8.7)
9.4 (4.2–17.7)
Indian continent
28; 4.7
5.0 (3.0–7.8)
3.4 (1.1–7.8)
5.9 (1.9–13.2)
Central Asia
11; 1.9
1.1 (0.3–2.8)
1.4 (0.2–4.9)
5.9 (1.9–13.2)
South east Asia
20; 3.4
2.8 (1.3–5.0)
2.1 (0.4–5.9)
8.2 (3.4–16.2)
Mixed
24; 4.1
3.6 (1.9–6.1)
3.4 (1.1–7.8)
7.1 (2.6–14.7)
Physical activity
(30 min/day–5 days/week)
No
343; 57.9
58.5 (53.2–63.6)
58.9 (50.5–67.0)
54.1 (43.0–65.0)
NS
Yes
249; 42.1
41.6 (36.4–46.8)
41.1 (33.0–49.5)
45.9 (35.0–57.0)
Sleep duration (h)
≤6
201; 34.0
29.6 (25.0–34.6)
41.1 (33.0–49.5)
40.0 (29.5–51.2)
a *
>6–8
308; 52.0
54.9 (49.6–60.1)
47.3 (39.0–55.7)
48.2 (37.3–59.3)
>8
83; 14.0
15.5 (11.9–19.7)
11.6 (6.9–18.0)
11.8 (5.8–20.6)
Sitting hours/day
<4
149; 25.2
27.7 (23.2–32.6)
18.5 (12.6–25.8)
25.9 (17.0–36.5)
NS
4–5
182; 30.7
29.4 (24.7–34.4)
32.9 (25.3–41.1)
32.9 (23.1–44.0)
6–8
155; 26.2
26.6 (22.1–31.5)
28.8 (21.6–36.8)
20.0 (12.1–30.1)
>8
106; 17.9
16.3 (12.7–20.6)
19.9 (13.7–27.3)
21.2 (13.1–31.4)
Smoking Habits
Non smoker
491; 82.9
82.8 (78.5–86.6)
84.3 (77.3–89.7)
81.2 (71.2–88.8)
NS
Smoker
101; 17.1
17.2 (13.4–21.5)
15.8 (10.3–22.7)
18.8 (11.2–28.8)
Daily fruit or vegetable intake (at least
one portion)
No
191; 32.3
33.0 (28.1–38.1)
33.6 (26.0–41.8)
27.1 (18.0–37.8)
NS
Yes
401; 67.7
67.0 (61.9–71.9)
66.4 (58.2–74.0)
72.9 (62.2–82.0)
N: total number of subjects in all subgroups; n: number of people in subgroup; Prehypertension was defined as systolic blood pressure
(SBP): 120 to 139 mm Hg, and/or diastolic blood pressure (DBP) 85 to 89 mm Hg; Hypertension was defined as SBP ≥140, and/or
DBP ≥90 mm Hg or taking blood pressure lowering treatment; a Non-hypertensive vs. 3. Results Pre-hypertensive; b Non-hypertensive vs. hyper-
tensive; c Prehypertensive vs hypertensive; *** p-value <0 001; ** p-value < 0 01; * p-value < 0 05; NS non-significant Table 3. Association between demographic, lifestyle, vital signs, and anthropometric characteristics with prehypertension
and hypertension in the female participants. N: total number of subjects in all subgroups; n: number of people in subgroup; Prehypertension was defined as systolic blood pressure
(SBP): 120 to 139 mm Hg, and/or diastolic blood pressure (DBP) 85 to 89 mm Hg; Hypertension was defined as SBP ≥140, and/or
DBP ≥90 mm Hg or taking blood pressure lowering treatment; a Non-hypertensive vs. Pre-hypertensive; b Non-hypertensive vs. hyper-
tensive; c Prehypertensive vs. hypertensive; *** p-value <0.001; ** p-value < 0.01; * p-value < 0.05; NS non-significant. Int. J. Environ. Res. Public Health 2021, 18, 11371 6 of 14 As shown in Table 3, general obesity, abdominal obesity, and upper-torso obesity
were significantly higher among people with elevated blood pressure. The mean BMI
of hypertensive men and women was significantly higher than the mean BMI of both
prehypertensive and normotensive men and women. In addition, the means of the pre-
hypertensive groups were also significantly higher than that of the corresponding nor-
motensive groups. Only 25.9% (95% CI: 18.1–35.0) of hypertensive men, and 25.9% (95% CI:
18.1–35.0) of hypertensive women had normal WC. Similar findings were noted when
waist to height ratio, waist to hip ratio, and neck circumference results were compared. Ethnicity, physical activity, and, to some extent, sleep duration were also different when
comparing the three groups. However, gender differences were obvious in the case of
these factors. Men descending from southeast Asia and those from African tribes had
a higher percentage of hypertension than expected according to the total distribution of
ethnicities, although the difference did not reach statistical significance. In the case of
women, those descending from central Asia, southeast Asia, and mixed origin showed a
higher percentage of hypertension than expected, while those from Arabic tribes had a
significantly lower percentage (p < 0.01). The effect of physical activity was statistically
apparent in men, but not in women, with a higher percentage than expected of physically
inactive men having normal blood pressure. g
p
In contrast, women with a sleep duration of ≤6 h had a lower percentage of normal
blood pressure than prehypertensive women. 3. Results No significant difference was found between
the three groups of men or women with respect to sitting hours, smoking habits, nor
daily intake of fruit and vegetables. Very few people reported eating the recommended
minimum amount of three portions daily of fruit and vegetables. Therefore, at least one
portion daily was used in our analysis. After adjusting for the effect of age, the results are presented in Table 4 for prehyper-
tension and hypertension and Table 5 for hypertension alone. Table 4. Unadjusted and age-adjusted odds ratio (OR), and confidence interval (CI) for anthropometric measurements and
lifestyle habits covariates associated with people with prehypertension and hypertension combined. Variable
Men
Women
Unadjusted OR, 95% CI
Age Adjusted OR, 95% CI
Unadjusted OR, 95% CI
Age Adjusted OR, 95% CI
High Body Mass Index (kg/m2)
<25
Ref
Ref
Ref
Ref
25–<30
1.70 (1.20–2.39) *
1.58 (1.11–2.24)
1.31 (0.86–1.97)
1.12 (0.73–1.73)
≥30
3.61 (2.40–5.44) **
3.36 (2.22–5.09) **
3.43 (2.25–5.20) **
2.65 (1.68–4.19) **
High Waist Circumference (cm)
Normal
Ref
Ref
Ref
Ref
Abdominal obesity Level 1
1.68 (1.15–2.45) *
1.58 (1.08–2.32)
1.48 (0.87–2.52)
1.33 (0.77–2.28)
Abdominal obesity Level 2
2.97 (2.03–4.35) **
2.69 (1.81–3.99) **
3.44 (2.32–5.11) **
2.76 (1.78–4.30) **
Elevated Waist to Height ratio
> 0.5
2.26 (1.62–3.17) **
2.02 (1.42–2.87) **
2.55 (1.75–3.74) **
1.95 (1.28–2.96) *
Elevated Waist to Hip ratio
> 0.95
1.88 (1.27–2.79) *
1.60 (1.06–2.42) *
2.30 (1.60–3.31) **
1.92 (1.31–2.80) *
High Neck Circumference (cm)
3.86 (1.99–4.11) **
2.65 (1.84–3.83) **
2.66 (1.88–3.75) **
2.22 (1.55–3.19) **
≥37 cm Men; ≥34 Women
Ethnic origin
Arabian tribes
Ref
Ref
Ref
Ref
African tribes
1.86 (0.78–4.44)
1.72 (0.71–4.13)
1.33 (0.71–2.51)
1.34 (0.71–2.55)
Mediterranean countries
1.96 (0.77–4.99)
1.95 (0.76–4.96)
1.02 (0.51–2.05)
0.89 (0.43–1.81)
Indian continent
0.96 (0.55–1.68)
0.92 (0.52–1.61)
0.93 (0.42–2.07)
1.02 (0.45–2.29)
Central Asia
1.24 (0.31–5.00)
1.52 (0.38–6.19)
2.94 (0.85–10.21)
3.38 (0.96–11.87)
Southeast Asia
0.99 (0.32–3.07)
1.01 (0.32–3.17)
1.68 (0.69–4.13)
1.48 (0.59–3.67)
Mixed
0.41 (0.15–1.18)
0.47 (0.16–1.34)
1.42 (0.62–3.25)
1.48 (0.63–3.46)
Low Physical activity
1.45 (1.08–1.95) *
1.41 (1.04–1.90) *
0.95 (0.68–1.32)
0.89 (0.63–1.25)
(<30 min/day–5 days/week)
Sleep duration (h)
>6–8
Ref
Ref
Ref
Ref
≤6
1.02 (0.75–1.38)
0.99 (0.73–1.35)
1.58 (1.10–2.27) **
1.48 (1.02–2.14) *
>8
0.82 (0.49–1.77)
0.94 (0.49–1.80)
0.87 (0.52–1.45)
1.01 (0.59–1.71) Table 4. Unadjusted and age-adjusted odds ratio (OR), and confidence interval (CI) for anthropometric me
lifestyle habits covariates associated with people with prehypertension and hypertension combined. Table 4. 3. Results Unadjusted and age-adjusted odds ratio (OR), and confidence interval (CI) for anthropometric measurements and
lifestyle habits covariates associated with people with prehypertension and hypertension combined. d age-adjusted odds ratio (OR), and confidence interval (CI) for anthropometric measurements and
es associated with people with prehypertension and hypertension combined. Int. J. Environ. Res. Public Health 2021, 18, 11371 7 of 14 Table 4. Cont. Table 4. Cont. Table 4. Cont. Variable
Men
Women
Unadjusted OR, 95% CI
Age Adjusted OR, 95% CI
Unadjusted OR, 95% CI
Age Adjusted OR, 95% CI
Sitting hours/day
<4
Ref
Ref
Ref
Ref
4–5
1.39 (0.86–2.25)
1.33 (0.81–2.16)
1.46 (0.93–2.29)
1.64 (1.03–2.61) *
6–8
1.34 (0.84–2.16)
1.34 (0.83–2.15)
1.25 (0.78–2.01)
1.74 (1.05–2.72) *
>8
1.39 (0.83–2.32)
1.37 (0.82–2.29)
1.63 (0.97–2.72)
1.98 (1.16–3.38) *
Smoking Habits
0.94 (0.69–1.27)
0.97 (0.71–1.31)
0.98 (0.63–1.52)
1.00 (0.65–1.58)
* p-value < 0.05; ** p-value < 0.001; High Waist Circumference (cm); Normal ≤94 cm Men; ≤80 cm Women; Abdominal obesity
Level 1 > 94–102 cm Men; >80–88 cm women; Abdominal obesity Level 2 > 102 cm Men; >88 cm Women. * p-value < 0.05; ** p-value < 0.001; High Waist Circumference (cm); Normal ≤94 cm Men; ≤80 cm Women; Abdominal obesity
Level 1 > 94–102 cm Men; >80–88 cm women; Abdominal obesity Level 2 > 102 cm Men; >88 cm Women. Table 5. Unadjusted and age-adjusted odds ratio (OR), and confidence interval (CI) for anthropometric measurements and
lifestyle habits covariates associated with hypertension. Table 5. Unadjusted and age-adjusted odds ratio (OR), and confidence interval (CI) for anthropometric measurements and
lifestyle habits covariates associated with hypertension. 3. Results Variable
Men
Women
Unadjusted OR, 95% CI
Age Adjusted OR, 95% CI
Unadjusted OR, 95% CI
Age Adjusted OR, 95% CI
High Body Mass Index (kg/m2)
<25
Ref
Ref
Ref
Ref
25–<30
2.00 (1.12–3.59) *
1.55 (0.84–2.85)
1.56 (0.80–3.01)
1.15 (0.57–2.32)
≥30
6.76 (3.73–12.3) **
5.38 (2.90–9.99) **
6.00 (3.29–10.9) **
3.18 (1.59–6.35) **
High Waist Circumference (cm)
Normal
Ref
Ref
Ref
Ref
Abdominal obesity Level 1
2.18 (1.17–4.06) *
1.64 (0.85–3.13)
2.48 (1.03–6.02) *
1.84 (0.74–4.59)
Abdominal obesity Level 2
6.49 (3.75–11.23) **
4.54 (2.54–8.07) **
7.17 (1.03–14.18) **
4.09 (1.94–8.65) **
Elevated Waist to Height ratio
> 0.5
3.81 (2.07–7.03) **
2.44 (1.28–4.67) **
4.76 (2.44–9.28) **
2.62 (1.27–5.41) **
Elevated Waist to Hip ratio
> 0.95
2.67 (1.58–4.51) *
1.59 (0.89–2.85)
4.13 (2.24–7.60) **
3.03 (1.61–5.71) **
High Neck Circumference (cm)
3.56 (1.89–6.69) **
2.73 (1.42–5.28) **
3.92 (2.35–6.53) **
2.78 (1.63–4.76) **
≥37 cm Men; ≥34 Women
Ethnic origin
Arabian tribes
Ref
Ref
Ref
Ref
African tribes
2.36 (0.77–7.20)
2.08 (0.64–6.77)
1.67 (0.68–4.09)
1.92 (0.75–4.95)
Mediterranean countries
1.83 (0.50–6.65)
1.90 (0.49–7.41)
1.99 (0.84–4.71)
1.69 (0.65–4.41)
Indian continent
1.50 (0.71–3.16)
1.53 (0.70–3.33)
1.59 (0.56–4.49)
2.38 (0.80–7.11)
Central Asia
0.92 (0.09–8.93)
1.59 (0.16–15.81)
7.15 (1.85–27.62) *
11.54 (2.76–48.18) **
South east Asia
2.20 (0.58–8.38)
2.48 (0.61–10.06)
4.00 (1.45–11.05) *
3.51 (1.22–10.13) *
Mixed
0.61 (0.13–2.88)
0.89 (0.18–4.37)
2.64 (0.96–7.30)
3.24 (1.04–10.08) *
Low Physical activity (<30
min/day–5 days/week)
1.64 (1.05–2.56) *
1.51 (0.95–2.42)
0.84 (0.52–1.35)
0.70 (0.42–1.17)
Sleep duration (h)
>6–8
Ref
Ref
Ref
Ref
≤6
1.14 (0.72–1.81)
1.00 (0.62–1.64)
1.54 (0.92–2.56)
1.25 (0.73–2.16)
>8
1.64 (0.79–4.08)
1.76 (0.73–4.23)
0.86 (0.41–1.83)
1.15 (0.52–2.52)
Sitting hours/day
<4
Ref
Ref
Ref
Ref
4–5
1.81 (0.84–3.90)
1.67 (0.74–3.75)
1.20 (0.65–2.24)
1.46 (0.76–2.81)
6–8
1.47 (0.68–3.15)
1.71 (0.76–3.84)
0.81 (0.40–1.61)
1.12 (0.54–2.33)
>8
1.79 (0.80–3.99)
1.90 (0.81–4.42)
1.39 (0.69–2.80)
2.01 (0.95–4.28)
Smoking Habits
1.24 (0.79–1.93)
1.54 (0.96–2.48)
1.12 (0.61–2.06)
1.20 (0.63–2.278)
* p-Value < 0.05; ** p-Value < 0.001; High Waist Circumference (cm); Normal ≤94 cm Men; ≤80 cm Women; Abdominal obesity Level 1 >
94–102 cm Men; >80–88 cm women; Abdominal obesity Level 2 > 102 cm Men; >88 cm Women. * p-Value < 0.05; ** p-Value < 0.001; High Waist Circumference (cm); Normal ≤94 cm Men; ≤80 cm Women; Abdominal obesity Level 1 >
94–102 cm Men; >80–88 cm women; Abdominal obesity Level 2 > 102 cm Men; >88 cm Women. 4. Discussion In this study, we aimed to investigate the association between lifestyle practices,
ethnic origin, as well as different measures of obesity and higher than desirable blood
pressure to identify the factors associated with hypertension and prehypertension among
Saudi men and women. To avoid bias and ensure a good representation of the Saudi
population, the sample was randomly collected from attendees of PHCCs in Jeddah whose
inhabitants cover all socioeconomic sectors and ethnicities living in Saudi Arabia [26]. Both prehypertension and hypertension were common in our study population, which
is close to previously published prevalence data for hypertension on Saudi populations,
although it should be kept in mind that our study included non-diabetic persons only
while previous studies included all people [12,14,27]. Only one of the previous studies,
however, reported the prevalence of prehypertension as 66.1%, 48.1% and 54.9% in men,
women and all people, respectively [27]. The latter study was conducted in Alkharj city
which is located in central Saudi Arabia, south of the capital Riyadh, with a population
descending from Arabic tribes, and composed of mixed urban (military and civilian), rural,
and adjacent nomadic communities [27]. In view of the reported racial disparity in the
prevalence of hypertension and prehypertension [22], our study is the first to present the
prevalence of prehypertension in the largest city on the western side of Saudi Arabia, the
population of which reflects the different ethnicities living in the country as noted from our
results. The lower prevalence in our study could be due to the exclusion of diabetic people
from our analysis. y
The prevalence of prehypertension was studied in other Gulf countries. In Oman, the
prevalence of prehypertension in a population sampled from a national screening program
of chronic non communicable diseases in primary health care institutions was reported to
be 45% [28], while in prediabetic adults the prevalence was reported to be 54.1% [29]. In a
pilot study among healthy adults in the United Arab Emirates (UAE), the prevalence of
prehypertension was 42.9% among men and 16.9% among women [30], which is close to
our own results. In Kuwait [31], and Bahrain [32], the prevalence of prehypertension in
college students was reported to be 39% and 37%, respectively, emphasizing the effect of
age on estimated prevalence. An earlier national study [14] investigated the association of various sociodemo-
graphic and lifestyle factors and hypertension only. 3. Results After adjusting for age, all measures of obesity (BMI, abdominal obesity level 2, neck
circumference, elevated waist to hip and to height ratios) were found to be associated with
elevated blood pressure in men and women. In addition, low physical activity was found
to be associated with elevated blood pressure in men, while sleep duration of ≤6 h and
sitting for ≥4 h were associated with elevated blood pressure in women. Ethnicity, and
smoking were not associated with elevated blood pressure, Table 4. Similarly, after adjusting for age, all measures of obesity (BMI, abdominal obesity level
2, neck circumference, elevated waist to hip and waist to height ratios) were also found to
be associated with hypertension in both men and women. In addition, descending from
central Asia, and, to a lesser extent, from southeast Asia and being of mixed origin were Int. J. Environ. Res. Public Health 2021, 18, 11371 8 of 14 associated with hypertension among women. However, none of other investigated lifestyle
factors showed a significant association with hypertension, as shown in Table 5. associated with hypertension among women. However, none of other investigated lifestyle
factors showed a significant association with hypertension, as shown in Table 5. 4. Discussion Saudi Arabia is a large country, with
each of its regions having its own characteristic mixture of ethnicities, dietary and lifestyle
practices. The national study was carried out on the population as a whole without dis-
tinguishing between the different regions. Furthermore, the study included people with
diabetes. This might have led to incorrect conclusions due to the effect of diabetes on diet
and lifestyle practices. y
p
Therefore, our study is the first in the Saudi kingdom to investigate the association
between lifestyle factors and increased blood pressure (prehypertension and hypertension)
among non-diabetic people in a city of mixed ethnicities. Many modifiable and non-modifiable factors have previously been reported to increase
the risk of high blood pressure. Age, ethnicity, and gender are all non-modifiable factors. Aging and being male were found to increase the prevalence of elevated blood pressure,
as prehypertension and hypertension in our study in accordance with previous Saudi
studies [12,14,27], as well as studies in other populations [28–30,33–37]. Therefore, data
were analyzed for men and women separately, and age was adjusted for when calculating
the association and risk assessment with various previously known risk factors. p
y
It was interesting to note that the effect of ethnicity was apparent among female
participants, but not significant among men, during initial analysis of data. Racial dif-
ferences in the risk of pre-hypertension and hypertension have long been reported, even
for people living in the same country [22,38]. After adjusting for age, women descending Int. J. Environ. Res. Public Health 2021, 18, 11371 9 of 14 from central Asia, and, to a lesser extent, from southeast Asia and of mixed origin had a
significantly increased risk of hypertension [OR (CI): 11.54 (2.76–48.18), 3.51 (1.22–10.13),
3.24 (1.04–10.08) respectively]. from central Asia, and, to a lesser extent, from southeast Asia and of mixed origin had a
significantly increased risk of hypertension [OR (CI): 11.54 (2.76–48.18), 3.51 (1.22–10.13),
3.24 (1.04–10.08) respectively]. p
y
Obesity, measured by different indices, has long been recognized as an indepen-
dent and important risk factor for the development of hypertension in different popula-
tions [39–43]. After adjusting for age, general obesity (BMI ≥30 kg/m2) was associated
with prehypertension and hypertension to a greater extent for men than women (OR 3.36
for prehypertension, and 5.38 for hypertension among men, and 2.65 for prehypertension,
and 3.18 for hypertension among women). 4. Discussion Measures of abdominal obesity (level 2 high
WC, high WC: height, and WC: hip ratios), as well as neck circumference, were associated
with an increased risk of prehypertension and hypertension among both men and women
to a similar extent. Obesity was associated with hypertension in an earlier Saudi study [12]
and a more recent national survey [14]. Gender difference in the association between
anthropometric measurements and hypertension was reported earlier in a large Chinese
study on older adults, indicating the need to develop gender-specific strategies for the
male and female elderly in the primary and secondary prevention of hypertension [44]. y
p
y
y p
yp
The gender difference in risk factors became more apparent when the effects of lifestyle
factors were investigated. g
Prospective epidemiological evidence has indicated that sedentary behavior, defined
as any waking behavior characterized by an energy expenditure ≤1.5 metabolic equiva-
lents while in a sitting or reclining posture [45], including office work, is associated with
increased risk of various clinical and population health problems, such as type 2 diabetes
and cardiovascular disease, hence increasing the risk of mortality [46,47]. On the other
hand, increased physical activity has been reported to be associated with lower blood
pressure [48,49]. In this study, prehypertension, but not hypertension as such, was as-
sociated with ≥4 h/day of sitting among women and with low physical activity (<the
recommended 30 min/day–5 days/week) among men. The association between elevated
blood pressure and sedentary behavior has been reported in various studies carried out
on people of different ethnicities and age groups, including children [50–56]. Moreover,
a recent systematic review and meta-analysis examined the associations between time
spent in sedentary behaviors and blood pressure in both adults and children, concluding
that for each hourly increase in self-reported sedentary behavior, there was an associated
small increase in systolic and diastolic blood pressure of 0.06 (95% CI, 0.01–0.11) and 0.20
(95% CI, 0.10–0.29) mm Hg, respectively. In addition, there was a 2% elevation in the risk
for hypertension (odds ratio, 1.02; 95% CI, 1.003–1.03) for each hourly increase in sedentary
behavior [57]. Decreased physical activity and sedentary behavior have been linked to
becoming overweight and obese [58–60]. Indeed, sedentary behavior has been reported to
be more independently and strongly associated with becoming overweight and obese than
physical activity [59,61]. 4. Discussion The association between sedentary behavior and obesity might
explain the noted association with increased blood pressure in our study. Another gender difference in the effect of lifestyle practices was noted when the
association between sleeping hours and blood pressure was examined. Prehypertension
was found to be associated with a shorter sleep duration of ≤6 h among women, but not
men. In the CARDIA sleep study, higher systolic and diastolic blood pressure were reported
to be associated with a short sleep duration and lower sleep maintenance (the percent
of the time during the sleep period spent sleeping) after adjustments for confounders
and the exclusion of participants on antihypertensive drug treatments [62]. Moreover,
extensive evidence for the association between a short sleep duration and prehypertension
and hypertension was found in a recent review which included both cross-sectional and
longitudinal epidemiologic studies [63]. However, two Saudi studies linked hypertension
to a longer sleep duration [64,65]. Both studies investigated the association between sleep
duration and various factors and health characteristics in Saudi adults without excluding
people with diabetes. In contrast, our approach was different, as we investigated the Int. J. Environ. Res. Public Health 2021, 18, 11371 10 of 14 10 of 14 association between lifestyle factors, including sleep duration, and high blood pressure in
a non-diabetic population. association between lifestyle factors, including sleep duration, and high blood pressure in
a non-diabetic population. The first study investigated the association between sleeping hours and BMI, hyperten-
sion, and hyperglycemia, concluding that people with hypertension slept for >8 h/night [64]. This study was carried out in the city of Jeddah about five years prior to our study, but the
sample collection was not representative of the population due to the use of two malls for
the recruitment of participants. In addition, they used different cut-offs to define a short
and long sleep duration, defining a short sleep duration as <7 h/night, and a longer sleep
duration as >7 h/night, with >8 h/night as a subgroup, and no adjustment for age was
carried out, even though they reported its effect. The second Saudi study was conducted
in the City of Hail in the North, measuring blood pressure only and using an electronic
questionnaire to collect data on sleep duration as well as all other variables, including
weight and height [65]. 4. Discussion They reported that a longer sleep duration during the weekend
only was associated with hypertension without adjusting for the effect of age. A recent
meta-analysis showed that in pooled data, after adjusting for age and gender, compared
with a duration of seven hours of sleep, both shorter and longer sleep duration were found
to be associated with hypertension, with a sleep duration of five hours or less having the
largest OR [66]. However, when gender specific data were analyzed, women with a short
sleep duration (sleep time ≤6 h vs. 7 h) had a significantly higher risk of hypertension,
while men showed an insignificant association, in a similar manner to our findings [66]. g
g
It has been reported that tobacco smoking is one of the main preventable causes of
hypertension and myocardial infarction [67–69]. In our study there was no association
between high blood pressure and smoking habits, after adjusting for age. This was similar
to findings from the national study [14] Even though cigarette smoking acutely increases
blood pressure, mainly through the stimulation of the sympathetic nervous system, the
available data do not present clear evidence supporting a direct causal relationship between
chronic smoking and elevated blood pressure [70]. This is supported by the evidence that
no lower blood pressure values have been observed after chronic smoking cessation [71]. Indeed, one study reported lower blood pressure levels among smokers compared to
former smokers [72], and two other studies reported increased blood pressure after smoking
cessation [73,74]. However, hypertensive smokers have been reported to be more likely to develop
severe forms of hypertension compared to nonsmokers [69,70]. Therefore, treating physi-
cians usually advise their hypertensive patients to quit smoking, which might explain the
lack of association between smoking and hypertension in this study, since many of the
nonsmokers were former smokers. Our study has many points of strengths in addition to a few limitations. The main
strength lies in the avoiding of bias in the sample selection by recruiting participants
randomly from randomly selected PHCCs representing the different geographical regions
of the city, and hence the different sociodemographic classes and ethnicities. In addition,
well-trained medical students were involved in data collection, using well-standardized
methods in a clinical setting of the health care centers. Furthermore, the accuracy of
the laboratory results was assured by performing all biochemical measurements in one
accredited laboratory. 4. Discussion The first limitation of our study lies in its cross-sectional design, which allows only
an association between studied variables, but not causation, to be suggested. Another
limitation is due to the exclusion of all people with diabetes, leading to the exclusion
of most people in the older age group; hence only a few individuals > 64 years of age
were included. 5. Conclusions and B.M.E.; writing—review and editing, all authors; visualization, S.B. and J.T.; supervision, S.B.,
H.J., R.A.-R., G.M.A. and J.A.-A.; project administration, S.B. and G.M.A.; funding acquisition, S.B.,
G.M.A. and J.T. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by King Abdulaziz University, grant number (2-140-1434-HiCi) Institutional Review Board Statement: Ethical approval was obtained from the Committee on Ethics
of Human Research at Faculty of Medicine King Abdulaziz University, part of dysglycemia risk score
study (Reference No. 338-10) and Committee on Research Ethics at the Ministry of Health, Kingdom
of Saudi Arabia. Institutional Review Board Statement: Ethical approval was obtained from the Committee on Ethics
of Human Research at Faculty of Medicine King Abdulaziz University, part of dysglycemia risk score
study (Reference No. 338-10) and Committee on Research Ethics at the Ministry of Health, Kingdom
of Saudi Arabia. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: The datasets analyzed for this study can be found at king Abdulaziz
university repository at http://www.kau.edu.sa/GetFile.aspx?id=306527&fn=RS (accessed on 26
August 2021). Acknowledgments: We thank the Deanship of Research in King Abdulaziz University in the highly
cited program for supporting this work. Acknowledgments: We thank the Deanship of Research in King Abdulaziz University in the highly
cited program for supporting this work. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. 5. Conclusions In conclusion, our study showed that age, ethnicity, obesity, as well as various lifestyle
factors, are associated with prehypertension and hypertension in a gender-specific manner. After adjusting for age, all measures of obesity (BMI, abdominal obesity level 2, neck Int. J. Environ. Res. Public Health 2021, 18, 11371 11 of 14 11 of 14 circumference, elevated waist to hip, and to height ratios) were found to be associated with
prehypertension and hypertension in men and women. In addition, hypertension among
women only was significantly increased for those descending from central Asia, and to a
lesser extent for those from southeast Asia or of mixed origin. On the other hand, high
blood pressure was found to be associated with low physical activity in men, and with a
sleep duration of ≤6 h and sitting for ≥4 h in women. Therefore, hypertension may be
prevented in the Saudis by increasing physical activity, especially because such an increase
can counter the effect of sedentary behavior according to a number of experimental studies
in overweight/obese and hypertensive adults, which reported reductions in blood pressure
when sitting is interrupted with intermittent light-intensity activity [75–78]. Moreover,
increasing physical activity will help in reducing weight and decreasing the prevalence of
obesity [79], which is another modifiable factor that has been found to be associated with
hypertension in our study. Author Contributions: Conceptualization, all authors; methodology, S.B., H.J., R.A.-R., G.M.A.,
J.A.-A., A.B. and J.T.; software, H.J., R.A.-R., G.M.A., J.A.-A., L.A., S.E. and B.M.E.; validation, S.B.,
H.J., R.A.-R., G.M.A., J.A.-A., S.E. and J.T.; formal analysis, S.B., L.A., S.E. and H.J.; investigation,
S.B., H.J., R.A.-R., G.M.A., J.A.-A., A.B. and J.T.; resources, S.B., H.J., R.A.-R., G.M.A., J.A.-A. and
A.B.; data curation, S.B., H.J., R.A.-R., G.M.A. and J.A.-A.; writing—original draft preparation, S.B. and B.M.E.; writing—review and editing, all authors; visualization, S.B. and J.T.; supervision, S.B.,
H.J., R.A.-R., G.M.A. and J.A.-A.; project administration, S.B. and G.M.A.; funding acquisition, S.B.,
G.M.A. and J.T. All authors have read and agreed to the published version of the manuscript. Author Contributions: Conceptualization, all authors; methodology, S.B., H.J., R.A.-R., G.M.A.,
J.A.-A., A.B. and J.T.; software, H.J., R.A.-R., G.M.A., J.A.-A., L.A., S.E. and B.M.E.; validation, S.B.,
H.J., R.A.-R., G.M.A., J.A.-A., S.E. and J.T.; formal analysis, S.B., L.A., S.E. and H.J.; investigation,
S.B., H.J., R.A.-R., G.M.A., J.A.-A., A.B. and J.T.; resources, S.B., H.J., R.A.-R., G.M.A., J.A.-A. and
A.B.; data curation, S.B., H.J., R.A.-R., G.M.A. and J.A.-A.; writing—original draft preparation, S.B. hmed, A.; Mahmoud, M. The prevalence of hypertension in Saudi Arabia. Saudi Med. J. 1992, 13, 548–551. 7.
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62. Knutson, K.L.; Van Cauter, E.; Rathouz, P.J.; Yan, L.L.; Hulley, S.B.; Liu, K.; Lauderdale, D.S. Association between sleep and blood
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[PubMed]
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https://openalex.org/W2886150863
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https://ora.ox.ac.uk/objects/uuid:33f3df17-a8b2-4662-ac61-d8f89ec9b8c1/files/mdb4beeb181e75d3289a4b6cebc0cb1b5
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English
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A review of the effects of unilateral hearing loss on spatial hearing
|
Hearing research
| 2,019
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cc-by
| 14,385
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a r t i c l e i n f o . . . . . . . . . . . 26
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Contents lists available at ScienceDirect Hearing Research * Corresponding author.
E-mail address: andrew.king@dpag.ox.ac.uk (A.J. King). Hearing Research 372 (2019) 17e28 Hearing Research 372 (2019) 17e28 a r t i c l e i n f o . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
2. The importance and limitations of binaural processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
3. Prevalence of unilateral hearing loss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a r t i c l e i n f o . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
4. Effects of unilateral hearing loss on the developing auditory system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
5. Adaptation to unilateral hearing loss in the mature auditory system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
6. Perceptual training for hearing-impaired listeners . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a r t i c l e i n f o Article history:
Received 26 March 2018
Received in revised form
5 July 2018
Accepted 9 August 2018
Available online 11 August 2018
Keywords:
Sound localization
Spatial release from masking
Plasticity
Binaural
Monaural spectral cue
Auditory cortex Article history:
Received 26 March 2018
Received in revised form
5 July 2018
Accepted 9 August 2018
Available online 11 August 2018
Keywords:
Sound localization
Spatial release from masking
Plasticity
Binaural
Monaural spectral cue
Auditory cortex Article history:
Received 26 March 2018
Received in revised form
5 July 2018
Accepted 9 August 2018
Available online 11 August 2018 The capacity of the auditory system to extract spatial information relies principally on the detection and
interpretation of binaural cues, i.e., differences in the time of arrival or level of the sound between the
two ears. In this review, we consider the effects of unilateral or asymmetric hearing loss on spatial
hearing, with a focus on the adaptive changes in the brain that may help to compensate for an imbalance
in input between the ears. Unilateral hearing loss during development weakens the brain's represen-
tation of the deprived ear, and this may outlast the restoration of function in that ear and therefore
impair performance on tasks such as sound localization and spatial release from masking that rely on
binaural processing. However, loss of hearing in one ear also triggers a reweighting of the cues used for
sound localization, resulting in increased dependence on the spectral cues provided by the other ear for
localization in azimuth, as well as adjustments in binaural sensitivity that help to offset the imbalance in
inputs between the two ears. These adaptive strategies enable the developing auditory system to
compensate to a large degree for asymmetric hearing loss, thereby maintaining accurate sound locali-
zation. They can also be leveraged by training following hearing loss in adulthood. Although further
research is needed to determine whether this plasticity can generalize to more realistic listening con-
ditions and to other tasks, such as spatial unmasking, the capacity of the auditory system to undergo
these adaptive changes has important implications for rehabilitation strategies in the hearing impaired. © 2018 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/). Keywords:
Sound localization
Spatial release from masking
Plasticity
Binaural
Monaural spectral cue
Auditory cortex Contents
1. Introduction . . . . . . . . . . . . a r t i c l e i n f o . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
2. The importance and limitations of binaural processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
3. Prevalence of unilateral hearing loss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
4. Effects of unilateral hearing loss on the developing auditory system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
5. Adaptation to unilateral hearing loss in the mature auditory system . . . . a r t i c l e i n f o . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
6. Perceptual training for hearing-impaired listeners . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
References . . . . . https://doi.org/10.1016/j.heares.2018.08.003
0378-5955/© 2018 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 1. Introduction potential mates or prey or avoiding and escaping from approaching
predators. This is particularly the case if the source lies beyond the
detection range of the other senses, either because it is located
outside the visual field or is too far away to be registered by other
sensory receptors. The basis for directional hearing relies princi-
pally on the fact that animals have two ears that are physically
separated on either side of the head, or in the case of some insects,
on other parts of the body. This means that, depending on the
location of the sound source, the signals reaching each ear may An ability to localize and segregate different sound sources is
extremely important for most species that can hear, often playing a
crucial role in guiding behavioral responses, such as seeking out D.P. Kumpik, A.J. King / Hearing Research 372 (2019) 17e28 18 i
1
i
l
d
l
i
(A)
l l
l diff
Research 372 (2019) 17 28 Fig. 1. Binaural cues to sound source location. (A) Interaural level differences as a
function of sound azimuth and frequency. (B) Interaural time differences as a function
of sound azimuth and frequency. Negative values indicate azimuths and corresponding
binaural cue values on the left of the midline. Data for both cues are derived from
head-related transfer function measurements (0 elevation) published in the CIPIC
database by Algazi et al. (2001). (Copyright (c) 2001 The Regents of the University of
California. All Rights Reserved). g
(
) differ in their time of arrival or intensity, giving rise to binaural
spatial cues (reviewed by Blauert, 1997; Schnupp et al., 2011). A large number of studies have demonstrated the importance of
binaural
cues
for
sound-source
localization,
as
well
as
for
improving the perception of target sounds in the presence of other,
interfering sounds (Bronkhorst, 2000), and a great deal is known
about how these cues are processed in the brain (reviewed by
Grothe et al., 2010). By eliminating binaural cues, or at least altering
the relationship between the interaural acoustic differences and
directions in space, unilateral or asymmetric hearing loss can have
very disruptive effects on spatial hearing. Furthermore, monaural
deprivation in infancy can induce maladaptive changes in the brain
that may persist even if hearing in the affected ear is restored,
resulting in longer-term deficits in spatial hearing (Kaplan et al.,
2016). 2. The importance and limitations of binaural processing Because cochlear hair cells are tuned to different sound fre-
quencies, with their topographically organized outputs producing
tonotopic maps throughout the core or lemniscal regions of the
central
auditory
pathway,
sound-source
location
has
to
be
computed through the sensitivity of neurons to the physical cues
generated by the geometry of the head and external ears. For sound
sources located to one side of the midline, frequency-dependent
interaural level differences (ILDs) may be generated by a combi-
nation of the spectral filtering effects produced by the external ears
and the attenuation at the far ear due to the acoustic shadow cast by
the head (Fig. 1A). In addition, the difference in path length from
the sound source to each ear produces an interaural time difference
(ITD) whose magnitude is determined by both the distance be-
tween the ears and the angle subtended by the source relative to
the head (Fig. 1B). Fig. 1. Binaural cues to sound source location. (A) Interaural level differences as a
function of sound azimuth and frequency. (B) Interaural time differences as a function
of sound azimuth and frequency. Negative values indicate azimuths and corresponding
binaural cue values on the left of the midline. Data for both cues are derived from
head-related transfer function measurements (0 elevation) published in the CIPIC
database by Algazi et al. (2001). (Copyright (c) 2001 The Regents of the University of
California. All Rights Reserved). Because of the tonotopic organization of the auditory system,
these binaural comparisons mostly take place within frequency-
specific channels. For simple periodic sounds, such as pure tones,
the temporal fine structure is represented by the phase-locked
discharges of auditory neurons at relatively low frequencies only
(e.g., Sumner and Palmer, 2012). Moreover, interaural phase dif-
ferences become spatially ambiguous as the sound frequency is
increased (Mills, 1972; Blauert, 1997). Conversely, the ILDs gener-
ated by the shadowing effect of the head are the dominant locali-
zation cue when the wavelength of the sound is less than the
distance between the two ears and therefore for adult humans at
frequencies above ~1700 Hz (Fig. 1A). This provides the basis for the
duplex theory of sound localization (Strutt, 1907), whereby ITDs
and ILDs are utilized for localizing low-frequency and high-
frequency sounds, respectively. the source is varied (Blauert,1997; Carlile et al., 2005). 1. Introduction However, as described in the following sections, there is
growing evidence that the plasticity of central auditory processing
can help to partially compensate for loss of hearing in one ear,
leading to some recovery in the ability to localize sound (e.g. Knudsen et al., 1984; Keating et al., 2013, 2015, 2016). In this article,
we review the effects of asymmetric hearing loss on spatial pro-
cessing, both during development and in later life, and consider the
factors that may promote adaptive changes in the brain and their
potential clinical relevance. 4. Effects of unilateral hearing loss on the developing
auditory system Experimental studies of the effects of unilateral hearing loss on
spatial hearing have focussed primarily on the consequences of
conductive loss (reviewed in Keating and King, 2013). It is impor-
tant to note that sensorineural hearing loss can produce spec-
trotemporal processing deficits that would be expected to affect
neural sensitivity to spatial localization cues (Moore, 1996; Felix
and Portfors, 2007; Trujillo and Razak, 2013). Nevertheless,
inducing a conductive hearing loss in one ear has the great
advantage from an experimental perspective that it is, in principle,
fully reversible. For example, monaural occlusion can be used in
both humans and animals to produce a temporary imbalance in
input between the two ears. From a clinical standpoint, under-
standing how the brain responds to conductive hearing loss can
provide insight into the consequences of otitis media with effusion
and other disorders that affect sound transmission through the
external or middle ear. g (
g
)
Although spatial release from masking can occur in the absence
of subjective sound localization, two key processes that support
this phenomenon, better-ear listening and binaural unmasking, are
explicitly dependent upon the interaural disparities that arise
when a sound source is located to one side of the head. Better-ear
listening can improve the signal-to-noise-ratio (SNR) at one ear for
a target sound if the masker is attenuated due to the shadowing
effect of the head. In realistic speech-in-noise scenarios, such as
when multiple spatially-separated maskers are present, the better
ear may fluctuate over time and frequency. Consequently, better-
ear effects are thought to result from the auditory system's ability
to “glimpse” these short-term changes in SNR (e.g., Brungart and
Iyer, 2012; Schoenmaker and van de Par, 2017). In contrast to
better-ear effects, binaural unmasking involves a comparison of
information at the two ears (Licklider, 1948; Durlach, 1963). Detection thresholds for bilaterally presented pure tones in noise
can be up to 15 dB lower when the phase of either signal is fully
inverted in one ear, a measure known as the binaural masking level
difference. Similar manipulations are known to improve the intel-
ligibility of masked speech (measured using the binaural intelligi-
bility level difference; Levitt and Rabiner, 1967). 3. Prevalence of unilateral hearing loss Estimates of the prevalence of unilateral hearing loss, in which
the impairment is restricted to one ear, vary with numerous factors,
including the age of the subjects and, of course, the type and extent
of the hearing loss (e.g. Bess et al., 1998; Lee et al., 2011; Berninger
and Westling, 2011). For example, minimal sensorineural hearing
loss in one ear (15e40 dB HL) has been reported in 3% of sampled
school-age children (Bess et al., 1998). In terms of the potential
impact on spatial hearing, it is just as important to consider
asymmetric hearing loss, where both ears might be affected but to
differing degrees. This is particularly the case in young children,
where the changing incidence of either unilateral or bilateral otitis
media with effusion with age is likely to provide highly variable
experience of spatial cues for the majority of individuals (Hogan
et al., 1997; Whitton and Polley, 2011). Furthermore, treatments
for hearing loss may actually exacerbate asymmetric hearing, such
as when individuals with severe to profound bilateral deafness
receive a cochlear implant in one ear only or sequentially in the two
ears. This may also be the case if there is a delay in providing a
device to the affected ear when hearing loss is unilateral. Fig. 2. Monaural spectral cues to sound source elevation. Pinna gain for the right ear is
shown as a function of sound frequency and elevation for sounds presented at 0 az-
imuth. Data are derived from head-related transfer function measurements published
in the CIPIC database by Algazi et al. (2001). (Copyright (c) 2001 The Regents of the
University of California. All Rights Reserved). (or target detection) thresholds in the presence of interfering
sounds when the target and masking sounds are spatially sepa-
rated. Spatial release from masking is one process that can support
auditory stream segregation, including the capacity to perceive a
particular speaker's voice in “cocktail-party” situations, where
other, interfering, sounds are simultaneously present (Cherry,1953;
Middlebrooks, 2017). The improvement in speech intelligibility that
results from the spatial separation of the sound sources varies in
magnitude with the nature of the masker, with a greater benefit
being obtained with informational masking than with energetic
masking (Arbogast et al., 2002). 2. The importance and limitations of binaural processing Spectral cues
are also important in the horizontal plane as they provide the basis
for determining whether sound sources are located in front or
behind the listener, and therefore for resolving the cones of
confusion that are inherent in the way binaural cues vary with
spatial location (Blauert, 1997; Schnupp et al., 2011). Although
these cues otherwise appear to contribute little to localization in
the horizontal plane, which instead relies principally on ITDs and
ILDs (Macpherson and Middlebrooks, 2002), we shall show in the
following that the relative weighting of the cues used to localize
sound sources in azimuth can change with experience, particularly
following hearing loss in one ear. Because of their unusual ear asymmetry, barn owls are able to
use the two binaural cues for localizing sounds at any angle relative
to the head, relying on ILDs in the vertical plane while ITDs provide
the principal basis for localization in the horizontal plane (Knudsen
and Konishi, 1979). In most other species, however, the skull is
symmetrical, with the values of both binaural cues varying pre-
dominantly in the horizontal plane. Vertical localization relies
instead on spectral localization cues (Fig. 2), i.e., frequency-
dependent changes in the level of the sound as the location of In addition to providing a basis for localizing sounds, the ability
to extract interaural information facilitates target detection in noisy
environments (Darwin, 2006; Eramudugolla et al., 2008; Maddox
and Shinn-Cunningham, 2012), a phenomenon known as spatial
release from masking. This refers to the change in speech reception D.P. Kumpik, A.J. King / Hearing Research 372 (2019) 17e28 19 Fig. 2. Monaural spectral cues to sound source elevation. Pinna gain for the right ear is
shown as a function of sound frequency and elevation for sounds presented at 0 az-
imuth. Data are derived from head-related transfer function measurements published
in the CIPIC database by Algazi et al. (2001). (Copyright (c) 2001 The Regents of the
University of California. All Rights Reserved). localization and spatial release from masking depend on binaural
processing and will therefore be impaired, particularly if the target
sound is located on that side, if hearing is lost in one ear. 4. Effects of unilateral hearing loss on the developing
auditory system For example, Slattery and Middlebrooks (1994) compared the
localization ability of monaural subjects who had congenital deaf-
ness in one ear with that of normal-hearing controls wearing a
monaural earplug to simulate asymmetric hearing loss. Monaural
occlusion in the controls severely disrupted sound localization in
the horizontal plane, with the subjects displaying a large lateral
response bias towards the open ear, and also affected vertical
localization, particularly on the side of the plugged ear. In contrast, The capacity of the developing auditory system to compensate
for asymmetric hearing loss by becoming more dependent on the
spectral cues generated by the intact ear has been demonstrated
most clearly by rearing ferrets with one ear occluded with earplugs
that attenuated acoustical inputs by 15e45 dB in a frequency-
dependent fashion and delayed them by ~110 ms (Keating et al.,
2013, 2016). The use of an animal model affords more control
over the age of onset and duration of the hearing loss than is
possible in studies in people. In these experiments, monaural oc-
clusion began at around 4 weeks of age, corresponding to the onset
of auditory function in this altricial species, and continued for
several months until the animals were fully grown. During this
time, brief intermittent periods of normal hearing were provided
by removing the earplug, in order to more closely mimic the fluc-
tuating periods of hearing loss associated with otitis media with
effusion. Fig. 3. Binaural masking level difference (BMLD) in 19 patients before and after sur-
gery to correct congenital unilateral hearing loss resulting from an abnormal external
and/or middle ear on one side. The BMLD (N0S0 minus N0Sp) is the difference in
detection threshold of a tone presented either in phase or with the phase reversed
between the ears in the presence of broadband noise, which was always presented in
phase at the two ears. Some subjects had post-operative MLDs in the normal range,
whereas others showed a persistent deficit in binaural processing. Modified with
permission from Wilmington et al. (1994). The animals were trained to perform a free-field sound locali-
zation task in which noise bursts were presented from one of 12
loudspeakers positioned at 30 intervals around the perimeter of
the testing chamber (Fig. 5A). 4. Effects of unilateral hearing loss on the developing
auditory system The
physiological changes induced by unilateral or asymmetric stimu-
lation can be interpreted in terms of competitive interactions tak-
ing place in the developing brain between each ear. From a clinical
perspective, they likely underpin the condition of amblyaudia or
“lazy ear”, the persistent deficit in binaural processing experienced
by people with a developmental history of asymmetric hearing loss
(Snik et al., 1994; Kaplan et al., 2016). The consequences have been
found to include impairments in sound localization and binaural
unmasking, which can outlast restoration of function to the pre-
viously deprived ear (Clements and Kelly, 1978; Beggs and
Foreman, 1980; Pillsbury et al., 1991; Wilmington et al., 1994;
Moore et al., 1999; Gray et al., 2009) (Fig. 3). electrophysiological recordings, and observed impaired binaural
integration, with greater reorganization in the primary auditory
cortex (A1) than in the inferior colliculus (IC). Furthermore, they
found that this plasticity is more pronounced in infancy than in
older animals. Other electrophysiological studies have also re-
ported that abnormal binaural processing is present after correc-
tion of the unilateral hearing loss (Clopton and Silverman, 1977;
Silverman and Clopton, 1977; Brugge et al., 1985) or following
stimulation via bilateral cochlear implants (Tillein et al., 2016). The
physiological changes induced by unilateral or asymmetric stimu-
lation can be interpreted in terms of competitive interactions tak-
ing place in the developing brain between each ear. From a clinical
perspective, they likely underpin the condition of amblyaudia or
“lazy ear”, the persistent deficit in binaural processing experienced
by people with a developmental history of asymmetric hearing loss
(Snik et al., 1994; Kaplan et al., 2016). The consequences have been
found to include impairments in sound localization and binaural
unmasking, which can outlast restoration of function to the pre-
viously deprived ear (Clements and Kelly, 1978; Beggs and
Foreman, 1980; Pillsbury et al., 1991; Wilmington et al., 1994;
Moore et al., 1999; Gray et al., 2009) (Fig. 3). although two of the monaural patients tested gave similar results to
the controls, the other three showed little or no lateral response
bias and localized sounds on their deaf and hearing sides equally
well. Slattery and Middlebrooks (1994) proposed that these lis-
teners had learned to use the spectral cues of their intact ear to
judge the lateral angle of a sound source, but also noted that the
head-shadow effect may have influenced their performance. 4. Effects of unilateral hearing loss on the developing
auditory system Subsequent work in monaural listeners has confirmed this (Van
Wanrooij and Van Opstal, 2004; Agterberg et al., 2014). Thus,
relative to binaural controls, the horizontal localization judgments
of monaural humans are much more affected by stimulus level,
suggestive of a dependence on the attenuating effects of the head. Furthermore, in some cases, performance was found to be impaired
by degrading spectral cues either by filling the concha of the intact
ear with wax or by using low-frequency sounds where those cues
provide little directional information. Monaural subjects appear to
be quite variable, however, in their capacity to use spectral cues to
localize in azimuth (Van Wanrooij and Van Opstal, 2004; Agterberg
et al., 2014). Apart from individuals with total deafness in one ear, an
important consideration is whether plasticity in the processing of
spectral cues can enhance the localization accuracy of subjects with
partial hearing loss and who may therefore have access to binaural
cues that provide conflicting spatial information. Human listeners
with a normal history of binaural hearing during childhood who
then experience impaired hearing in one ear, due either to acquired
conductive hearing loss (Agterberg et al., 2012) or the presence of
an earplug (Van Wanrooij and Van Opstal, 2007), can use spectral
cues to localize low-level broadband sounds that are insufficiently
loud to reach the affected ear. However, based on the degradation
in performance observed at higher sound levels when the input to
the impaired ear is further reduced by covering it with a muff,
Agterberg et al. (2012) concluded that listeners with acquired
unilateral conductive hearing loss are also able to use their
abnormal binaural cues to localize sounds in azimuth (Fig. 4). Although these findings are indicative of maladaptive plasticity
in binaural processing following unilateral hearing loss, other
changes can take place that help to compensate for the impaired
spatial hearing that would otherwise be observed. As previously
mentioned, monaural spectral cues normally appear to contribute
little to lateral location judgments (Macpherson and Middlebrooks,
2002). However, several studies have reported that some human
listeners with single-sided deafness or severe-to-profound hearing
loss in one ear can localize broadband or high-pass noise stimuli
accurately in the horizontal plane (Newton, 1983; Slattery and
Middlebrooks, 1994;
Van
Wanrooij
and
Van
Opstal,
2004;
Rothpletz et al., 2012; Agterberg et al., 2014; Firszt et al., 2017). 4. Effects of unilateral hearing loss on the developing
auditory system A number of studies in animals have examined the effects of
unilateral hearing loss during development on the morphology
(Coleman and O'Connor, 1979; Webster and Webster, 1979; Moore
et al., 1989), connectivity (Moore et al., 1989) and response prop-
erties (Clopton and Silverman, 1977; Silverman and Clopton, 1977;
Moore and Irvine, 1981; Brugge et al., 1985; Popescu and Polley,
2010; Polley et al., 2013; Keating et al., 2013, 2015) of neurons at
different levels of the auditory system. The results of many (though
not all) of these studies are consistent with unilateral hearing loss
causing a weakening of the representation of the deprived ear and a
strengthening of the representation of the intact ear. Similarly,
chronic stimulation of one ear via a cochlear implant during early
life has been shown to result in a pronounced reorganization of
cortical responses in humans (Gordon et al., 2013) and cats (Kral
et al., 2013) in favor of the stimulated ear. Much less attention has been given to the role of spectral cues in
spatial release from masking. Because the spectral filtering pro-
vided by the head, and particularly the external ears, is direction
dependent, this will contribute to the better-ear effect at high
sound frequencies. Indeed, there is some evidence that speech
intelligibility in the presence of spatially-separated masking noise
improves if natural spectral cues are available than when they are
not
(Rychtarikova
et
al.,
2011). Nevertheless,
both
sound In terms of the consequences of unilateral or asymmetric
hearing loss on spatial hearing, it is important to ask what effect
this shift in aural preference has on neural sensitivity to binaural
cues. Popescu and Polley (2010) addressed this by rearing rats with
one ear canal ligated, which was reversed prior to carrying out D.P. Kumpik, A.J. King / Hearing Research 372 (2019) 17e28 20 electrophysiological recordings, and observed impaired binaural
integration, with greater reorganization in the primary auditory
cortex (A1) than in the inferior colliculus (IC). Furthermore, they
found that this plasticity is more pronounced in infancy than in
older animals. Other electrophysiological studies have also re-
ported that abnormal binaural processing is present after correc-
tion of the unilateral hearing loss (Clopton and Silverman, 1977;
Silverman and Clopton, 1977; Brugge et al., 1985) or following
stimulation via bilateral cochlear implants (Tillein et al., 2016). 4. Effects of unilateral hearing loss on the developing
auditory system These data
were obtained without using the subject's bone-anchored device (BCD off) (A), and in the BCD off condition with an additional muff over the impaired ear to further alter binaural
cues (B). The gains of responses (obtained from the slopes of the regression lines fitted to the data) to stimuli with levels of 55 dB SPL (solid gray regression lines) and 65 dB SPL
(solid black regression lines) decreased significantly when the impaired ear was covered with the muff, indicating that the subjects were relying on binaural cues for localization,
whereas this was not the case at the lower level of 45 dB SPL (gray dashed regression lines), which was unlikely to be audible at the deprived ear. g ¼ response gain. Reproduced
with permission from Agterberg et al. (2012). Fig. 4. Unaided sound-localization responses for one subject with a unilateral conductive hearing loss in the left ear. The stimulus was broadband noise (0.5e20 kHz). These data
were obtained without using the subject's bone-anchored device (BCD off) (A), and in the BCD off condition with an additional muff over the impaired ear to further alter binaural
cues (B). The gains of responses (obtained from the slopes of the regression lines fitted to the data) to stimuli with levels of 55 dB SPL (solid gray regression lines) and 65 dB SPL
(solid black regression lines) decreased significantly when the impaired ear was covered with the muff, indicating that the subjects were relying on binaural cues for localization,
whereas this was not the case at the lower level of 45 dB SPL (gray dashed regression lines), which was unlikely to be audible at the deprived ear. g ¼ response gain. Reproduced
with permission from Agterberg et al. (2012). substantial degree to the asymmetric hearing loss (King et al.,
2000; Keating et al., 2013, 2015) (Fig. 5B and C). achieved via a partial compensatory adjustment in ILD sensitivity
(Keating et al., 2015) (Fig. 6). Both forms of adaptation can be
observed in the same animals, with largely separate populations of
A1 neurons showing adaptive plasticity in the processing of
monaural spectral cues and binaural cues (Keating et al., 2016). Following removal of the earplug, these animals were able to
localize sounds as accurately as the controls (Fig. 5C). This lack of an
after-effect argues against the basis for adaptation being a sys-
tematic remapping of sensitivity to the altered binaural cues. 4. Effects of unilateral hearing loss on the developing
auditory system To
examine the role of pinna cues at the non-occluded ear, Keating
et al. (2013) randomized the spectrum of the broadband noise
bursts across trials so that it was not possible to determine whether
spectral features were due to the filtering effects of the head and
ears or were instead properties of the stimulus itself. When tested
with the ear plugged, sound localization performance in ferrets
raised with one ear occluded declined as the amount of spectral
randomization was increased, but this effect largely disappeared
once the earplug was removed (Fig. 5D). In other words, the ani-
mals' horizontal localization behavior was guided by spectral cues
in the asymmetric hearing loss condition, but not when normal
binaural inputs were available. This was confirmed by calculating
the mean stimulus spectrum preceding responses to each of the 12
loudspeaker locations, which revealed high-frequency spectral
features that matched the directional transfer function of the intact
ear. Electrophysiological recordings from these animals showed
that A1 neurons carried more information about the spectral cues
available at the non-occluded ear, but again only when a conductive
hearing loss was applied to the previously occluded ear (Keating
et al., 2013) (Fig. 5E and F). It is unclear whether providing these animals with intermittent
episodes of normal hearing while they were being raised with one
ear occluded is required for the observed adaptation in their spatial
hearing abilities. However, it has been shown that providing cats
with brief periods of binocular vision during development can
reduce the amblyopia, or loss of visual acuity, that would otherwise
result from monocular deprivation (Mitchell et al., 2003, 2011). It is
therefore possible that some experience of normal hearing during
development may be necessary if spatial hearing abilities are to be
preserved following asymmetric hearing loss, which has implica-
tions for the timing of treatment in children with hearing disorders
(Gordon et al., 2013; Keating and King, 2013). 4. Effects of unilateral hearing loss on the developing
auditory system The performance of the animals was
assessed as the duration, level and spectral composition of the
stimulus were varied, by measuring both the accuracy and latency
of the initial head orienting response made following sound pre-
sentation
and
the
loudspeaker/reward
spout
subsequently
approached. As expected, acute monaural occlusion in normally-
reared control animals resulted in an immediate decline in locali-
zation accuracy (Fig. 5C). However, ferrets raised with an earplug
placed in one ear and tested with that ear still occluded were able
to localize broadband sounds reasonably well at all locations tested,
indicating that the developing auditory system had adapted to a Fig. 3. Binaural masking level difference (BMLD) in 19 patients before and after sur-
gery to correct congenital unilateral hearing loss resulting from an abnormal external
and/or middle ear on one side. The BMLD (N0S0 minus N0Sp) is the difference in
detection threshold of a tone presented either in phase or with the phase reversed
between the ears in the presence of broadband noise, which was always presented in
phase at the two ears. Some subjects had post-operative MLDs in the normal range,
whereas others showed a persistent deficit in binaural processing. Modified with
permission from Wilmington et al. (1994). D.P. Kumpik, A.J. King / Hearing Research 372 (2019) 17e28 21 Fig. 4. Unaided sound-localization responses for one subject with a unilateral conductive hearing loss in the left ear. The stimulus was broadband noise (0.5e20 kHz). These data
were obtained without using the subject's bone-anchored device (BCD off) (A), and in the BCD off condition with an additional muff over the impaired ear to further alter binaural
cues (B). The gains of responses (obtained from the slopes of the regression lines fitted to the data) to stimuli with levels of 55 dB SPL (solid gray regression lines) and 65 dB SPL
(solid black regression lines) decreased significantly when the impaired ear was covered with the muff, indicating that the subjects were relying on binaural cues for localization,
whereas this was not the case at the lower level of 45 dB SPL (gray dashed regression lines), which was unlikely to be audible at the deprived ear. g ¼ response gain. Reproduced
with permission from Agterberg et al. (2012). Fig. 4. Unaided sound-localization responses for one subject with a unilateral conductive hearing loss in the left ear. The stimulus was broadband noise (0.5e20 kHz). 5. Adaptation to unilateral hearing loss in the mature
auditory system Reversible manipulation of acoustic localization cues has been
widely used to probe the adaptive capabilities of the auditory
system in adulthood. Plasticity during development is clearly
important for calibrating neural circuits during the period when
these cues are naturally changing in value as the head and ears
grow (Schnupp et al., 2003). It also provides a potential means of
adjusting to recurring periods of hearing loss that may be experi-
enced during infancy (Hogan et al.,1997; Whitton and Polley, 2011). Although studies in barn owls (Knudsen et al., 1984; Knudsen,
1985) and rodents (Popescu and Polley, 2010; Polley et al., 2013)
have shown that changes in binaural processing in response to
asymmetric hearing loss are restricted to, or at least most pro-
nounced during, a sensitive period of development, there is now
overwhelming evidence in mammals that the adult brain can also This strategy of up-weighting spectral cues in a context-
dependent fashion following a history of asymmetric hearing loss
enables accurate sound localization to be maintained irrespective
of whether the hearing loss is present or not in the other ear. In fact,
the auditory system possesses an even greater capacity for ac-
commodating abnormal spatial cues. If access to spectral localiza-
tion cues is minimized by using narrowband noise bursts as stimuli,
ferrets raised with one ear occluded still exhibit adaptive plasticity
in both their behavioral and cortical responses, but this is now D.P. Kumpik, A.J. King / Hearing Research 372 (2019) 17e28 22 p
,
J
g /
g
(
) 5. Adaptation to asymmetric hearing loss during infancy can be achieved by reweighting auditory spatial cues. (A) Schematic of setup used for measuring sound localization in
horizontal plane by adult ferrets. Twelve loudspeakers were located at 30 intervals around the perimeter of the apparatus. 0 is straight ahead, with negative numbers
ating locations to the animal's left. A trial was initiated by the animal licking a spout at the center of the chamber. This triggered the presentation of a burst of broadband noise
a flat spectrum from one of the loudspeakers; the animal received a water reward for making a correct approach-to-target response. (B) Average joint distributions of stimulus
response location for ferrets raised wearing an earplug in the left ear (interspersed with brief periods of normal hearing); the size of the circles represents the proportion o
for each stimulus-response combination. 5. Adaptation to unilateral hearing loss in the mature
auditory system Thus,
different strategies for recovering sound localization accuracy in
the presence of asymmetric hearing loss are also present in
adulthood, with individual subjects varying in the extent to which
they adapted by cue reweighting or cue remapping (Keating et al.,
2016). Several studies have shown that horizontal localization by adult
humans can adapt to varying degrees to asymmetric hearing loss
induced by occluding one ear, resulting in a partial recovery in their
ability to localize sound (Bauer et al., 1966; Butler, 1987; Kumpik
et al., 2010; Irving and Moore, 2011; Keating et al., 2016). An
important question is whether this plasticity is driven solely by
training
on
the
localization
task
or
whether
other
factors
contribute. Although listeners with normal hearing can learn
within a few hours to reinterpret the relationship between auditory
localization cues and directions in space (e.g., Mendonça et al.,
2013; Shinn-Cunningham et al., 1998; Zahorik et al., 2006), the
spacing of the trials seems to be important for adaptation to
hearing loss in one ear (Musicant and Butler, 1980; Kumpik et al.,
2010). For example, Kumpik et al. (2010) observed steady im-
provements in performance in subjects who wore an earplug all
day (except during showering or sleep) if the sound localization
training was distributed across several days, but not in a second
group who completed a similar number of trials compressed into
one day. This implies that a period of memory consolidation may be
required for adaptation to asymmetric hearing loss. The behavioral plasticity observed in ferrets raised with a uni-
lateral conductive hearing loss is mirrored by changes in the pro-
cessing of sound localization cues in A1 (Keating et al., 2013, 2015). However, adaptive changes in the auditory spatial tuning of neu-
rons in the superior colliculus have also been described in
monaurally-deprived animals (King et al., 1988, 2000), so the site of
plasticity remains unclear. The ability of adult ferrets to compen-
sate with training to temporary loss of hearing in one ear requires a
functioning auditory cortex (Nodal et al., 2012), but is also impaired
if the layer V neurons in A1 that project to the IC are selectively
eliminated (Bajo et al., 2010). with error bars showing bootstrapped 95% confidence intervals. Acutely plugging one ear (‘Plug’) in the normally-raised control ferrets caused a substantial drop in localization
accuracy. Significantly higher scores were achieved by the juvenile-plugged ferrets, and these animals localized just as accurately as the control group when the earplug was
removed (‘No plug’). (D) Effect of disrupting spectral cues by increasing the degree of spectral randomization in the stimuli on localization accuracy by juvenile-plugged animals
with and without an earplug in place. (E) Recordings were made bilaterally in the primary auditory cortex (A1) of these animals. (F) Neurons in juvenile-plugged animals were more
sensitive to the monaural spatial cues provided to the intact ear and less sensitive to the other available cues; this is indicated by the higher weighting index (mean ± 95% confidence
intervals) in juvenile-plugged animals than in the control group (whose mean values are indicated by the horizontal dashed lines). Increased weighting of spectral cues in juvenile-
plugged animals was observed only when a virtual earplug was introduced to the previously occluded ear during the recordings. Adapted with permission from Keating et al. (2013). 5. Adaptation to unilateral hearing loss in the mature
auditory system Thus, although the auditory cortex
plays a critical role in spatial hearing and in the experience-
dependent plasticity that allows the brain to compensate for
asymmetric reversible hearing loss, its descending projections
appear to play a specific role in retraining the auditory system. Experiments in monaurally-plugged adult ferrets have shown
that the extent of the recovery in localization accuracy is deter-
mined by the frequency of training (Kacelnik et al., 2006). These
animals adapted more quickly and more extensively when pro-
vided with daily training than when the training sessions were
more spread out, even though the same overall number of trials
were included. The ferret experiments also demonstrated that the
improvements in localization accuracy were specific to auditory
training, and that neither vision nor feedback about the accuracy of
the response were required for some adaptation to take place
(Kacelnik et al., 2006). It is likely, however, that other sensory,
motor and cognitive factors may promote learning (Strelnikov et al.,
2011; Carlile et al., 2014) when abnormal auditory cues are expe-
rienced. For example, a greater improvement in auditory localiza-
tion accuracy has been observed in human listeners wearing an
earplug if performance feedback is provided, and especially if the
auditory stimuli are accompanied by spatially-congruent visual
cues (Strelnikov et al., 2011). 5. Adaptation to unilateral hearing loss in the mature
auditory system These data were obtained with the earplug in place; note the similarity in the accuracy of the localization responses on the plugged
non-plugged sides. (C) Percentage correct scores for control and juvenile-plugged groups, with individual animals denoted by symbols. Horizontal lines indicate mean values Fig. 5. Adaptation to asymmetric hearing loss during infancy can be achieved by reweighting auditory spatial cues. (A) Schematic of setup used for measuring sound localization in
the horizontal plane by adult ferrets. Twelve loudspeakers were located at 30 intervals around the perimeter of the apparatus. 0 is straight ahead, with negative numbers
indicating locations to the animal's left. A trial was initiated by the animal licking a spout at the center of the chamber. This triggered the presentation of a burst of broadband noise
with a flat spectrum from one of the loudspeakers; the animal received a water reward for making a correct approach-to-target response. (B) Average joint distributions of stimulus
and response location for ferrets raised wearing an earplug in the left ear (interspersed with brief periods of normal hearing); the size of the circles represents the proportion of
trials for each stimulus-response combination. These data were obtained with the earplug in place; note the similarity in the accuracy of the localization responses on the plugged
and non-plugged sides. (C) Percentage correct scores for control and juvenile-plugged groups, with individual animals denoted by symbols. Horizontal lines indicate mean values, D.P. Kumpik, A.J. King / Hearing Research 372 (2019) 17e28 23 learn to utilize abnormal spatial cues (reviewed by Mendonça
2014). is occluded. Perceptual learning studies carried out in listeners with
normal hearing have shown that sensitivity to binaural spatial cues
can improve with training (Wright and Fitzgerald, 2001; Kumpik
et al., 2009; Sand and Nilsson, 2014) and, as mentioned in the
previous section, binaural plasticity represents part of the basis for
adaptation to asymmetric hearing loss during infancy. Although
Kumpik et al. (2010) found no changes in ITD or ILD sensitivity over
a week long period of monaural occlusion in adult humans, during
which performance on a free-field localization task gradually
improved, exposing subjects to altered cues only during training
sessions did result in remapping of both binaural cues onto
appropriate locations (Keating et al., 2016) (Fig. 7C and D). 6. Perceptual training for hearing-impaired listeners Nevertheless, reweighting of different spatial cues is not the
only means of learning to localize sounds accurately when one ear th
id
bl
id
f
l
i
t di
th t
l
h
d
f i
d
i d
d
tili
daptation to asymmetric hearing loss during infancy by remapping the altered binaural cues onto new locations in space. (AeC) Joint distributions o
expressed as degrees (deg) azimuth, for a control ferret with normal hearing (A) and a control (B) and juvenile-plugged (JP) ferret (C) wearing an earplug
represents the number of trials (n) corresponding to each stimulus-response combination. (D) Mean unsigned error for control and earplugged ferrets, norm
orrespond to perfect and chance performance, respectively. Error bars show bootstrapped 95% confidence intervals. Controls wearing an earplug (n ¼ 6 ferre
an normal hearing controls (n ¼ 4; P < 0.001, bootstrap test). While wearing an earplug, juvenile-plugged ferrets (n ¼ 2) made smaller errors than acutely pl
, bootstrap test). (E) Mean binaural interaction (±s. e.m.) as a function of ILD across neurons recorded in A1 of control ferrets under normal hearing cond
eparately for left (n ¼ 142 units, black) and right (n ¼ 177 units, gray) A1. Best ILDs for each hemisphere are indicated by arrows. (F) Binaural interaction funct
uvenile-plugged ferrets under normal hearing conditions, which are shifted, relative to controls, in the appropriate direction to compensate for the hea
uring development. Adapted with permission from Keating et al. (2015). D.P. Kumpik, A.J. King / Hearing Research 372 (2019) 17e28 D.P. Kumpik, A.J. King / Hearing Research 372 (2019) 17e28 24 Fig. 6. Adaptation to asymmetric hearing loss during infancy by remapping the altered binaural cues onto new locations in space. (AeC) Joint distributions of stimulus and
response, expressed as degrees (deg) azimuth, for a control ferret with normal hearing (A) and a control (B) and juvenile-plugged (JP) ferret (C) wearing an earplug in the left ear. Grayscale represents the number of trials (n) corresponding to each stimulus-response combination. (D) Mean unsigned error for control and earplugged ferrets, normalized so that
0 and 1 correspond to perfect and chance performance, respectively. Error bars show bootstrapped 95% confidence intervals. Controls wearing an earplug (n ¼ 6 ferrets) made larger
errors than normal hearing controls (n ¼ 4; P < 0.001, bootstrap test). 6. Perceptual training for hearing-impaired listeners Compared with the large body of work that has examined the
effects of sound localization training in normal-hearing listeners
with unperturbed or perturbed hearing, attempts to translate such
training to clinical populations have only recently gathered pace. This is perhaps because of a prior emphasis on providing speech
recognition training (Henshaw and Ferguson, 2013; Fu et al., 2015),
and also possibly because the potential benefits of functional
plasticity in bilateral and bimodal artificial or amplified hearing for
spatial masking release and sound localization have only recently
started to be recognized. Recently, Firszt et al. (2015) provided a
rich free-field sound localization training regime with visuospatial
feedback and showed that adults with severe to profound unilateral
hearing loss can be trained to more accurately localize broadband
sounds with complex spectral and temporal structure in the hori-
zontal plane, and that this improvement generalized to the locali-
zation of monosyllabic words. This finding is somewhat analogous
to those of the monaural ear-plugging studies described above with
normal-hearing listeners, although the relative contribution of
changes in the weights given to head shadow versus spectral cues
was not quantified in that study, and so the extent to which the
improvements might generalize to real-world spatial scenes is
unclear. As with monaural deprivation during development, when
broadband sounds are used as stimuli, adaptation of auditory
localization behavior to asymmetric hearing loss in adulthood is
based on subjects learning to rely more than before on the un-
changed spectral localization cues provided by the normal ear
(Kacelnik et al., 2006; Kumpik et al., 2010; Keating et al., 2016)
(Fig. 7A and B). These findings therefore support the growing body
of evidence from studies in which spectral localization cues are
altered by mechanically reshaping the external ear (Hofman et al.,
1998; Van Wanrooij and Van Opstal, 2007; Carlile et al., 2014;
Trapeau et al., 2016; Watson et al., 2017) or by presenting virtual
acoustic
space
stimuli using
non-individualized
head-related
transfer functions (Zahorik et al., 2006; Parseihian and Katz, 2012)
for considerable plasticity in the way these cues are processed in
the brain. 6. Perceptual training for hearing-impaired listeners While wearing an earplug, juvenile-plugged ferrets (n ¼ 2) made smaller errors than acutely plugged controls
(P < 0.001, bootstrap test). (E) Mean binaural interaction (±s. e.m.) as a function of ILD across neurons recorded in A1 of control ferrets under normal hearing conditions. Data are
plotted separately for left (n ¼ 142 units, black) and right (n ¼ 177 units, gray) A1. Best ILDs for each hemisphere are indicated by arrows. (F) Binaural interaction functions (mean ± s. e.m.) in juvenile-plugged ferrets under normal hearing conditions, which are shifted, relative to controls, in the appropriate direction to compensate for the hearing loss expe-
rienced during development. Adapted with permission from Keating et al. (2015). Given the considerable evidence from earplugging studies that a
key step in compensating for an imbalance in inputs between the
two ears is to change the weighting of different spatial cues, with
the auditory brain becoming more dependent on the unchanged
spectral cues available at the non-affected ear, it is important to ask
how clinically relevant this might be. It is clear that at least some people who are deaf in one ear do indeed utilize monaural spectral
cues for localization in the horizontal plane (e.g. Newton, 1983;
Slattery and Middlebrooks, 1994; Van Wanrooij and Van Opstal,
2004). Furthermore,
the
improvement
in
sound
localization
sometimes reported in blind individuals has been attributed to
their greater sensitivity to spectral cues corresponding to lateral D.P. Kumpik, A.J. King / Hearing Research 372 (2019) 17e28 25 human listeners can relearn to localize sound after introducing an asymmetric hearing loss by occluding one ear. (A) Sound localization performan
aining session for one subject who wore an earplug in the right ear during the localization tests. Scores for each session (dots) were fitted using linear
lope values, which quantified the rate of adaptation. Relative to flat-spectrum noise (blue), much less adaptation occurred with random-spectrum n
efulness of spectral cues to sound location. (B) Adaptation rate is shown for flat- and random-spectrum stimuli for different subjects (gray lines; n ¼ 1
rovements in localization performance with training. Mean adaptation rates across subjects (±bootstrapped 95% confidence intervals) are shown in
ulus types. Dotted black lines indicate adaptation rates observed in a previous study (Kumpik et al., 2010). (C) Mean error magnitude plotted as a fu
ne subject when pure tones were used as the stimuli. 6. Perceptual training for hearing-impaired listeners Data are plotted separately for low- (1 kHz, dark blue) and high-frequency (8 kHz, light blue
was associated with a reduction in error magnitude, producing negative values for the change (D) in error magnitude. (D) D error for low- and hig
ach subject (gray lines; n ¼ 11). Mean values for D error across subjects (±bootstrapped 95% confidence intervals) are shown in blue. Although ther
fferences for the adaptation observed at the two tone frequencies, almost all values are <0, indicating that error magnitude declined over the trainin
ws D error values that would have been observed if human listeners had adapted as well as ferrets reared with a unilateral earplug (Keating et al., 20
(2016). Fig. 7. Adult human listeners can relearn to localize sound after introducing an asymmetric hearing loss by occluding one ear. (A) Sound localization performance (% correct) as a
function of training session for one subject who wore an earplug in the right ear during the localization tests. Scores for each session (dots) were fitted using linear regression (lines)
to calculate slope values, which quantified the rate of adaptation. Relative to flat-spectrum noise (blue), much less adaptation occurred with random-spectrum noise (pink), which
limits the usefulness of spectral cues to sound location. (B) Adaptation rate is shown for flat- and random-spectrum stimuli for different subjects (gray lines; n ¼ 11). Positive values
indicate improvements in localization performance with training. Mean adaptation rates across subjects (±bootstrapped 95% confidence intervals) are shown in blue and pink for
the two stimulus types. Dotted black lines indicate adaptation rates observed in a previous study (Kumpik et al., 2010). (C) Mean error magnitude plotted as a function of training
session for one subject when pure tones were used as the stimuli. Data are plotted separately for low- (1 kHz, dark blue) and high-frequency (8 kHz, light blue) tones. Improved
performance was associated with a reduction in error magnitude, producing negative values for the change (D) in error magnitude. (D) D error for low- and high-frequency tones
plotted for each subject (gray lines; n ¼ 11). Mean values for D error across subjects (±bootstrapped 95% confidence intervals) are shown in blue. Although there are pronounced
individual differences for the adaptation observed at the two tone frequencies, almost all values are <0, indicating that error magnitude declined over the training sessions. 6. Perceptual training for hearing-impaired listeners Dotted
red line shows D error values that would have been observed if human listeners had adapted as well as ferrets reared with a unilateral earplug (Keating et al., 2015). Adapted from
Keating et al. (2016). sound locations (Doucet et al., 2005; Voss et al., 2011), providing
further evidence for compensatory plasticity in the use of spectral
localization cues. can help hearing-impaired subjects to understand target speech in
the presence of spatially-separated masking speech (Carr Levy
et al., 2015), spectral cues are seriously distorted by the use of
microphones that do not sit inside the auditory canal, such as in
behind-the-ear hearing aids (Moore and Popelka, 2016). There is
some indication that inclusion of algorithms that preserve pinna
cues can improve horizontal localization and speech perception in
noise in hearing aid users (Kuk et al., 2013; Xu and Han, 2014;
Korhonen et al., 2015; Gomez and Seeber, 2017). However, this However, whether listeners provided with hearing devices can
benefit in the affected ear in a similar fashion is more questionable. However, whether listeners provided with hearing devices can
benefit in the affected ear in a similar fashion is more questionable. For one thing, the progressive loss of high-frequency hearing in
age-related sensorineural hearing loss will restrict the availability
of spectral cues. Although modern hearing aids can have band-
widths of up to 10 kHz or more (Kuk and Baekgaard, 2009), which For one thing, the progressive loss of high-frequency hearing in
age-related sensorineural hearing loss will restrict the availability
of spectral cues. Although modern hearing aids can have band-
widths of up to 10 kHz or more (Kuk and Baekgaard, 2009), which D.P. Kumpik, A.J. King / Hearing Research 372 (2019) 17e28 26 will depend on whether the hearing aids provide sufficient
amplification for individual listeners at the high frequencies where
most of the directional information is available in these cues. with acquired unilateral conductive hearing loss: improved directional hearing
with a bone-conduction device. Hear. Res. 286, 9e18. with acquired unilateral conductive hearing loss: improved directional hearing
with a bone-conduction device. Hear. Res. 286, 9e18. Agterberg, M.J., Hol, M.K., Van Wanrooij, M.M., Van Opstal, A.J., Snik, A.F., 2014. Single-sided deafness and directional hearing: contribution of spectral cues and
high-frequency hearing loss in the hearing ear. Front. Neurosci. 8, 188. 6. Perceptual training for hearing-impaired listeners The finding that listeners with one ear plugged can be trained to
partially recover their sound localization accuracy by learning to
remap the distorted ILDs and ITDs onto appropriate spatial loca-
tions (Keating et al., 2016) potentially offers much greater scope for
utilizing adaptive plasticity to promote improvements in spatial
hearing in the hearing impaired. Indeed, the results of this study
raise the possibility of adopting targeted training strategies based
on the residual hearing abilities of individual patients and therefore
the localization cues they have available. This is also relevant to
patients with cochlear implants whose limited spatial hearing
abilities can be improved if they adapt their ILD sensitivity to the
range of values provided by the output of the implants (Dorman
et al., 2014; Dorman et al., 2015) and by enhancing the availabil-
ity of ILDs at low frequencies (Brown, 2014). Recent studies have
started to examine the effects of training on cochlear implant users
who have either been implanted bilaterally or retain access to
binaural information as a result of having one good ear. There is
some indication that sound localization training can promote
binaural hearing, both with unilateral implantation when the other
ear is preserved (Nawaz et al., 2014) and with bilateral cochlear
implants (Tyler et al., 2010). Furthermore, a training paradigm in
which auditory and visual stimuli were randomly interleaved has
been shown to improve the auditory localization accuracy and
cortical coding of ILDs in adult ferrets fitted with bilateral cochlear
implants following deafening in infancy (Isaiah et al., 2014). Algazi, V.R., Duda, R.O., Thompson, D.M., Avendano, C., 2001. The CIPIC HRTF
database. In: Proceedings of the IEEE Workshop on Applications of Signal
Processing to Audio and Electroacoustics. Mohonk Mountain House, New Paltz,
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Towards an Improved Measurement of the Proton Magnetic Moment
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(Received May 24, 2016) The BASE collaboration performed the most precise measurement of the proton magnetic moment. By applying the so-called double Penning-trap method with a single proton a fractional precision of
3.3 parts-per-billion was reached. This article describes the primary limitations of the last measure-
ment and discusses improvements to reach the sub-parts-per-billion level. KEYWORDS: proton, antiproton, magnetic moment, g-factor, double trap method, CPT,
Penning trap author(s) and the title of the article, journal citation, and DOI.
This article is available under the terms of the Creative Commons Attribution 4.0 License. Any further distribution of this work must maintain attribution to the JPS Conf. Proc.
, 011018 (2017)
https://doi.org/10.7566/JPSCP.18.011018
18
Proc. 12th Int. Conf. Low Energy Antiproton Physics (LEAP2016)
Proceedings of the 12th International Conference on Low Energy Antiproton Physics (LEAP2016)
Downloaded from journals.jps.jp by 128.141.46.242 on 03/23/18 JPS Conf. Proc. , 011018 (2017)
https://doi.org/10.7566/JPSCP.18.011018
18
Proc. 12th Int. Conf. Low Energy Antiproton Physics (LEAP2016)
Proceedings of the 12th International Conference on Low Energy Antiproton Physics (LEAP2016)
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https://doi.org/10.7566/JPSCP.18.011018
18
Proc. 12th Int. Conf. Low Energy Antiproton Physics (LEAP2016) 1.
Introduction Invariance under the combined transformation of charge (C), parity (P), and time (T) is an exact
symmetry of the relativistic quantum field theories involved in the Standard Model of particle physics. Consequently the fundamental properties of matter and antimatter conjugates are predicted to be
identical, apart from signs. On cosmological scales, on the other hand, a dominance of matter over
antimatter is observed. This unexplained imbalance between antimatter and matter provides a strong motivation for
the experiments of the BASE collaboration [1, 2] which contribute to stringent CPT tests by com-
paring the fundamental properties of protons and antiprotons, in particular charge-to-mass ratios
(q/m)p/(q/m)¯p and magnetic moments µp,¯p = gp,¯p/2 · µN, where µN = qℏ/(2mp,¯p) is the nuclear
magneton with charge q and the mass m of the proton/antiproton, respectively. In 2014 we measured
the g-factor of a single trapped proton with a fractional precision of 3.3 parts-per-billion (p.p.b.) [3] gp = 5.585 694 700(14)stat(12)syst . (1) (1) Our measurement improved the 42 year old MASER based value [4] by a factor of 2.5 and is the most
precise direct measurement of gp. The experiment was carried out by applying the double Penning-trap method [5]. The g-factor
is obtained by measuring the ratio of the particle’s free cyclotron frequency νc = 1/(2π) q/m B0
and its spin precession frequency νL = gp/2 · νc, the Larmor frequency. The precision frequency
measurements are carried out in a “precision trap” with homogeneous magnetic field, while the spin
state analysis, essential for the Larmor frequency determination, is conducted in an “analysis trap” Towards an Improved Measurement of the Proton
Magnetic Moment Georg Schneider1,2, Nathan Leefer3, Andreas Mooser2, Klaus Blaum4, Takashi Higuchi2,5,
Yasuyuki Matsuda5, Hiroki Nagahama2,5, Wolfgang Quint6, Stefan Sellner2, Christian
Smorra2,7, Jochen Walz1,3, and Stefan Ulmer2 1Institut f¨ur Physik, Johannes Gutenberg-Universit¨at Mainz, 55099 Mainz, Germany
2Ulmer Initiative Research Unit, RIKEN, Saitama 351-0198, Japan
3Helmholtz-Institut Mainz, 55099 Mainz, Germany
4Max-Planck-Institut f¨ur Kernphysik, 69117 Heidelberg, Germany
5Graduate School of Arts and Sciences, University of Tokyo, Tokyo 153-8902, Japan
6GSI-Helmholtzzentrum f¨ur Schwerionenforschung, 64291 Darmstadt, Germany
7CERN, 1211 Geneva 23, Switzerland E-mail: georg.schneider@uni-mainz.de E-mail: georg.schneider@uni-mainz.de
(Received May 24, 2016) JPS Conf. Proc.
, 011018 (2017)
https://doi.org/10.7566/JPSCP.18.011018
18
Proc. 12th Int. Conf. Low Energy Antiproton Physics (LEAP2016)
Proceedings of the 12th International Conference on Low Energy Antiproton Physics (LEAP2016)
Downloaded from journals.jps.jp by 128.141.46.242 on 03/23/18 ■■■
011018-1 ©2017 The Author(s) ©2017 The Author(s) Proceedings of the 12th International Conference on Low Energy Antiproton Physics (LEAP2016)
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Downloaded from journals.jps.jp by 128.141.46.242 on 03/23/18 011018-2 JPS Conf. Proc. , 011018 (2017)
18 with a strong superimposed magnetic inhomogeneity [6]. The fractional precision achieved in these
measurements was limited by the residual magnetic field inhomogeneity in the precision trap and by
nonlinear magnetic field drifts. Both contributed to a broadening of the measured g-factor resonance
and led to systematic shifts [3]. In this article we discuss the previous limitations and describe the
implementation of experimental improvements which will allow an order of magnitude increase in
precision for the measured g-factor. Proceedings of the 12th International Conference on Low Energy Antiproton Physics (LEAP2016)
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Penning trap and frequency detection , 011018 (2017)
18 due to the E × B drift of the particle. due to the E × B drift of the particle. due to the E × B drift of the particle. The invariance theorem [9] allows the determination of the free cyclotron frequency νc by mea-
surement of ν+, νz, and ν− The invariance theorem [9] allows the determination of the free cyclotron frequency νc by mea-
surement of ν+, νz, and ν−
√ νc =
√
ν2
+ + ν2z + ν2
−. (5) (5) The axial frequency νz of the particle can be measured by applying a non-destructive image current
detection technique. The particle induces small image currents on the order of a few femto-Ampere
which are detected with a tuned superconducting LC circuit, the axial detector. The detector acts
like a thermal bath that cools the particle resistively. In thermal equilibrium the particle shorts the
Johnson-Nyquist noise of the detector and appears as a dip at νz in the fast Fourier transform (FFT)
spectrum of the detector’s time signal. This is shown in Fig. 2. (a). 633650 633670 633690
frequency (Hz)
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power (dBm)
633650 633670 633690
frequency (Hz)
power (dBm)
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-106
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-118
-120
(a)
(b)
νz
νl
νr
Fig. 2. (a) In thermal equilibrium the proton shorts the Johnson-Nyquist noise of the axial detector which
results in a single dip in the FFT spectrum. (b) A rf-drive is applied to one of the trap electrodes which couples
the modified cyclotron mode to the axial mode. As a result a double-dip can be observed. From the combined
measurements of both the single and the double-dip, the modified cyclotron frequency can be determined. 633650 633670 633690
frequency (Hz)
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power (dBm)
(a)
νz 633650 633670 633690
frequency (Hz)
power (dBm)
-104
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(b)
νl
νr Fig. 2. (a) In thermal equilibrium the proton shorts the Johnson-Nyquist noise of the axial detector which
results in a single dip in the FFT spectrum. (b) A rf-drive is applied to one of the trap electrodes which couples
the modified cyclotron mode to the axial mode. As a result a double-dip can be observed. 2.
Penning trap and frequency detection Our experiment makes use of two cylindrical Penning traps which consist of five electrodes [7] and
a superimposed magnetic field, see Fig. 1. Dimensions of the electrodes and applied voltages are
chosen such that an ideal quadrupole potential is realized in the center of the trap. The trajectory of a B
V0
Vcorr
Vcorr
sapphire ring
ring electrode
correction electrode
correction electrode
7 mm B
V0
Vcorr
Vcorr
sapphire ring
ring electrode
correction electrode
correction electrode
7 mm
Fig. 1. Schematic cylindrical Penning trap with characteristic voltages on five trap electrodes. The gold-
plated copper electrodes are separated by sapphire rings. The ring voltage V0 and the correction voltage Vcorr
lead to a quadrupole field in the trap center. This static electric field together with a superimposed magnetic
field allows stable storage of charged particles. sapphire ring Fig. 1. Schematic cylindrical Penning trap with characteristic voltages on five trap electrodes. The gold-
plated copper electrodes are separated by sapphire rings. The ring voltage V0 and the correction voltage Vcorr
lead to a quadrupole field in the trap center. This static electric field together with a superimposed magnetic
field allows stable storage of charged particles. single particle in such a Penning trap is described by the superposition of three independent harmonic
oscillator motions [8], the axial motion as a result of the electrostatic potential at single particle in such a Penning trap is described by the superposition of three independent harmonic
oscillator motions [8], the axial motion as a result of the electrostatic potential at νz = 1
2π
√
2qC2V0
m
,
(2) (2) where V0 is the voltage on the ring electrode and C2 is a trap specific geometric parameter, the
modified cyclotron motion at where V0 is the voltage on the ring electrode and C2 is a trap specific geometric parameter, the
modified cyclotron motion at ν+ = 1
2
(
νc +
√
ν2c −2ν2z
)
,
(3) (3) and the magnetron motion with frequency and the magnetron motion with frequency ν−= 1
2
(
νc −
√
ν2c −2ν2z
)
,
(4) (4) 2
■■ Proceedings of the 12th International Conference on Low Energy Antiproton Physics (LEAP2016)
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Penning trap and frequency detection From the combined
measurements of both the single and the double-dip, the modified cyclotron frequency can be determined. The modified cyclotron frequency ν+ and the magnetron frequency ν−are also measured with
the axial detector by using sideband coupling [8]. A quadrupole radiofrequency (rf) drive at approxi-
mately νrf = νz+ν−(or νrf = ν+−νz) is applied to the trap electrodes, which leads to a coupling of the
axial mode to the magnetron or cyclotron mode, respectively. As a result the single dip at νz splits into
a double-dip νr and νl as shown in Fig. 2. (b) for the modified cyclotron mode. ν+ can be calculated
with the measurement of the frequencies νr, νl and the axial frequency νz from an independent single
dip measurement (6) ν+ = νl + νr + νrf −νz ,
(6) and likewise for the magnetron frequency ν− and likewise for the magnetron frequency ν− and likewise for the magnetron frequency ν− (7) ν−= −νl −νr + νrf + νz . (7) Mode coupling is not only crucial for frequency measurements but also for changing the particle’s en-
ergy. While the coupling drive is applied, energy is exchanged between the modes and an equilibrium
is reached with ⟨nz⟩= ⟨n±⟩[8]. This leads to Mode coupling is not only crucial for frequency measurements but also for changing the particle’s en-
ergy. While the coupling drive is applied, energy is exchanged between the modes and an equilibrium
is reached with ⟨nz⟩= ⟨n±⟩[8]. This leads to ⟨E−⟩= ν−
νz
⟨Ez⟩
and
⟨E+⟩= ν+
νz
⟨Ez⟩. (8) (8) For typical axial detector temperatures of ⟨Tz⟩= 6(2) K a modified cyclotron measurement results in
an average energy of ⟨E+⟩≈kB · 270 K and ⟨E−⟩≈kB · 0.1 K after the sideband coupling. For typical axial detector temperatures of ⟨Tz⟩= 6(2) K a modified cyclotron measurement results in
an average energy of ⟨E+⟩≈kB · 270 K and ⟨E−⟩≈kB · 0.1 K after the sideband coupling. 3
■ Proceedings of the 12th International Conference on Low Energy Antiproton Physics (LEAP2016)
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18 3.
Continuous Stern-Gerlach effect and double trap method The Larmor frequency νL is proportional to the energy that is needed to change the spin state in
a magnetic field. A measurement of the spin transition rate as a function of the frequency of an
external spin-flip driving field yields the Larmor frequency. To determine the spin state the so-called
continuous Stern-Gerlach effect [10] is used which couples the spin state to the axial motion in the
presence of a magnetic inhomogeneity called “magnetic bottle”, which is deliberately superimposed
on the trap. The magnetic bottle adds Vm = −µzBz to the electric potential energy, which results in a
modified axial frequency ˜νz = 1
2π
√
2qC2V0
m
± 2µzB2
m
≈νz ±
1
4π2
µzB2
mνz
. (9) (9) This allows the determination of the spin state by measuring the axial frequency. Here B2 describes
the strength of the magnetic bottle according to Bz = B0 + B2z2. The observable axial frequency
change is then given by ∆νz, SF ≈
1
2π2
µzB2
mνz
≈170 mHz . (10) (10) Since µz and m are fixed by the particle species, and also νz is fixed by experimental constraints,
only B2 can be used to change the size of this frequency jump. In our experiment we use a magnetic
bottle with B2 = 300 000 Tm−2 resulting in a jump of 170 mHz at an axial frequency of 750 kHz. The magnetic bottle also couples the cyclotron energy E+ to the axial motion, being one of the main
challenges for spin state detection. As a result, the axial frequency shifts by Since µz and m are fixed by the particle species, and also νz is fixed by experimental constraints,
only B2 can be used to change the size of this frequency jump. In our experiment we use a magnetic
bottle with B2 = 300 000 Tm−2 resulting in a jump of 170 mHz at an axial frequency of 750 kHz. The magnetic bottle also couples the cyclotron energy E+ to the axial motion, being one of the main
challenges for spin state detection. As a result, the axial frequency shifts by ∆νz, n+→n+±1 = 1
2π
ℏν+
mνz
B2
B0
≈60 mHz
(11) (11) each time the cyclotron quantum number changes by ∆n+ = 1. The rate p+ of these changes scales
linearly as indicated by Fig. 3. 3.
Continuous Stern-Gerlach effect and double trap method (b) with the absolute quantum number n+ or equivalently, the energy
of the cyclotron motion which can be calculated by perturbation theory and the corresponding matrix
element [11]. Figure 3. (a) shows a sequence of simulated axial frequency measurements. In the
presence of cyclotron quantum jumps the spin-flip can only be observed in case of a low cyclotron
energy. zν
170 mHz
low energy
< 40 µeV
high energy
> 180 µeV
spin-fip
spin-fip
axial frequency
measurements
0.08
0.06
0.04
0.02
0.00
0
50 100
150
rate p+ (1/s)
modifed cyclotron energy (µeV)
(a)
(b)
= 0.033 s–1
K–1
= 0.38 s–1
neV–1
dp+
dE+
Fig. 3. (a) Using the continuous Stern-Gerlach effect the spin state is coupled to the axial motion. This results
in a discrete frequency change of the axial motion due to a spin-flip. The ability to detect such a spin-flip by
successive frequency measurements (dots represent simulated data) is limited by the amount of background
noise which is a result of the energy fluctuations in the cyclotron motion. The left side shows a particle with
low cyclotron energy and the underlying spin-flip is observable, while in case of high cyclotron energy on the
right side one cannot conclude that a spin-flip has occurred. (b) The rate p+ at which cyclotron quantum jumps
occur increases with the energy of the particle’s modified cyclotron motion. zν
170 mHz
low energy
< 40 µeV
high energy
> 180 µeV
spin-fip
spin-fip
axial frequency
measurements
(a) 0.08
0.06
0.04
0.02
0.00
0
50 100
150
rate p+ (1/s)
modifed cyclotron energy (µeV)
(b)
= 0.033 s–1
K–1
= 0.38 s–1
neV–1
dp+
dE+ (a) Fig. 3. (a) Using the continuous Stern-Gerlach effect the spin state is coupled to the axial motion. This resu Fig. 3. (a) Using the continuous Stern-Gerlach effect the spin state is coupled to the axial motion. This results
in a discrete frequency change of the axial motion due to a spin-flip. The ability to detect such a spin-flip by
successive frequency measurements (dots represent simulated data) is limited by the amount of background
noise which is a result of the energy fluctuations in the cyclotron motion. The left side shows a particle with
low cyclotron energy and the underlying spin-flip is observable, while in case of high cyclotron energy on the
right side one cannot conclude that a spin-flip has occurred. Proceedings of the 12th International Conference on Low Energy Antiproton Physics (LEAP2016)
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Continuous Stern-Gerlach effect and double trap method (b) The rate p+ at which cyclotron quantum jumps
occur increases with the energy of the particle’s modified cyclotron motion. Fig. 3. (a) Using the continuous Stern-Gerlach effect the spin state is coupled to the axial motion. This results
in a discrete frequency change of the axial motion due to a spin-flip. The ability to detect such a spin-flip by
successive frequency measurements (dots represent simulated data) is limited by the amount of background
noise which is a result of the energy fluctuations in the cyclotron motion. The left side shows a particle with
low cyclotron energy and the underlying spin-flip is observable, while in case of high cyclotron energy on the
right side one cannot conclude that a spin-flip has occurred. (b) The rate p+ at which cyclotron quantum jumps
occur increases with the energy of the particle’s modified cyclotron motion. 4■■ Proceedings of the 12th International Conference on Low Energy Antiproton Physics (LEAP2016)
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18 011018-5 While a large magnetic bottle B2 is essential for the determination of the spin state, it leads
to systematic shifts of the measured motional frequencies. In our case, the magnetic bottle would
limit the precision of the g-factor measurement at the p.p.m. level, if we carried out all frequency
measurements and the spin state determination in a single trap. For that reason the double Penning-
trap method [5] is used, which utilizes two traps as shown in Fig. 4. The so-called precision trap (PT)
is used to carry out precise frequency measurements and to drive spin-flips. The second trap is the
analysis trap (AT) with the strong magnetic bottle superimposed to identify the spin state. Due to the
spatial separation the magnetic field in the PT of the previous setup was by a factor of around 75 000
times more homogeneous than in the AT. This allows measurements at the p.p.b. level. 3.
Continuous Stern-Gerlach effect and double trap method –10
0
10
z
ν
rν
lν
time (min)
0
20
40
60
80
1.89 T
1.17 T
magnetic
feld
Analysis trap
contains magnetic bottle
for spin fip detection
Precision trap
used for frequency
measurements
axial frequency
signal
axial frequency - ofset (Hz)
ferromagnetic ring electrode
leads to magnetic inhomogeneity
B2 = 300 000 T m–2
residual magnetic inhomogeneity
B2 = 4 T m–2
7 mm
Fig. 4. The double trap of the Mainz g-factor proton experiment with the analysis trap (AT) for the spin state
detection and the precision trap (PT) for precise frequency determination. time (min)
0
20
40
60
80
Analysis trap
contains magnetic bottle
for spin fip detection
axial frequency Precision trap
used for frequency
measurements ferromagnetic ring electrode
leads to magnetic inhomogeneity
B2 = 300 000 T m–2 Fig. 4. The double trap of the Mainz g-factor proton experiment with the analysis trap (AT) for the spin state
detection and the precision trap (PT) for precise frequency determination. For the g-factor measurement in the double trap scheme, the proton starts in the AT where the spin
state is detected. After moving to the PT, the free cyclotron frequency is measured. At the same time
a test frequency is applied close to the Larmor frequency to induce a spin-flip. Since free cyclotron
frequency measurement and the Larmor drive are carried out at the same time the magnetic field
cancels in the frequency ratio. Finally, the proton is once again transported to the AT where the
spin state is measured a second time. Together with the first spin state measurement, this determines
whether or not a spin-flip occurred in the PT. The process is repeated several hundred times for
different test frequencies in the PT. The resulting Larmor resonance from all test frequencies together
with the free cyclotron frequency measurements is then used to determine g. Proceedings of the 12th International Conference on Low Energy Antiproton Physics (LEAP2016)
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Towards sub-p.p.b. precision To improve the measurements to the sub-p.p.b. level careful analysis of the main limitations in our last
g-factor measurement from 2014 is required. In this section we will discuss how the width of the g-
factor resonance can be decreased by reducing the magnetic inhomogeneity in the PT and by reducing
fluctuations of the magnetic field. Furthermore, particle preparation can be made more efficient. This
reduces the measurement time per data-point and thus improves statistics. 5
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18 011018-6 After a double-dip measurement the measured values of the modified cyclotron energy are expo-
nentially distributed. The energy can be measured in the AT since the axial frequency is a function of
the cyclotron energy in the presence of the magnetic bottle. The coupling of E+ to the axial frequency
in the presence of the residual magnetic bottle in the PT induces line shape broadening. In order to
reduce B2 the distance between the PT and the AT was increased while keeping the total length of
the trap assembly constant. This corresponds to a decrease in B2 = 0.5 Tm−2, which is a factor of 8
smaller than in the 2014 measurement. Other limitations to the cyclotron frequency stability are magnetic field fluctuations. For that
reason a self-shielding coil [12] was implemented with its highest shielding in the center of the PT. Such a coil keeps the surrounding magnetic field flux constant and cancels external magnetic field
fluctuations. Our coil reduces magnetic field disturbances along the trap axis by around a factor of 50. Figure 5. shows the combined result of B2 reduction and installed shielding coil and thus improved
short term stability. Compared to the previous run the cyclotron stability was improved by a factor of
10, therefore enabling g-factor measurements below the p.p.b. level. fraction (a.u.)
–60 –40 –20 0 20 40 60
2014 setup for 3 p.p.b. measurement
new setup for sub-p.p.b. measurement
rel. frequency fuctuations in 10–9
rel. frequency fuctuations in 10–9
σ = 2 . 10 –8
σ = 2 . 10 –9
(a)
(b)
–60 –40 –20 0 20 40 60
Fig. 5. Proceedings of the 12th International Conference on Low Energy Antiproton Physics (LEAP2016)
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Towards sub-p.p.b. precision Proc. , 011018 (2017)
18 proton which meets the requirements of spin state detection it is repeatedly coupled to the resonator
until it is found in the lowest temperature bin. In our 2014 measurement this particle preparation time was around 100 minutes out of the 160
minutes for the entire measurement cycle and therefore was a major contribution to the time budget of
the measurement [3]. The preparation time is set by the temperature of the resonator Tres (∼number
of required cycles) and the coupling constant tcpl (∼time per cycle). So improving either of these
would decrease the particle preparation time significantly. 0.5
0.4
0.3
0.2
0.1
0 0.5
1.0 1.5 2.0
energy (meV)
probability
0.5
0.4
0.3
0.2
0.1
0.5
0.4
0.3
0.2
0.1
Tres = 2 K
strong feedback
Tres = 3.6 K
medium feedback
Tres = 6 K
no feedback
(a)
(b)
(c)
0 0.5
1.0 1.5 2.0
energy (meV)
0 0.5
1.0 1.5 2.0
energy (meV)
Fig. 6. The proton is coupled to a cyclotron resonator with temperature Tres. After waiting for a time tcpl the
particle is in one of the bins according to the Boltzmann distributed probability. By using active electronic feed-
back the effective temperature of the detector can be reduced. (a) represents the coldest achievable temperature
for the cyclotron detector with active feedback. 0.5
0.4
0.3
0.2
0.1
Tres = 3.6 K
medium feedback
(b)
0 0.5
1.0 1.5 2.0
energy (meV) 0.5
0.4
0.3
0.2
0.1
0 0.5
1.0 1.5 2.0
energy (meV)
probability
Tres = 2 K
strong feedback
(a) 0.5
0.4
0.3
0.2
0.1
Tres = 6 K
no feedback
(c)
0 0.5
1.0 1.5 2.0
energy (meV) (a) Fig. 6. The proton is coupled to a cyclotron resonator with temperature Tres. After waiting for a time tcpl the
particle is in one of the bins according to the Boltzmann distributed probability. By using active electronic feed-
back the effective temperature of the detector can be reduced. (a) represents the coldest achievable temperature
for the cyclotron detector with active feedback. For that reason a new superconducting resonator at 28.9 MHz was built, with a quality factor
Q = ν0/∆ν = 1400 with the resonator frequency ν0 and the full width at half maximum ∆ν. 5.
Conclusion The goal of our experiment is to measure the g-factor of the proton at the sub-p.p.b. level. Since the
previous measurement in 2014 with a relative precision of 3.3 p.p.b. several upgrades have been made,
including a smaller residual magnetic field inhomogeneity in the precision trap, the installation of a
self shielding coil and the implementation of a new cyclotron detector for faster particle preparation. These optimizations should allow to improve the previous result by around one order of magnitude. In the future, these techniques can be applied to the antiproton, providing a stringent sub-p.p.b. test of CPT symmetry. In the future, these techniques can be applied to the antiproton, providing a stringent sub-p.p.b. test of CPT symmetry. 4.
Towards sub-p.p.b. precision Through
active electronic feedback [17, 18] the actual temperature of the detector can be decreased to Tres =
2 K, compared to Tres,old = 5 K for the previously used cyclotron resonator with feedback in 2014
[19]. Figure 6. (a)-(c) shows three temperature measurements with and without feedback. Hereby
the proton is coupled to the thermal bath and its energy is measured subsequently. Repeating this
for several times one obtains a Boltzmann distribution which characterizes the thermal bath and
thus the temperature of the cyclotron detector. Besides the improved temperature also the coupling
time constant of the new cyclotron detector is by a factor of 2 better with tcpl = 60 s, compared
to tcpl = 120 s of the previous one. In total the new detector allows a factor of four times faster
particle preparation that reduces the total measurement cycle time by a factor of two which doubles
the amount of accumulated data within the same time window. 4.
Towards sub-p.p.b. precision (a) shows the cyclotron stability for the measurement in 2014 while (b) shows the result for the new
setup with smaller residual magnetic field in the PT and the self-shielding coil installed. The improvements
allow an increase of the g-factor’s measurement precision by at least one order of magnitude. rel. frequency fuctuations in 10–9 Fig. 5. (a) shows the cyclotron stability for the measurement in 2014 while (b) shows the result for the new
setup with smaller residual magnetic field in the PT and the self-shielding coil installed. The improvements
allow an increase of the g-factor’s measurement precision by at least one order of magnitude. Independent measurements of the axial stability show that the width of the cyclotron stability can
be explained solely by voltage fluctuations, since the modified cyclotron frequency is measured by
coupling to the axial mode. The stability is thus not limited by external magnetic field fluctuations or
coupling effects due to B2. To improve the cyclotron frequency stability further using the double-dip
technique, a stabilization of the axial frequency by developing improved power supplies for exam-
ple based on Josephson voltage references is required. Alternatively, a direct measurement of the
modified cyclotron frequency could be implemented, for example by using phase-sensitive detection
techniques [13, 14, 15]. Due to the scaling of the axial frequency stability with the cyclotron quantum number n+, a low
cyclotron energy (typically E+/kB < 1 K) is a crucial requirement for spin state detection. How-
ever, each measurement of the modified cyclotron frequency raises the energy of the mode drastically
(typically E+/kB ≈200 K) and the cyclotron energy needs to be reduced after each individual mea-
surement of ν+. This is achieved by tuning the cyclotron resonator to the modified cyclotron frequency
of the proton. The resonator then acts as a thermal bath at a temperature Tres [16], similar to the axial
detector in case of the axial mode. When the proton is decoupled from the thermal bath it acquires a
fixed but arbitrary cyclotron energy. The distribution of the individual energy values follows an expo-
nential distribution, its width being defined by the thermodynamic temperature. To prepare a single 6■■ Proceedings of the 12th International Conference on Low Energy Antiproton Physics (LEAP2016)
Downloaded from journals.jps.jp by 128.141.46.242 on 03/23/18 Proceedings of the 12th International Conference on Low Energy Antiproton Physics (LEAP2016)
Downloaded from journals.jps.jp by 128.141.46.242 on 03/23/18 011018-7 JPS Conf. Proceedings of the 12th International Conference on Low Energy Antiproton Physics (LEAP2016)
Downloaded from journals.jps.jp by 128.141.46.242 on 03/23/18 Acknowledgment We acknowledge financial support by the RIKEN Initiative Research Unit Program, RIKEN Presi-
dent Funding, RIKEN Pioneering Project Funding, RIKEN FPR Funding, the RIKEN JRA Program, 7
■■ Proceedings of the 12th International Conference on Low Energy Antiproton Physics (LEAP2016)
Downloaded from journals.jps.jp by 128.141.46.242 on 03/23/18 Proceedings of the 12th International Conference on Low Energy Antiproton Physics (LEAP2016)
Downloaded from journals.jps.jp by 128.141.46.242 on 03/23/18 011018-8 JPS Conf. Proc. , 011018 (2017)
18 the Grant-in-Aid for Specially Promoted Research (grant number 24000008) of MEXT, the Max-
Planck Society, the EU (ERC advanced grant number 290870-MEFUCO), the BMBF, the Helmholtz-
Gemeinschaft, and the CERN Fellowship programme. the Grant-in-Aid for Specially Promoted Research (grant number 24000008) of MEXT, the Max-
Planck Society, the EU (ERC advanced grant number 290870-MEFUCO), the BMBF, the Helmholtz-
Gemeinschaft, and the CERN Fellowship programme. Proceedings of the 12th International Conference on Low Energy Antiproton Physics (LEAP2016)
Downloaded from journals.jps.jp by 128.141.46.242 on 03/23/18 References [1] C. Smorra et al., Eur. Phys. J.-Spec. Top 224, 3055 (2015) [2] S. Ulmer et al., Nature 524, 196 (2015) [4] P. F. Winkler et al., Phys. Rev. A 5, 83 (1972) [5] A. Mooser et al., Phys. Lett. B 723, 78 (2013) y
[6] S. Ulmer et al., Phys. Rev. Lett. 106, 253001 (2011) [7] G. Gabrielse et al., Int. J. Mass Spectrom. Ion Processes 88, 319 (1989) [8] L. S. Brown et al., Rev. Mod. Phys. 58, 233 (1986) [9] L. S. Brown et al., Phys. Rev. A 25, 2423 (1982) [10] H. Dehmelt et al., Bull. Am. Phys. Soc. 18, 72 (1973) [11] A. Mooser et al., Phys. Rev. Lett. 110, 140405 (2013) [12] G. Gabrielse et al., J. Appl. Phys. 63, 5143 (1988) [13] J. K. Thompson et al., Nature 430, 58 (2004) [13] J. K. Thompson et al., Nature 430, 58 (2004) [14] S. Rainville et al., Science 303, 334 (2004) [14] S. Rainville et al., Science 303, 334 (2004) [15] S. Sturm et al., Phys. Rev. Lett. 107, 143003 (2011) [16] S. Djekic et al., Eur. Phys. J. D. 31, 451 (2004) [16] S. Djekic et al., Eur. Phys. J. D. 31, 451 (2004) [17] H. Dehmelt et al., Proc. Nat. Acad. Sci. 83, 5761 (1986) ] H. Dehmelt et al., Proc. Nat. Acad. Sci. 83, 576 [18] B. d’Urso et al., Phys. Rev. Lett. 90, 43001 (2003) [18] B. d’Urso et al., Phys. Rev. Lett. 90, 43001 (2003) [19] S. Ulmer et al., Rev. Sci. Instrum. 80, 123302 (2009) [19] S. Ulmer et al., Rev. Sci. Instrum. 80, 123302 (2009) [19] S. Ulmer et al., Rev. Sci. Instrum. 80, 1233 8
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Efficacy and toxicity of the combination chemotherapy of thalidomide, alkylating agent, and steroid for relapsed/refractory myeloma patients: a report from the Korean Multiple Myeloma Working Party (KMMWP) retrospective study
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© 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use,
distribution and reproduction in any medium, provided the original work is properly cited. Efficacy and toxicity of the combination chemotherapy of
thalidomide, alkylating agent, and steroid for relapsed/
refractory myeloma patients: a report from the Korean
Multiple Myeloma Working Party (KMMWP) retrospective
study
Jihyun Kwon1, Chang-Ki Min2, Kihyun Kim3, Jae-joon Han4, Joon Ho Moon5, Hye Jin Kang6,
Hyeon-Seok Eom7, Min Kyoung Kim8, Hyo Jung Kim9, Dok Hyun Yoon10, Jeong-Ok Lee11,
Won Sik Lee12, Jae Hoon Lee13, Je-Jung Lee14, Yoon-seok Choi15, Sung hyun Kim16 &
Sung-soo Yoon17 1Department of Internal Medicine, Chungbuk National University Hospital, Cheongju, Korea
2Department of Internal Medicine, Seoul St. Mary’s Hospital, The Catholic University of Korea, Korea
3Department of Internal Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Korea
4Departments of Hematology-Oncology, School of Medicine, Kyung Hee University, Seoul, Korea
5Department of Internal Medicine, Kyungpook National University Hospital, Daegu, Korea
6Department of Internal Medicine, Korea Cancer Center Hospital, Seoul, Korea
7Hematology-Oncology Clinic, National Cancer Center, Goyang-si, Korea
8Department of Internal Medicine, Yeungnam University Medical Center, Daegu, Korea
9Department of Internal Medicine, Hallym University Sacred Heart Hospital, Hallym University College of Medicine, Anyang, Korea
10Department of Oncology, Asan Medical Center, University of Ulsan, College of Medicine, Seoul, Korea
11Department of Internal Medicine, Seoul National University Bundang Hospital, Seongnam, Korea
12Department of Hemato-Oncology, Inje University Busan Paik Hospital, Busan, Korea
13Department of Internal Medicine, Gachon University Gil Hospital, Incheon, Korea
14Department of Hematology-Oncology, Chonnam National University Hwasun Hospital, Jeollanamdo, Korea
15Department of Internal Medicine, Chungnam National University Hospital, Daejeon, Korea
16Department of Internal Medicine, Dong-A Medical Center, Dong-A University College of Medicine, Busan, Korea
17Department of Internal Medicine, Seoul National University Hospital, Seoul, Korea Cancer Medicine Open Access ORIGINAL RESEARCH Efficacy and toxicity of the combination chemotherapy of
thalidomide, alkylating agent, and steroid for relapsed/
refractory myeloma patients: a report from the Korean
Multiple Myeloma Working Party (KMMWP) retrospective
study
Jihyun Kwon1, Chang-Ki Min2, Kihyun Kim3, Jae-joon Han4, Joon Ho Moon5, Hye Jin Kang6,
Hyeon-Seok Eom7, Min Kyoung Kim8, Hyo Jung Kim9, Dok Hyun Yoon10, Jeong-Ok Lee11,
Won Sik Lee12, Jae Hoon Lee13, Je-Jung Lee14, Yoon-seok Choi15, Sung hyun Kim16 &
Sung-soo Yoon17 Abstract Keywords
Alkylating agent, cyclophosphamide,
melphalan, multiple myeloma, thalidomide
Correspondence
Sung-soo Yoon, Professor of Seoul National
University, College of Medicine, 101
Daehak-ro, Chongno-gu, 110-744, Seoul,
Republic of Korea. Tel: 02-2072-3079;
Fax: 02-2072-2228;
E-mail: ssysmc@gmail.com
Funding Information
No funding information provided. Received: 11 August 2016; Revised: 27
September 2016; Accepted: 25 October 2016
Cancer Medicine 2017; 6(1):100–108
doi: 10.1002/cam4.970 y
Alkylating agent, cyclophosphamide,
melphalan, multiple myeloma, thalidomide
Correspondence
Sung-soo Yoon, Professor of Seoul National
University, College of Medicine, 101
Daehak-ro, Chongno-gu, 110-744, Seoul,
Republic of Korea. Tel: 02-2072-3079;
Fax: 02-2072-2228;
E-mail: ssysmc@gmail.com
Funding Information
No funding information provided. Received: 11 August 2016; Revised: 27
September 2016; Accepted: 25 October 2016
Cancer Medicine 2017; 6(1):100–108
doi: 10.1002/cam4.970 Alkylating agent, cyclophosphamide,
melphalan, multiple myeloma, thalidomide Alkylating agent, cyclophosphamide,
melphalan, multiple myeloma, thalidomide We analyzed the treatment responses, toxicities, and survival outcomes of pa-
tients with relapsed or refractory multiple myeloma who received daily thalido-
mide, cyclophosphamide, and dexamethasone (CTD) or daily thalidomide,
melphalan, and prednisolone (MTP) at 17 medical centers in Korea. Three-
hundred and seventy-six patients were enrolled. The combined chemotherapy
of thalidomide, corticosteroid, and an alkylating agent (TAS) was second-line
chemotherapy in 142 (37.8%) patients, and third-line chemotherapy in 135
(35.9%) patients. The response rate overall was 69.4%. Patients who were not
treated with bortezomib and lenalidomide before TAS showed a higher response
rate compared to those who were exposed to these agents. The estimated median
progression-free survival and overall survival times were 10.4 months and
28.0 months, respectively. The adverse events during TAS were generally toler-
able, but 39 (10.4%) patients experienced severe infectious complications. There
were no differences in terms of efficacy between CTD and MTP, but infectious
complications were more common in CTD group. TAS is an effective treatment
regimen which induces a high response rate in relapsed or refractory multiple
myeloma patients. Due to the high incidence of grade 3 or 4 infection, proper
management of infection is necessary during the TAS treatment, especially the
CTD. Correspondence
Sung-soo Yoon, Professor of Seoul National
University, College of Medicine, 101
Daehak-ro, Chongno-gu, 110-744, Seoul,
Republic of Korea. Tel: 02-2072-3079;
Fax: 02-2072-2228;
E-mail: ssysmc@gmail.com Method Clinical data including age, sex, prior therapies, stage
according to the International Staging System (ISS), the
results of cytogenetics or fluorescence in situ hybridiza-
tion, baseline laboratory data including white blood cell
count, hemoglobin, platelet count, serum calcium, creati-
nine, and prior treatment lines of antimyeloma therapy
were collected. We also analyzed monoclonal protein
subtypes, the presence of extramedullary plasmacytoma,
myeloma-related organ dysfunction such as osteolysis,
pathologic
fractures,
and
renal
replacement
by
hemodialysis. Our primary endpoints were the overall response rate
to CTD or MTP treatment, and the toxicity profile. Treatment response was evaluated according to the
International Myeloma Working Group (IMWG) response
criteria [25]. The overall response rate was defined as the
proportion of patients who showed a partial response or
better. Adverse events during TAS were assessed by the
National Cancer Institute–Common Terminology Criteria
for Adverse Events (NCI CTCAE), version 4.03 [26]. Secondary endpoints included progression-free survival
(PFS), overall survival (OS), and a comparison of usage
patterns and efficacy and toxicity levels between CTD and
MTP regimens. PFS was calculated from the time of TAS
to the time of disease progression or death, and OS was
calculated from the time of TAS to the time of death. The aforementioned results of clinical trials conducted
to test thalidomide-containing multiagent chemotherapy,
however, were carried out by targeting small numbers of
patients who had been treated only with conventional
chemotherapy. At present, more potent drugs such as
bortezomib and lenalidomide have been introduced, and
increasingly more patients are therefore treated with these
highly effective novel agents from the early phase of treat-
ment. It is not known that whether the combination of
thalidomide and an alkylating agent is still active in the
patients who suffered a recurrence later or failed to show
improvement with these newer agents. The objective of this multicenter retrospective study
was to investigate the using pattern, efficacy, and toxicity
of a combination of thalidomide, an alkylating agent, and
a steroid as chemotherapy in patients with relapsed or
refractory multiple myeloma in the era of novel agents. In addition, differences in clinical outcomes according to
the choice of the alkylating agent and steroid added to
thalidomide were evaluated. Differences in responses according to patient and disease
characteristics were compared by means of a chi-square
analysis. Method Seventeen medical centers in the Republic of Korea par-
ticipated in this multicenter retrospective study. We col-
lected the medical records of patients who were treated
with combination chemotherapy including thalidomide,
an alkylating agent, and a steroid (TAS) as a form of
chemotherapy. All enrolled patients had recurrent and/or
refractory multiple myeloma which was defined as relapsed
or progressed disease after at least one prior antimyeloma
regimen. Multiple myeloma (MM) remains an incurable hematologic
malignancy in spite of the development of various anti-
myeloma agents. In the early days, alkylating agents such
as melphalan or cyclophosphamide were used, but improv-
ing the prognosis of MM patients was not satisfactory
due to the low efficacy of therapy with an isolated alkylat-
ing agent [1]. By the 1990s, high-dose melphalan chemo-
therapy followed by autologous hematopoietic stem cell
transplantation (ASCT) was introduced. This strategy
improved the overall response and survival of MM patients,
but the benefits of high-dose chemotherapy and ASCT
were limited to younger patients due to the significant
toxicity of this therapy [2, 3]. TAS included two types of chemotherapy regimens: the
cyclophosphamide (intravenously 300 mg/m2 for 3 days
or orally 50 mg daily), thalidomide (orally 100 mg daily),
and dexamethasone (40 mg for 4 days) (CTD) or the
melphalan (orally 0.15 mg/kg for 7 days), thalidomide
(orally 100 mg daily), prednisolone (60 mg for 7 days)
(MTP) regimen. The decision to choose one of the two
regimens and to change the dose or schedule of chemo-
therapy was made by the treating physician in each case. Since the 2000s, there have been major developments
in the treatment of MM with the introduction of “novel
agents” represented by immunomodulatory drugs (IMiDs)
and proteasome inhibitors (PIs). Thalidomide, the first
IMiD used to treat myeloma, was demonstrated to have
good therapeutic effects in a number of clinical trials. Thalidomide monotherapy or combined therapy with a
steroid showed a response rate exceeding 60% in newly
diagnosed cases of multiple myeloma [4, 5] and 25–55%
in relapsed or refractory cases of myeloma [6–13]. Recently,
the combined regimen of thalidomide, an alkylating agent,
and a steroid is widely used; it is an active regimen with
a higher response rate than thalidomide or alkylating agent
monotherapy approaches. The response rate of this com-
bination treatment exceeded 80% in newly diagnosed cases
of myeloma [14–18], and 60–70% in relapsed or refractory
cases [19–24]. Thalidomide and Alkylators for Multiple Myeloma J. Kwon et al. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. Correspondence Correspondence
Sung-soo Yoon, Professor of Seoul National
University, College of Medicine, 101
Daehak-ro, Chongno-gu, 110-744, Seoul,
Republic of Korea. Tel: 02-2072-3079;
Fax: 02-2072-2228;
E-mail: ssysmc@gmail.com Cancer Medicine 2017; 6(1):100–108
doi: 10.1002/cam4.970 100 Thalidomide and Alkylators for Multiple Myeloma Patient characteristics The median number of chemotherapy regimens used
before receiving TAS was two (range 1–8). TAS was per-
formed as a second-line therapy in 142 (37.8%) patients
and as a third-line therapy in 135 (35.9%). patients Two
hundred and sixty-six (70.7%) patients were previously
treated with bortezomib and 83 (22.1%) patients had been
exposed to thalidomide before their chemotherapy, while
lenalidomide was used in only 13 (3.5%) patients. Patients
who underwent high-dose chemotherapy followed by ASCT
numbered 135, accounting for 35.9% of all patients enrolled
in this study. Baseline characteristics and the appearance of MM-related
organ dysfunctions are described in Table 1. In total, 376
patients were enrolled. Two hundred and thirty-six patients
were treated with CTD and 140 patients with MTP. The Table 1. Baseline characteristics and multiple myeloma-related organ
failure of patients. N (%)
Sex
Male
224 (69.6)
Female
152 (40.4)
Age
Younger than 70
245 (65.2)
70 or older
131 (34.8)
B2-microglobulin
<3.5 mg/dL
94 (25.0)
3.5–5.5 mg/dL
58 (15.4)
>5.5 mg/dL
86 (22.9)
Stage by ISS
I
53 (14.1)
II
81 (21.5)
III
90 (23.9)
Immunophenotype
Heavy chain
IgG
183 (48.7)
IgA
100 (26.6)
IgM
9 (2.4)
Light chain
kappa
208 (55.3)
lambda
154 (41.0)
Anemia (Hb <10 g/dL)
186 (49.8)
Leukopenia (WBC<4 × 103/mm3)
88 (23.7)
Thrombocytopenia (platelet <100 × 103/mm3)
56 (14.6)
Hypercalcemia (serum Ca>11.5 mg/dL)
14 (3.7)
Renal insufficiency (serum Cr>2.0 mg/dL)
43 (11.4)
Hemodialysis
20 (5.3)
Osteolytic lesions
194 (51.6)
History of fracture
104 (27.7)
Hyperviscosity syndrome
17 (4.5)
Extramedullary plasmacytoma
61 (16.2)
ISS, International Staging System. Method In case of the patients whose best responses to
TAS were not evaluable, they were excluded from the
analysis of predictive factors for overall response. The
median PFS and OS were estimated using the Kaplan–Meier 101 © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. Thalidomide and Alkylators for Multiple Myeloma J. Kwon et al. median age of the enrolled patients was 66 years old
(range 36–90). Data about the initial ISS stage and serum
level of B2-microglobulin were not available in 152 (40.4%)
and 138 (36.7%) patients. The most common subtype of
the monoclonal heavy chain protein was IgG, and 54
(14.4%) patients had only a light chain. Among the signs
of MM-related organ dysfunction, the most common
presentation was osteolytic lesions, followed by anemia. Hypercalcemia and hyperviscosity syndrome were relatively
rare. Results of chromosomal analyses or FISH tests were
not available in two thirds of all patients. In the remain-
ing patients, t(4;14) was reported in 40 (10.6%) patients,
t(14;16) in 17 (4.5%), del(17p) in 11 (2.9%), t(11;14) in
33 (8.8%), and del(13q) in 26 (6.9%) patients. method, and differences between subgroups of patients
were compared using log-rank tests. A multivariate survival
analysis was conducted using the Cox proportional hazards
model with forward selection with a cutoff of P < 0.05
to add new variables. SPSS software (version 21; IBM
Corp., Armonk, NY) was used for statistical analyses. This study was supported by the Korean Multiple
Myeloma Working Party (KMMWP, Protocol No. KMM158) and reviewed by the Institutional Review Board
of each participating medical institutes. method, and differences between subgroups of patients
were compared using log-rank tests. A multivariate survival
analysis was conducted using the Cox proportional hazards
model with forward selection with a cutoff of P < 0.05
to add new variables. SPSS software (version 21; IBM
Corp., Armonk, NY) was used for statistical analyses. This study was supported by the Korean Multiple
Myeloma Working Party (KMMWP, Protocol No. KMM158) and reviewed by the Institutional Review Board
of each participating medical institutes. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. ISS, International Staging System. Analysis of efficacy and survival rates Table 2. Response rates according to previous treatment history. and/or thalidomide resulted in a reduced response rate,
most of the subgroups showed response rates which
exceeded 60%. The overall response rate of patients who
were refractory to bortezomib treatment or progressed
after prior bortezomib therapy was 69.9%, which was
similar with that of bortezomib-sensitive patients. Response
rate of the subgroup of patients who were previously
treated with lenalidomide was noticeably low, at less than
half (ORR 46.2%), but there were few analyzed patients. Earlier treatment with TAS brought a better response rate. When this therapy was performed as a second-line therapy,
the response rate was significantly higher compared to
when it was used as a third-line therapy or a fourth-line
therapy (P = 0.018). A history of high-dose chemotherapy
followed by ASCT had no significant impact on the response
rate. and/or thalidomide resulted in a reduced response rate,
most of the subgroups showed response rates which
exceeded 60%. The overall response rate of patients who
were refractory to bortezomib treatment or progressed
after prior bortezomib therapy was 69.9%, which was
similar with that of bortezomib-sensitive patients. Response
rate of the subgroup of patients who were previously
treated with lenalidomide was noticeably low, at less than
half (ORR 46.2%), but there were few analyzed patients. Earlier treatment with TAS brought a better response rate. When this therapy was performed as a second-line therapy,
the response rate was significantly higher compared to
when it was used as a third-line therapy or a fourth-line
therapy (P = 0.018). A history of high-dose chemotherapy
followed by ASCT had no significant impact on the response
rate. Figure 1. Progression-free survival (PFS). (A) Presence of thrombocyto
penia or not, (B) being on hemodialysis or not, (C) overall response (CR,
VGPR, and PR) to study therapy or no response (SD or PD). Upon a median follow-up of 22.6 months, 197 (52.4%)
patients had died. Median PFS was 10.4 months, and
median OS was 28.0 months. In the multivariate analysis,
thrombocytopenia (HR: 1.665, P = 0.002), hemodialysis
(HR: 2.008, P = 0.016), and the overall response rates
to therapy (HR: 2.871, P < 0.001) were found to be
significant risk factors for PFS (Fig. 1). Patients who
achieved CR, inter alia, showed long PFS of 24.9 months. Analysis of efficacy and survival rates Prognostic factors for OS included age over 70 years (HR:
1.494, P = 0.017), hemodialysis (HR: 2.234, P = 0.017),
the presence of extramedullary plasmacytoma (HR: 1.852,
P = 0.001), previous use of bortezomib (HR: 1.467,
P = 0.038), grade 3 or 4 infection during TAS (HR:
2.005, P = 0.001), and an overall response to TAS (HR:
2.806, P < 0.001) (Fig. 2). Analysis of efficacy and survival rates The median duration of treatment was 5.3 months (median:
six cycles). At the time of the analysis, 13 (3.5%) patients
were treated with TAS. The most common cause of cessa-
tion was progression of the disease (156 patients, 43.0%),
followed by the need for a resting period (62 patients, 17.1%). six cycles). At the time of the analysis, 13 (3.5%) patients
were treated with TAS. The most common cause of cessa-
tion was progression of the disease (156 patients, 43.0%),
followed by the need for a resting period (62 patients, 17.1%). The overall response rate was 69.4% (261 patients). CR
was confirmed in 61 (16.2%) patients, 5 of whom showed
stringent CR. Forty-three (11.4%) patients showed VGPR,
and PR was reported in 157 (41.8%) patients . The best
response to TAS was not evaluable in 20 (5.3%) patients. Age, sex, and initial ISS stage did not influence the response
to TAS. The subtype of the immunoglobulin heavy chain
was not correlated with the response, but patients with
a kappa light chain showed higher response rates than those
with a lambda light chain (78.3% vs. 66.2%, P = 0.009). Patients with anemia (66.7% vs. 80.7%, P = 0.002) and
thrombocytopenia (57.7% vs. 76.6%, P = 0.005) showed
significantly lower response rates compared to those without
these conditions. Other MM-related instances of organ
failure, including hyperviscosity symptoms, renal insuffi-
ciency, osteolytic lesions and/or pathologic fractures, and
extramedullary plasmacytoma had no direct correlation
with a poor response to TAS. A history of antimyeloma therapy also affected the
response to TAS (Table 2). While exposure to bortezomib © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. 102 Thalidomide and Alkylators for Multiple Myeloma J. Kwon et al. Figure 1. Progression-free survival (PFS). (A) Presence of thrombocyto
penia or not, (B) being on hemodialysis or not, (C) overall response (CR,
VGPR, and PR) to study therapy or no response (SD or PD). Table 2. Response rates according to previous treatment history. Overall response (CR,
VGPR, or PR) N (%)
P
Previous treatment history
Bortezomib
Exposure
177 (70.0)
0.016
No exposure
84 (81.6)
Thalidomide
Exposure
52 (65.0)
0.041
No exposure
209 (75.7)
Lenalidomide
Exposure
6 (46.2)
0.032
No exposure
255 (74.3)
High-dose therapy and ASCT
Done
90 (69.2)
0.099
Not done
166 (76.1)
Lines of chemotherapy
2nd
111 (82.2)
0.0181
3rd
87 (68.0)
4th
39 (65.0)
5th≤
17 (65.4)
1Pearson’s chi-square. Toxicity Fifty-six patients stopped TAS due to treatment-related
toxicity (15.6%). Adverse events during TAS are described
in Table 3. Most reported cases were mild, at grade 1 or
2. Among nonhematologic adverse events, somnolence and
fatigue were the most common symptoms (114 patients,
30.3%). Peripheral neuropathy was reported in 108 (28.7%)
patients, but only 11 (2.9%) patients complained of severe
symptoms of grade 3 or 4. Hematologic toxicity was
reported in 66 (17.6%) patients, and 26 (6.9%) patients
had grade 3 or 4 events. The most common hematologic
adverse event was neutropenia (44 patients, 11.1%). Among 103 © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. Thalidomide and Alkylators for Multiple Myeloma J. Kwon et al. J. Kwon et al. Thalidomide and Alkylators for Multiple Myeloma
Figure 2. Overall survival (OS). (A) Age younger than 70 years old versus 70 years old or older, (B) being on hemodialysis versus no hemodialysis, (C)
presence of extramedullary plasmacytoma versus not, (D) previous exposure to bortezomib versus no exposure, (E) grade 3 or 4 infection during
treatment versus no or mild infection, (F) overall response (CR, VGPR, and PR) to study therapy or no response (SD or PD). Figure 2. Overall survival (OS). (A) Age younger than 70 years old versus 70 years old or older, (B) being on hemodialysis versus no hemodialysis, (C)
presence of extramedullary plasmacytoma versus not, (D) previous exposure to bortezomib versus no exposure, (E) grade 3 or 4 infection during
treatment versus no or mild infection, (F) overall response (CR, VGPR, and PR) to study therapy or no response (SD or PD). the serious adverse events at grade 3 or 4, the incidence
of infection was highest (39 patients, 10.4%), which was
the major cause of the cessation of treatment. Three
patients died during TAS due to infection; these were
one case of pneumonia and two cases of septic shock. All of these three patients were treated with CTD
regimen. Comparison of MTP and CTD The median age was older in the MTP group (68 years,
range 42–90) compared to the CTD group (64 years,
36–84) (P < 0.001). Besides, the MTP group contained
more elderly patients who were older than 70 (62, 44.3%)
compared to the CTD group (69, 29.2%, P = 0.003). © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. 104 Thalidomide and Alkylators for Multiple Myeloma J. Kwon et al. Table 3. Adverse events. Table 3. Adverse events. Adverse events
N (%)
All grade
Grade 3 or 4
Dose reduction of
therapy
Discontinuation
of therapy
Nonhematologic
Peripheral neuropathy
108 (28.7)
11 (2.9)
18 (4.8)
17 (4.5)
Venous thrombosis
9 (2.4)
1 (0.3)
0 (0.0)
3 (0.8)
Infection
65 (17.3)
39 (10.4)
2 (0.5)
35 (9.3)
Fatigue & somnolence
114 (30.3)
31 (8.1)
25 (6.6)
24 (6.4)
Dizziness, postural hypotension
44 (11.7)
5 (1.3)
8 (2.1)
5 (1.3)
Gastrointestinal complications
76 (20.2)
2 (0.5)
5 (1.3)
8 (2.1)
Hematologic
Neutropenia
44 (11.7)
21 (5.6)
9 (2.4)
19 (5.1)
Anemia
31 (8.2)
3 (0.8)
0 (0.0)
2 (0.5)
Thrombocytopenia
17 (4.5)
9 (2.4)
2 (0.5)
3 (0.8) Patients in the CTD subgroup had more osteolytic lesions
(136 patients, 57.6% vs. 58 patients, 41.4%, P = 0.011)
and instances of extramedullary plasmacytoma (49 patients,
20.7% vs. 12 patients, 8.6%, P = 0.004). On the other
hand, a history of fracture was more common in the
MTP group than the CTD group (47 patients, 33.6% vs. 57 patients, 24.2%, P = 0.011). There were no significant
differences in the treatment histories between the CTD
and MTP groups. Table 4. Comparison of response rate and survival outcome according
to the treatment regimens. Subtype of TAS
MTP
CTD
P value
N = 140
N = 236
Treatment response
N of patients
(%)
N of patients
(%)
Overall response rate
91 (65.0)
170 (72.0)
0.1211
Complete response rate
26 (18.6)
35 (14.8)
0.3731
Survival
Months
(95% CI)
Months
(95% CI)
Median progression-
free survival (PFS)
12.6
(11.0–14.2)
8.7
(6.7–10.6)
0.1782
Median overall survival
(OS)
33.5
(20.1–46.9)
26.9
(21.7–32.0)
0.5882
MTP, melphalan, thalidomide and prednisolone; CTD, cyclophospha-
mide, thalidomide, and dexamethasone. 1Pearson’s chi-square. 2Kaplan–Meier survival analysis, log-rank. The overall response rates of these two group were
similar, 65.0% for the MTP group and 72.0% for the
CTD group (P = 0.121). Comparison of MTP and CTD Patients who had stopped TAS
due to treatment-related toxicity numbered 40 (16.9%)
in the CTD group and 16 (11.4%) in the MTP group. Grade 3 or 4 infection was more common in the CTD
group (30 patients, 12.7%) than in the MTP group (nine
patients, 6.4%), while grade 3 or 4 peripheral neuropathy
was more common in the MTP group (eight patients,
5.7%) than in the CTD group (three patients, 1.3%). There was no significant difference in the PFS and the
OS values between these two groups. (Table 4). introduced recently. Moreover, numerous novel antimy-
eloma agents including a new class of proteasome inhibi-
tors along with IMiD and anti-CD138 monoclonal
antibodies are currently in development. The role of
thalidomide and an alkylating agent has been reduced by
degrees, especially as an induction therapy. Similarly in
Korea, thalidomide and/or bortezomib-containing regimen
are used as an induction therapy for transplantation-eligible
patients. For transplant-ineligible patients, bortezomib–
melphalan–prednisolone is typical first-line therapy, and
lenalidomide began to use as a second-line treatment
recently. Therefore, a combination of thalidomide and an
alkylating agent is mainly used in patients who have
relapsed or who have reached refractory status. However,
whether this combined therapy is still effective for patients
who were heavily treated with various novels agent earlier
is not clear. introduced recently. Moreover, numerous novel antimy-
eloma agents including a new class of proteasome inhibi-
tors along with IMiD and anti-CD138 monoclonal
antibodies are currently in development. The role of
thalidomide and an alkylating agent has been reduced by
degrees, especially as an induction therapy. Similarly in
Korea, thalidomide and/or bortezomib-containing regimen
are used as an induction therapy for transplantation-eligible
patients. For transplant-ineligible patients, bortezomib–
melphalan–prednisolone is typical first-line therapy, and
lenalidomide began to use as a second-line treatment
recently. Therefore, a combination of thalidomide and an
alkylating agent is mainly used in patients who have
relapsed or who have reached refractory status. However,
whether this combined therapy is still effective for patients
who were heavily treated with various novels agent earlier
is not clear. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. Thalidomide and Alkylators for Multiple Myeloma Thalidomide and Alkylators for Multiple Myeloma J. Kwon et al. Hematologic toxicity levels were generally mild, while
grade 3 or 4 neutropenia was reported in 5.9% of all
patients. Infectious complications were reported in 18%
of all patients, and the incidence of severe cases (grade
3 or 4) was 10.5%. It is important to note that the major-
ity of these patients with severe infections, including three
cases in which the patients died, stopped TAS permanently. We suggest that the presence of infectious complications
has important clinical relevance for the combined therapy
of thalidomide, a steroid, and an alkylating agent, and
that the management of the infection affects the clinical
course of the patient. The incidence of peripheral neu-
ropathy was quite high, at 29.5%, while the majority of
cases of peripheral neuropathy were mild. Venous throm-
bosis was rarely observed, at 2.5%, explained partly by
the overall risk of overt thromboembolism of Asian patients
is lower than in a western population and by the fact
that most physicians use a prophylactic anticoagulant or
an antiplatelet agent. Our study found a considerable response rate of the
combination therapy of thalidomide, a steroid and an
alkylating agent, similar or superior to previous results
[19–24]. The response rate of patients who were previ-
ously treated with bortezomib or thalidomide is significantly
lower compared those who had never been exposed to
these agents. The diminished efficacy of the combined
therapy of thalidomide, a steroid, and an alkylating agent
followed by bortezomib appears to be due mainly to a
heavy treatment history. It has been suggested that the
acquisition of drug resistance and the refractoriness of
myeloma cells during prior chemotherapy are the main
causes of the failure of salvage chemotherapy [27]. Also,
our data indicate that cases with a large number of prior
treatments are associated with a poor response to TAS. Nevertheless, the response rate of patients with previous
treatment history with bortezomib or thalidomide remains
considerably high at approximately 60% or more. Besides,
bortezomib treatment failure did not compromise the
effects of subsequent TAS therapy. As a result, we could
regard TAS therapy as a good salvage treatment option
for the patients who failed to prior therapy including
bortezomib. The patients exposed to lenalidomide showed
a relatively low response rate of 46%, but we cannot
prove any correlations because of small number of patients. Thalidomide and Alkylators for Multiple Myeloma Despite some researchers revealed that no cross-resistance
between different kinds of IMiDs was observed [28, 29],
we need more investigation to verify the optimal sequence
of several IMiDs including treatment regimens. We investigated differences in the treatment behavior,
efficacy, and toxicities between the CTD and MTP regi-
mens despite the fact that a direct comparison of the
two regimens was not possible due to the limitations of
this retrospective study. MTP was more frequently used
for older patients, likely because it is advantageous given
its oral administration of melphalan in comparison with
the intravenous administration of cyclophosphamide. There
are no differences in response and survival rates between
the MTP and CTD regimens; therefore, the choice of the
alkylating agent and steroid added to thalidomide appeared
to be less important. The interruption of treatment due
to toxicity was more common in the CTD group due to
the higher incidence of severe infectious complications in
CTD than in the MTP group. The immunosuppressive
effect of cyclophosphamide is considered to be the main
factor behind the high incidence of infection during CTD. In previous reports, researchers suggested that intravenous
cyclophosphamide had an immunosuppressive effect [34],
and a decrease in the number of CD4-positive T cells
and regulatory T cells was observed in 35 patients treated
with the CTD regimen [35]. Therefore, prophylaxis and
appropriate management of infections are needed for
patients who are treated with thalidomide with a steroid
and an alkylating agent, especially in conjunction with
intravenous cyclophosphamide. Variable factors, including renal failure with hemodi-
alysis, thrombocytopenia, and the degree of the response
to TAS have been defined as independent prognostic fac-
tors for PFS. Deterioration of renal function is known
to be associated with inferior survival and a high incidence
of treatment toxicity in previous reports [30, 31]. Severe
renal failure does not interfere with the effectiveness of
drugs in itself, but this condition reflects a high tumor
burden of myeloma. Thrombocytopenia, which is associ-
ated with decreased bone marrow function, decreases
tolerability to antimyeloma agents in patients. Patients
with renal failure or thrombocytopenia must receive treat-
ment at a reduced dose and on a disrupted schedule;
hence, the benefits of the treatment are greatly limited
in these patients. The presence of extramedullary plas-
macytoma
retains
prognostic
significance
for
OS. Extramedullary plasmacytoma is related to a high tumor
burden and underlying refractoriness of tumor cells. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd. Discussion Our multicenter, retrospective study showed that a
combination of thalidomide, a steroid, and an alkylat-
ing agent is an active chemotherapy regimen in cases
of relapsed, refractory multiple myeloma. ORR is near
70% of all cases, and the CR rate was 16.2%. Toxicity
levels were generally tolerable, and the incidences of
severe
neurotoxicity
and
thromboembolism
were
extremely low. It was well known that a combination of thalidomide,
a steroid, and an alkylating agent is effective in both
newly diagnosed and relapsed refractory multiple myeloma
patients, but antimyeloma agents more potent than tha-
lidomide, such as bortezomib and lenalidomide, have been 105 References 1. Pasquali, S. D. M., and J. Vela. 1998. Combination
chemotherapy versus melphalan plus prednisone as
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thalidomide in patients with relapsed or refractory
multiple myeloma: a systematic comparison. Eur. J. Haematol. 79:93–99. None declared. Thalidomide and Alkylators for Multiple Myeloma It
has been shown that the response to thalidomide was
relatively poor in relation to extramedullary plasmacytoma
despite the significant reduction of monoclonal protein
[32, 33]. Garcia-Sanz et al. showed that patients with
extramedullary myelomatous lesions had shorter survival
after CTD treatment [19]. In conclusion, our data showed that the combined
treatment of thalidomide with a steroid and an alkylating
agent plays a significant role as a salvage therapy for
patients with relapsed or refractory myeloma. The previ-
ous use of bortezomib, lenalidomide, and high-dose
chemotherapy and ASCT may have contributed to the
relatively low response rates found here. This regimen is
generally tolerable, but proper care of infectious 106 Thalidomide and Alkylators for Multiple Myeloma J. Kwon et al. complications is important with regard to the prognosis
of the patient. The efficacy levels of MTP and CTD were
identical, but the CTD regimen led to an increase in
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(cyclophosphamide, thalidomide, dexamethasone) 108 © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.
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https://openalex.org/W2329199314
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English
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A Case of Acute Hemorrhagic Pancreatitis
|
The Boston medical and surgical journal/Boston medical and surgical journal
| 1,905
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public-domain
| 4,459
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Clinical Department. p
Blood. — A leucocyte count made two hours after
onset of symptoms showed 37,000 per cubic millimeter. U i
Abo t
120
d l t
i
th
d REFERENCES. REFERENCES. 1 Crile. Blood-Pressure in Surgery : an Experimental and Clinical
Research. The Cartwright prize essay for 1903. 2 Keen. Therapeutic Gazette, 1904, 3. s., xx, 217. 3Cohen. Journal American Med. Assoc, 1903, xli, 1145. 4Maag. Centralbl. f. Chirurgie, 1901, xxviii, 20. 6 Sick. Centralbl. f. Chirurgie, 1903, xxx2, 981. «Starling & Lane. Lancet, 1903, ii, 1397. REFERENCES. 1 Crile. Blood-Pressure in Surgery : an Experimental and Clinical
Research. The Cartwright prize essay for 1903. 2 Keen. Therapeutic Gazette, 1904, 3. s., xx, 217. 3Cohen. Journal American Med. Assoc, 1903, xli, 1145. 4Maag. Centralbl. f. Chirurgie, 1901, xxviii, 20. 6 Sick. Centralbl. f. Chirurgie, 1903, xxx2, 981. «Starling & Lane. Lancet, 1903, ii, 1397. BY
ERNEST L. HUNT, M.D., WORCESTER, MASS.,
Assistant Pathologist to Worcester City Hospital. BY
ERNEST L. HUNT, M.D., WORCESTER, MASS.,
Assistant Pathologist to Worcester City Hospital. The Boston Medical and Surgical Journal as published by
The New England Journal of Medicine. Downloaded from nejm.org at UNIVERSITY OF CALGARY on September 15, 2016.
For personal use only. No other uses without permission. From the NEJM Archive. Copyright © 2010 Massachusetts Medical Society Heart sounds rapid and feeble. No
murmurs. No cardiac enlargement or displacement. Lungs negative. Upper limit of liver dullness at sixth
rib,
lower not
determined
because
of tenderness. There is extreme tenderness over entire epigastrium,
extending over left hypochondrium around the costal
margin to the back, where there is a point of especial
tenderness to the left of the twelfth dorsal vertebra. Palpation of upper abdomen unsatisfactory because
of muscular spasm. Blood. — A leucocyte count made two hours after
onset of symptoms showed 37,000 per cubic millimeter. Urine. — About
120
cc. passed later in the day. Analysis as follows : Pale; acid ¡specific gravity, 1,028;
albumin,
a large trace;
sugar, absent; sediment,
considerable, showing under the microscope numerous
hyaline
and finely granular casts,
a few coarsely
granular casts, very little free fat,
a few large and
small round cells, some of which are fatty, and numer-
ous squamous epithelial cells. following
an attack of
" grippe."
Mother is well,
except for varicose ulcers of leg. One brother subject
to malaria and severe dyspeptic attacks. Three sisters
died in youth of typhoid and another of
" bone-
cancer."
Two sisters are living at present, the elder,
forty-nine years old, is a diabetic of several months'
standing.1
The younger, forty-five
years old,
was
operated
on four years ago for uterine trouble, and
has been in delicate health since. their progress to the surface and frequently de-
termines their pointing externally behind the
angle of the jaw. In six cases which occurred at
the Children's Hospital between November and
May, 1904, the duration varied between ten days
and four weeks. Chronic, slowly accumulating
" cold " abscesses due to cervical caries are not
included, of course, in this category. The grave
condition of exhaustion frequently
seen in
a
patient with an acute or subacute retro-pharyn-
geal abscess is due to several factors: to the en-
forced starvation for many days, to sleeplessness,
to toxemia, and to insufficient oxygénation of
the blood in cases where the respiration is embar-
rassed. If now, preliminary to operative inter-
ference, the head is thrown back and the mouth
held wide open, the tendency is for the base of the
tongue and the posterior wall of the pharynx to
become still more closely approximated, unless,
indeed, the tongue is drawn strongly forward,
which perhaps is neglected in the operator's
effort to get a clear view. The Boston Medical and Surgical Journal as published by
The New England Journal of Medicine. Downloaded from nejm.org at UNIVERSITY OF CALGARY on September 15, 2016.
For personal use only. No other uses without permission. From the NEJM Archive. Copyright © 2010 Massachusetts Medical Society. Thus a sudden tem-
porarily complete asphyxiation may ensue, which,
with the frenzied struggles of the unreasonable
child, may result in cardiac collapse. Perhaps,
also, the stimulation of the sensory terminations
of the superior laryngeal nerve in the mouth and
pharynx by the necessary manipulations may
cause a reflex inhibition of the heart and respira-
tion through the pneumogastric and sympathetic
nerves, which contributes to the fatal result. I
i
il
th
d
b Personal
history. —• Typhoid
at
age
of
sixteen. Two miscarriages, the more recent being fifteen years
ago. Menstruation has been irregular since, varying
from three to six weeks. Has been subject to frequent
attacks of indigestion and epigastric pain for many
years. Is habitually
constipated. Sour
foods
in
particular
are poorly borne. Four years ago
was
treated by a stomach specialist for chronic gastritis,
and for
a few days was fed by rectum because of
nausea and vomiting. Later was
sent
to hospital,
where she was kept on a liquid diet and gradually
improved. There she was told that she had a small
uterine
fibroid. Diphtheria
one
year
ago. Four
months ago had a sudden attack of severe epigastric
pain with vomiting and marked local
tenderness. Was sick for three weeks, then resumed her customary
household duties. The epigastric
soreness persisted
more or less, however, and from time to time she has
been treated for it since. ll
l ,
In cases similar to the one reported above, a
preliminary tracheotomy would
seem to be
a
conservative procedure. It
could
be quickly
done under cocaine or even without anesthesia,
and although the child's struggles might be ex-
hausting they would, at least, not be accompanied
by the element of suffocation. The incision of
the abscess might then be deferred, if necessary,
until the general condition had improved, or, if
artificial respiration were required, the opportu-
nity would be most favorable. following
an attack of
" grippe."
Mother is well,
except for varicose ulcers of leg. One brother subject
to malaria and severe dyspeptic attacks. Three sisters
died in youth of typhoid and another of
" bone-
cancer."
Two sisters are living at present, the elder,
forty-nine years old, is a diabetic of several months'
standing.1
The younger, forty-five
years old,
was
operated
on four years ago for uterine trouble, and
has been in delicate health since. Personal
history. —• Typhoid
at
age
of
sixteen. Two miscarriages, the more recent being fifteen years
ago. Menstruation has been irregular since, varying
from three to six weeks. Has been subject to frequent
attacks of indigestion and epigastric pain for many
years. Is habitually
constipated. Sour
foods
in
particular
are poorly borne. Four years ago
was
treated by a stomach specialist for chronic gastritis,
and for
a few days was fed by rectum because of
nausea and vomiting. Later was
sent
to hospital,
where she was kept on a liquid diet and gradually
improved. There she was told that she had a small
uterine
fibroid. Diphtheria
one
year
ago. Four
months ago had a sudden attack of severe epigastric
pain with vomiting and marked local
tenderness. Was sick for three weeks, then resumed her customary
household duties. The epigastric
soreness persisted
more or less, however, and from time to time she has
been treated for it since. Present illness. — The patient felt
as well as usual
this morning until nine o'clock, when she was seized by
violent pain in the epigastrium radiating to left hypo-
chondrium and scapular region. Vomiting and retch-
ing was constant, but without relief of pain. At first,
she raised partially digested food, but after that only
an occasional mouthful of greenish fluid. No blood
has been raised. When seen, twenty minutes after
the onset, she was upon her knees on the floor, strain-
ing violently in effort to vomit, but raising nothing. She was very pale, beads of perspiration stood upon
her forehead, and the radial pulse was thread-like and
barely perceptible. Morphine sulphate
J-
gr. was
immediately given hypodermically, and patient placed
on the bed. An additional \ gr. brought slight relief. Physical examination. — Well developed and fairly
nourished. Pulse small
and compressible,
120 per
minute. Temperature,
95.5°
F. Skin pale,
moist
and cool. Pupils equal and now contracted. Tongue
lightly coated. A
CASE
OF
ACUTE
HEMORRHAGIC
PAN-
CREATITIS. onset of symptoms showed 37,000 per cubic millimeter. Urine. — About
120
cc. passed later in the day. Analysis as follows : Pale; acid ¡specific gravity, 1,028;
albumin,
a large trace;
sugar, absent; sediment,
considerable, showing under the microscope numerous
hyaline
and finely granular casts,
a few coarsely
granular casts, very little free fat,
a few large and
small round cells, some of which are fatty, and numer-
ous squamous epithelial cells. D i
th
d
th
hi
t d y p
p
Urine. — About
120
cc. passed later in the day. Analysis as follows : Pale; acid ¡specific gravity, 1,028;
albumin,
a large trace;
sugar, absent; sediment,
considerable, showing under the microscope numerous
hyaline
and finely granular casts,
a few coarsely
granular casts, very little free fat,
a few large and
small round cells, some of which are fatty, and numer-
ous squamous epithelial cells. D i
th
d
th
hi
t d The Boston Medical and Surgical Journal as published by
The New England Journal of Medicine. Downloaded from nejm.org at UNIVERSITY OF CALGARY on September 15, 2016.
For personal use only. No other uses without permission. From the NEJM Archive. Copyright © 2010 Massachusetts Medical Society DIAGNOSIS, OPERATION, RECOVERY. A
il 21
P
i
h
d
ll
i
d op ate,
o
g
At ten a.m. a suds enema was given and in the effort
to pass it the pain came on more severely than before. Three quarters of
a grain of morphine, hypodermi-
cally, in a single dose, brought partial relief. From
now on the abdomen distended rapidly and tenderness
became general, with small areas especially marked,
one such being just to the right of the umbilicus. There is dull tympany where percussion can be prac-
ticed. ,
,
g
,
April 21,
1904. Patient has gradually improved
since leaving the hospital. Complains a little of fine
darting pains in neighborhood of scar, and is some-
what
constipated. Gets
out
of doors frequently. Stools are colored normally and yellowish tint has long
since faded from sclerotics. There has been consider-
able gain in weight. Repeated examinations of the
urine have failed to show recurrence of either albumin
or glycosuria. ,
,
g
,
April 21,
1904. Patient has gradually improved
since leaving the hospital. Complains a little of fine
darting pains in neighborhood of scar, and is some-
what
constipated. Gets
out
of doors frequently. Stools are colored normally and yellowish tint has long
since faded from sclerotics. There has been consider-
able gain in weight. Repeated examinations of the
urine have failed to show recurrence of either albumin
or glycosuria. During the day the pulse rate increased, and at
4.30 p.m. was barely perceptible at 140 per minute. Temperature, 97° F. Skin covered with cool perspira-
tion. Respirations short and quick and patient mani-
fests great anxiety. Operation
had been
advised
earlier in the day and was now insisted upon. The
patient was accordingly transferred to Worcester City
Hospital where she arrived at 6 p.m. Pulse, at arrival,
152 per minute. L F W
d During the day the pulse rate increased, and at
4.30 p.m. was barely perceptible at 140 per minute. Temperature, 97° F. Skin covered with cool perspira-
tion. Respirations short and quick and patient mani-
fests great anxiety. Operation
had been
advised
earlier in the day and was now insisted upon. The
patient was accordingly transferred to Worcester City
Hospital where she arrived at 6 p.m. Pulse, at arrival,
152 per minute. L F W
d DIAGNOSIS, OPERATION, RECOVERY. Respirations short and quick and patient mani-
fests great anxiety. Operation
had been
advised
earlier in the day and was now insisted upon. The
patient was accordingly transferred to Worcester City
Hospital where she arrived at 6 p.m. Pulse, at arrival,
152 per minute. Operation. — Begun at 8 p.m. by Dr. L. F. Wood-
ward, through whose courtesy the writer
uses the
following notes upon the
case while the patient re-
mained at the hospital:
Under light ether anesthesia a four-inch
incision
was made in the median line above the umbilicus. There
was
a considerable subcutaneous fat layer,
although the patient is rather slight. Upon incising
the peritoneum, several ounces of bloody fluid escaped. The incision
was now enlarged downward passing to
the left of the umbilicus, about
a quart of bloody
fluid, containing many fat droplets, escaping. The
omentum was heavily laden with fat, and thickly
dotted over with small, circular, flattened, white areas
averaging 2 mm. in diameter. Upon sponging out the
greater omental cavity, the blood was seen to well up
from the neighborhood of the foramen of Winslow. Hasty exploration of the anterior stomach wall and
gall bladder was negative. Patient being now in an
alarming condition, the abdomen was hastily closed
with mass sutures, irrigation with hot salt solution
being kept up until the last few sutures were tied. In the meantime, two quarts of salt solution was given
intravenously. Patent
was taken to the recovery-
room in poor condition. For two days following the operation, patient re-
mained in
a critical condition
vomiting frequently Evening temperature,
Jan. 11, second day. Morning temperature, 96.3° F. Pulse, 120 per minute and of better character. Patient
had less pain during the night, slept a little under the
opiate, and is more comfortable this morning. d
i
d Evening te pe atu e,
Jan. 11, second day. Morning temperature, 96.3° F. Pulse, 120 per minute and of better character. Patient
had less pain during the night, slept a little under the
opiate, and is more comfortable this morning. A
d
i
d
h
ff ,
Pathological report on section of omentum cut to
show whitish areas is :
" Fat necrosis of omentum."
(Dr. F. H. Baker.)
Thi i
h d
F b
i h
d (Dr. Baker.)
Thirtieth day, Feb. 10, 1904, weight 101 pounds,
White count, 5,200. Hemoglobin, 85%. DISCUSSION. The
case
was
a
most interesting one from
the standpoint
of
differential
diagnosis, the
complicated
anatomy of the upper
abdomen
admitting several possibilities, and the striking
severity of the initial symptoms unquestionably
located the trouble in that region. f pe
Operation. — Begun at 8 p.m. by Dr. L. F. Wood-
ward, through whose courtesy the writer
uses the
following notes upon the
case while the patient re-
mained at the hospital:
U d
h
f
i
h
i
i i The remarkable elevation of the white-count
occurring but two hours after the onset of symp-
toms practically ruled out uncomplicated biliary
or renal colic and argued strongly against intes-
tinal
obstruction
in
which
the
leucocytosis
develops more gradually and rarely attains so
high a degree unless peritonitis has supervened. The persistent and stationary character of the
pain
as well as its location was against the two
former diseases. Perforation of an ulcer of the
posterior stomach wall, pylorus or duodenum or
acute pancreatitis were the most probable condi-
tions. The history of frequent attacks of epi-
gastric pain with nausea and vomiting was con-
sistent with any of them but absence of hemate-
mesis, prior symptoms of hyperacidity
or the
characteristic increase of pain with the ingestion
of food and beginning of the digestive process
were all against a gastric ulcer. To the absence
of tympany with obliteration of liver dullness
but little importance could be ascribed for, in
the case of duodenal ulcer, the perforation could
be retroperitoneal and if of the posterior gastric
wall, the gas might beretained in the lesser omental
cavity. The
association
of high
leucocytosis
with subnormal temperature would be unusual
in either perforative lesion, for in such cases the
white count depends upon the inflammatory reac-
tion
and
is
associated with
rise
of temper-
ature. osp ta
Under light ether anesthesia a four-inch
incision
was made in the median line above the umbilicus. There
was
a considerable subcutaneous fat layer,
although the patient is rather slight. Upon incising
the peritoneum, several ounces of bloody fluid escaped. The incision
was now enlarged downward passing to
the left of the umbilicus, about
a quart of bloody
fluid, containing many fat droplets, escaping. The
omentum was heavily laden with fat, and thickly
dotted over with small, circular, flattened, white areas
averaging 2 mm. in diameter. DIAGNOSIS, OPERATION, RECOVERY. Patient,
A. B.,
female, forty-eight
years old,
married. First seen by the writer Jan. 10, 1904, at
her home, in answer to an urgent summons referred
to him by Dr. J. M. B. Farnham. F
il
hi t
B
i
C
ti
t
H
b
d q
p
During the day the morphine
was repeated
as
needed. The pulse remaining of poor quality, ¿¡ gr. of strychnine sulphate was given every four hours. Calomel in ^
gr. doses
was ordered hourly during
the night. Sips of water were taken from time to
1 Personal observation y
Family history. — Born in
Connecticut. Husband
well, but has lived apart from patient for several years. One son, twenty-seven, well. Causes of grandparents'
death
not known. Father died of
acute diabetes time, but no diet allowed. The abdomen distended
moderately and the tenderness became more diffuse. The patient lay
on the right side constantly, and
dependent flank showed slight dullness to percussion. Evening temperature, 96.5° F. M
i Twenty-third day. Sat up in bed. Has been tak-
ing light solid and semi-solid diet without trouble. Complains still of soreness in left hypochondrium
and there is a faint yellowish tinge to sclerotics. On
palpation of abdomen there is resistance above um-
bilicus on both sides of scar, but no definite mass. P h l
i
l
i
f time, but no diet allowed. The abdomen distended
moderately and the tenderness became more diffuse. The patient lay
on the right side constantly, and
dependent flank showed slight dullness to percussion. Evening temperature, 96.5° F. Jan. 11, second day. Morning temperature, 96.3° F. Pulse, 120 per minute and of better character. Patient
had less pain during the night, slept a little under the
opiate, and is more comfortable this morning. At ten a.m. a suds enema was given and in the effort
to pass it the pain came on more severely than before. Three quarters of
a grain of morphine, hypodermi-
cally, in a single dose, brought partial relief. From
now on the abdomen distended rapidly and tenderness
became general, with small areas especially marked,
one such being just to the right of the umbilicus. There is dull tympany where percussion can be prac-
ticed. During the day the pulse rate increased, and at
4.30 p.m. was barely perceptible at 140 per minute. Temperature, 97° F. Skin covered with cool perspira-
tion. DISCUSSION. Upon sponging out the
greater omental cavity, the blood was seen to well up
from the neighborhood of the foramen of Winslow. Hasty exploration of the anterior stomach wall and
gall bladder was negative. Patient being now in an
alarming condition, the abdomen was hastily closed
with mass sutures, irrigation with hot salt solution
being kept up until the last few sutures were tied. In the meantime, two quarts of salt solution was given
intravenously. Patent
was taken to the recovery-
room in poor condition. F
d
f ll
i
th
ti
i p
For two days following the operation, patient re-
mained in
a critical condition, vomiting frequently
very small quantities of dark greenish fluid and com-
plaining of pain in upper abdomen. Small amounts
of milk and lime water were taken, the vomiting not
appearing to be excited by it. Blood-count on first
day after operation showed 24,000 whites. O
h
hi d
b
l
d b
ll y
p
,
On the third day, bowels were moved by ox-gall
enema and pulse had improved decidedly. Cathe-
terized specimen of urine at this time gave the fol-
lowing analysis: High color; acid;
specific gravity,
1,028; slightest possible trace of albumin; 2.38% of
sugar. Sediment
showed
rare
hyalin
and finely
granular casts, few leucocytes and large round cells. No blood. h
f
h d
h
b
f The family tendency to diabetes was suggestive
of, the repeated attacks of epigastric pain and
nausea were consistent with, the sudden terrific
onset, the location of the pain and its persistent
tearing quality, the extreme prostration with
subnormal temperature, the severe retching, and
later
the dullness in the dependent flank were
well nigh typical of acute hemorrhagic pancrea-
titis
to which diagnosis the writer committed
himself on the evening of the first day. On the fourth day there was but a trace of sugar. Occasional eructations of gas and fluid occurred. S i h
d
th
t
th d
th
d g
Stitches were removed on the tenth day, the wound
having healed by first intention. Liquid nourishment
still
continued
because
of nausea. Casts
and
all
traces of sugar have disappeared from the urine. cology and toxicology of paraffin; the danger
attending its use; the anatomical changes fol-
lowing its injection into animal
tissues;
the
technique of injection; finally, whether
hard
or soft paraffin
or vaseline should
be
used. ABLATION
OF
BENIGN
MAMMARY
TUMORS. H. Morestin 3 advocates the following method
for the removal of benign, movable, sharply-
circumscribed tumors of the breast. The ad-
vantage
is
the inconspicuous operation
scar
resulting. The technique consists of making the
skin incision in the axilla
along the anterior
border of the hair growth; then through this
opening, to tunnel through the subcutaneous fat
to the tumor, which in all of the writer's cases
was in the upper half of the breast and generally
in
the
outer quadrant. When
reached, the
capsule
was opened with scissors, the tumor
seized with forceps, enucleated and removed. The canal was drained and the wound sutured. Guinard recommends for larger
tumors
than
can be removed as above an incision in the sub-
mammary sulcus. BY
HERBERT
L.
BURRELL, M.D.,
BOSTON,
AND
H.
W.
CUSHING,
M.D., BOSTON. Harris 4 in concluding his paper read before
the Chicago Surgical Society in December, 1903,
emphasizes the following points:
(1) DISCUSSION. He recommends for use a mixture of vaseline and
hard paraffin having a melting point of 42° to 43°. This mixture is "plastic" as vaseline, and has
also the stability of hard paraffin. The forma-
tion of emboli results from injection of that
material in a liquid state. When the mixture is
so firm that it escapes from the needle point as a
fine thread the breaking loose of fine particles
which can escape into the blood current is impos-
sible. This requires
an especially constructed
syringe. The white-count of 37,000 is, so far as the writer
is aware, the largest reported in cases known to
be acute pancreatitis although 19,000 to 24,000
have been observed. Should experience prove
this early leucocytosis to be a constant feature
of the disease its diagnostic value can hardly be
overestimated. Unfortunately, the operation did not absolutely
settle the question of diagnosis as the pancreas
itself was not exposed. It did, however, afford
strong confirmatory evidence in the widespread
fat necrosis with free blood in the peritoneal
cavity, two conditions which are only associated
in pancreatic disease. The later appearance of
glycosuria though transitory
is best explained
by the assumption of a pancreatic lesion. syringe. A special chapter is devoted to the employment
of this mixture in different organs and tissues
and the special technique. This is abundantly
illustrated; two hundred and twenty-five biblio-
graphical references complete the work. y
assu pt o
pancreatic
In the light of the favorable outcome of the
case, question may perhaps be raised as to the
expediency of the operation. Obviously, all that
it accomplished was the removal of the bloody
fluid and clots from the abdomen where if, as is
likely, it contained enzymes of the pancreatic
secretion it would doubtless have done harm. Evidently the bleeding had
ceased sometime
before the operation, and that further hemor-
rhage did not take place was a matter of good
fortune hardly attributable to the operation. It
may therefore be fairly urged that non-operative
treatment would have had a like favorable result. To the writer's mind, however, operation
was
positively indicated
as
a diagnostic procedure. Indeed, it should have been resorted to several
hours sooner for had a perforation, which was
the most probable alternate diagnosis, existed,
the best chance for the patient lay in timely
operation. INHALATION OF GASOLINE VAPORS. Sollmann * in an article on this subject gives
the following conclusions: Gasoline
or (petro-
leum ether) when inhaled has
a comparatively
slight
anesthetic
action. The
anesthesia
is,
however, very dangerous, since the substance
also produces convulsions, weakening of the heart,
vasomoter depression and paralysis of the respi-
ration. The kidneys
are also irritated. The
effects are produced very promptly and recovery
under artificial respiration
is also prompt. The
cause of the convulsions is not known. They
do not occur in frogs. p
o o
g points:
(1) In penetrating wounds of the abdomen,
there are absolutely no known symptoms which
indicate injury to any of the viscera
except
those noted above in connection with the urin-
ary tract, stomach and, occasionally, the lower
bowel. (2) Except those relating to general shock,
all symptoms following such wounds indicate
either internal hemorrhage or peritonitis. (3) hemorrhage
peritonitis. (3) To wait for symptoms of perforation of
the intestine means to wait until peritonitis has
developed, therefore, p
,
,
(4) Every bullet or stab wound which pene-
trates the abdominal cavity should be operated
on at the earliest possible moment in order to
anticipate the advent of peritonitis. i The Boston Medical and Surgical Journal as published by
The New England Journal of Medicine. Downloaded from nejm.org at UNIVERSITY OF CALGARY on September 15, 2016.
For personal use only. No other uses without permission. From the NEJM Archive. Copyright © 2010 Massachusetts Medical Society The Boston Medical and Surgical Journal as published by
The New England Journal of Medicine. Downloaded from nejm.org at UNIVERSITY OF CALGARY on September 15, 2016.
For personal use only. No other uses without permission. From the NEJM Archive. Copyright © 2010 Massachusetts Medical Society. The Boston Medical and Surgical Journal as published by PARAFFIN INJECTION. This subject, which during the past few years
has
aroused
considerable attention,
has
been
exhaustively treated in
a monograph of
one
hundred and sixty-six pages by A. E. Stein.2
He discusses the history, chemistry, pharma- p
peritonitis. (5) No time should be wasted in attempting
to demonstrate the presence or absence of intes-
tinal perforation by such
means
as the rectal
|
https://openalex.org/W2272400168
|
https://europepmc.org/articles/pmc4644923?pdf=render
|
English
| null |
Efficiency of different protocols for enamel clean-up after bracket debonding: an in vitro study
|
Dental Press Journal of Orthodontics
| 2,015
|
cc-by
| 6,576
|
original article original article DOI: http://dx.doi.org/10.1590/2177-6709.20.5.078-085.oar DOI: http://dx.doi.org/10.1590/2177-6709.20.5.078-085.oar Objective: This study aimed to assess the efficiency of six protocols for cleaning-up tooth enamel after bracket debond-
ing. Methods: A total of 60 premolars were divided into six groups, according to the tools used for clean-up: 12-blade
bur at low speed (G12L), 12-blade bur at high speed (G12H), 30-blade bur at low speed (G30L), DU10CO ORTHO
polisher (GDU), Renew System (GR) and Diagloss polisher (GD). Mean roughness (Ra) and mean roughness depth (Rz) of
enamel surface were analyzed with a profilometer. Paired t-test was used to assess Ra and Rz before and after enamel clean-up. ANOVA/Tukey tests were used for intergroup comparison. The duration of removal procedures was recorded. The association
between time and variation in enamel roughness (∆Ra, ∆Rz) were evaluated by Pearson’s correlation test. Enamel topography
was assessed by scanning electron microscopy (SEM). Results: In Groups G12L and G12H, original enamel roughness did not
change significantly. In Groups G30L, GDU, GR and GD, a smoother surface (p < 0.05) was found after clean-up. In Groups
G30L and GD, the protocols used were more time-consuming than those used in the other groups. Negative and moderate
correlation was observed between time and (∆Ra, ∆Rz); Ra and (∆Ra, ∆Rz); Rz (r = - 0.445, r = - 0.475, p < 0.01). Conclu-
sion: All enamel clean-up protocols were efficient because they did not result in increased surface roughness. The longer the
time spent performing the protocol, the lower the surface roughness. Keywords: Orthodontic brackets. Dental enamel. Dental debonding. Objetivo: esse estudo objetivou avaliar a eficiência de seis protocolos de remoção de resina do esmalte após a descolagem
de braquetes. Métodos: sessenta (60) pré-molares foram divididos em seis grupos conforme as ferramentas utilizadas:
broca de 12 lâminas em baixa rotação (G12L), broca de 12 lâminas em alta rotação (G12H), broca de 30 lâminas em bai-
xa rotação (G30L), polidor DU10CO-ORTHO (GDU), Renew System (GR) e polidor Diagloss (GD). As médias de
rugosidade (Ra) e profundidade média de rugosidade (Rz) da superfície do esmalte foram analisadas com perfilômetro. Teste t pareado foi utilizado para avaliar Ra e Rz antes e depois da limpeza do esmalte; testes de ANOVA/Tukey foram
utilizados para avaliar a diferença intergrupos. A duração dos procedimentos de remoção da resina foi registrada. Ainda,
a associação entre o tempo e a variação da rugosidade do esmalte (ΔRa, ΔRz) foi avaliada pelo teste de correlação de
Pearson. How to cite this article: Sigilião LCF, Marquezan M, Elias CN, Ruellas AC,
Sant’Anna EF. Efficiency of different protocols for enamel clean-up after bracket
debonding: an in vitro study. Dental Press J Orthod. 2015 Sept-Oct;20(5):78-85.
DOI: http://dx.doi.org/10.1590/2177-6709.20.5.078-085.oar Efficiency of different protocols for enamel clean-up
after bracket debonding: an in vitro study Lara Carvalho Freitas Sigilião1, Mariana Marquezan2, Carlos Nelson Elias3,
Antônio Carlos Ruellas4, Eduardo Franzotti Sant’Anna4 Submitted: December 08, 2014 - Revised and accepted: February 05, 2015 DOI: http://dx.doi.org/10.1590/2177-6709.20.5.078-085.oar Efficiency of different protocols for enamel clean-up after bracket
debonding: an in vitro study. Dental Press J Orthod. 2015 Sept-Oct;20(5):78-85. DOI: http://dx.doi.org/10.1590/2177-6709.20.5.078-085.oar DOI: http://dx.doi.org/10.1590/2177-6709.20.5.078-085.oar A topografia do esmalte também foi avaliada, por microscopia eletrônica de varredura (MEV). Resultados: nos
grupos G12L e G12H, a rugosidade do esmalte original não se alterou significativamente. Nos Grupos G30L, GDU,
GR e GD, foi verificada superfície mais lisa após a limpeza (p < 0,05). Nos Grupos G30L e GD, os protocolos utilizados
foram mais demorados que nos demais grupos. Foi observada correlação negativa e moderada entre tempo, ΔRa e ΔRz
(r = -0.445, r = -0.475, p < 0,01). Conclusão: todos os protocolos de limpeza do esmalte foram eficientes, pois não resul-
taram no aumento da rugosidade superficial. Quanto maior o tempo gasto, menor a rugosidade da superfície. Objetivo: esse estudo objetivou avaliar a eficiência de seis protocolos de remoção de resina do esmalte após a descolagem
de braquetes. Métodos: sessenta (60) pré-molares foram divididos em seis grupos conforme as ferramentas utilizadas:
broca de 12 lâminas em baixa rotação (G12L), broca de 12 lâminas em alta rotação (G12H), broca de 30 lâminas em bai-
xa rotação (G30L), polidor DU10CO-ORTHO (GDU), Renew System (GR) e polidor Diagloss (GD). As médias de
rugosidade (Ra) e profundidade média de rugosidade (Rz) da superfície do esmalte foram analisadas com perfilômetro. Teste t pareado foi utilizado para avaliar Ra e Rz antes e depois da limpeza do esmalte; testes de ANOVA/Tukey foram
utilizados para avaliar a diferença intergrupos. A duração dos procedimentos de remoção da resina foi registrada. Ainda,
a associação entre o tempo e a variação da rugosidade do esmalte (ΔRa, ΔRz) foi avaliada pelo teste de correlação de
Pearson. A topografia do esmalte também foi avaliada, por microscopia eletrônica de varredura (MEV). Resultados: nos
grupos G12L e G12H, a rugosidade do esmalte original não se alterou significativamente. Nos Grupos G30L, GDU,
GR e GD, foi verificada superfície mais lisa após a limpeza (p < 0,05). Nos Grupos G30L e GD, os protocolos utilizados
foram mais demorados que nos demais grupos. Foi observada correlação negativa e moderada entre tempo, ΔRa e ΔRz
(r = -0.445, r = -0.475, p < 0,01). Conclusão: todos os protocolos de limpeza do esmalte foram eficientes, pois não resul-
taram no aumento da rugosidade superficial. Quanto maior o tempo gasto, menor a rugosidade da superfície. Palavras-chave: Braquetes ortodônticos. Esmalte dentário. Descolagem dentária. How to cite this article: Sigilião LCF, Marquezan M, Elias CN, Ruellas AC,
Sant’Anna EF. Contact address: Eduardo Franzotti Sant’Anna
Av. Professor Rodolpho Paulo Rocco, 325, Ilha do Fundão, Rio de Janeiro/RJ
E-mail: eduardo.franzotti@gmail.com INTRODUCTION The bond area was limited by marks made on the
base of the specimens to ensure that roughness as-
sessments were made in the same area. Direct bracket bonding to tooth surface became
possible with the advent of acid etching which revo-
lutionized the orthodontic practice.1 On comple-
tion of orthodontic treatment, the residual resin left
behind after bracket debonding must be cleaned ef-
ficiently and rapidly while preserving enamel sur-
face; in addition, enamel surface must be smoothed
and polished to prevent plaque accumulation. Several factors are involved in these procedures, in-
cluding the tools used for debonding, protocols for
residual resin removal, the type of adhesive used2
and the operator’s skill. Samples were randomly divided into six equal
groups (n = 10) to compare different protocols for
removal of adhesive remnant and enamel polishing
(Table 1). Sample size was calculated at a level of
significance set at 5% and test power of 80%, based
on data from a previous study.8 Teeth were cleaned with fine pumice slurry us-
ing a rubber cup in a low-speed handpiece for ap-
proximately 10 seconds, followed by rinsing and dry-
ing with moisture-free air spray. Subsequently, teeth
were etched for 20 seconds with 37% phosphoric
acid gel (Magic Acid Vigodent®, Rio de Janeiro, RJ,
Brazil), rinsed for 20 seconds and dried. Premolar
metal brackets (Morelli®, Sorocaba, SP, Brazil) were
bonded to teeth with Transbond XT (3M Unitek,
Monrovia, Calif, USA), following the manufacturer’s
instructions. Brackets were placed on teeth surfaces
and firmly pressed into position for the base to fit
perfectly, providing uniform resin layer in all spec-
imens. After removing excess resin from the edges
of bracket bases with the aid of a dental probe, teeth
were light-polymerized for 10 seconds on each side of
the bracket by means of a conventional LED curing
unit (Optilight Max - Gnatus®, Ribeirão Preto, SP,
Brazil). Specimens were then stored in artificial saliva
at 37 oC for 24 hours to facilitate maximum polymer-
ization and hydration of the material. Submitted: December 08, 2014 - Revised and accepted: February 05, 2015 » The authors report no commercial, proprietary or financial interest in the products
or companies described in this article. © 2015 Dental Press Journal of Orthodontics 78 Dental Press J Orthod. 2015 Sept-Oct;20(5):78-85 al article original article Sigilião LCF, Marquezan M, Elias CN, Ruellas AC, Sant’Anna EF INTRODUCTION Although there is no consensus in the literature
regarding this matter, one of the most common
methods of removing residual adhesive from the
enamel surface is using a tungsten carbide bur at low
speed.3-6 Several new and more conservative multiple
and one-step systems for enamel clean-up, such as
fiber-reinforced composites,7 polishers with diamond
particles, aluminum oxide rubber and sandblasting,6
have been developed and gained popularity among
orthodontists. However, many of these tools have not
been tested as a method of providing characteristics
similar to those of the original enamel. The aims of this study were to compare in vitro
enamel surface roughness by using six protocols for
removal of adhesive remnant and enamel polishing
after bracket debonding; assess the time spent to re-
move residual resin in each one of them; and assess
the correlation between roughness and removal time. Brackets were then removed by gently squeezing their
mesial and distal wings with How Reto pliers.9 Enamel
surfaces were evaluated under Olympus SZ40 stereomi-
croscope (Olympus, Japan) under 15X magnification.10
They were classified according to the Adhesive Remnant
Index (ARI)11: score 0 = no adhesive on enamel, score 1
= less than 50% adhesive on enamel, score 2 = more than
50% adhesive on enamel, score 3 = all adhesive remain-
ing on enamel. Teeth were included in the experiment
only if the most of resin remained on enamel surface af-
ter debonding (score 2 or 3), in order to allow adequate
evaluation of all finishing protocols. Fortunately, none of
the samples were excluded. Groups G12L, G12H, G30L
and GD had five specimens classified as ARI score 2 and
five specimens classified as ARI score 3. Groups GDU
and GR had four specimens classified as score 2 and six
specimens classified as score 3. © 2015 Dental Press Journal of Orthodontics RESULTS Results showed that all protocols tested for
removal of adhesive remnant from enamel did not
lead to increase in the original surface roughness
significantly. Quantitative and qualitative enamel evaluations were
performed. For quantitative evaluation, roughness was
measured at two time points: before bonding, to estab-
lish initial roughness; and after debonding and removal
of adhesive remnants with finishing and polishing pro-
tocols, to establish final roughness. A profilometer (Mi-
tutoyo Surftest SJ-400, Japan), with a cut-off value of
0.8 mm, was used to measure the roughness profile of
each surface. Two measurements were performed on
each specimen, parallel to one another, traversing the
entire 4-mm bonding surface. The mean value of the
two measurements of each specimen was recorded. This
process involved recording two roughness parameters:
1) Mean roughness (Ra), in µm, determined as the arith-
metic mean of all absolute distances of the roughness
profile from the center line within the measuring length;
and 2) Mean roughness depth (Rz) which describes the
average maximum peak-to-valley height of five consec-
utive sampling lengths.5,12 Variation in roughness was
calculated by the equations: ΔRa = final Ra – initial Ra
and ΔRz = final Rz – initial Rz. Ra results for measurements taken before bracket
bonding and after residual resin removal are summa-
rized in Table 2. Groups G12H and G12L, in which a
12-blade tungsten carbide bur was used at low and high
speed, respectively, showed no significant differences
before bonding and after debonding. Groups G30L,
GDU, GR and GD showed a smoother surface after
30-blade tungsten carbide bur (low speed), DU10CA
ORTHO points, 12-blade tungsten carbide bur (high
speed) + Renew™ Finishing System, and Diagloss
polisher were used, respectively (p < 0.05). Rz results for measurements taken before bracket
bonding and after residual resin removal are summa-
rized in Table 3. Groups G12H and G12L showed
no significant differences before bonding and after
debonding, and so did Group GDU. Groups G30L,
GR and GD showed a reduction in maximum peak-
to-valley height (p < 0.05). When ΔRa was compared by means of ANOVA/
Tukey tests, there was no statistically significant dif-
ference among the six groups (Table 4). All values
were negative because the final Ra value was lower
than the initial Ra value. When the six groups were
compared in terms of ΔRz, some statistical differ-
ences were observed (Table 4). RESULTS Groups G30L and
GD presented a decrease in vertical irregularities,
while the positive value of ΔRz for G12H implied
an increase in vertical irregularities. For qualitative evaluation of enamel surface,
scanning electron microscopy (Quanta Feg 250, FEI
Company, Oregon, USA) was performed to com-
pare enamel surface of experimental groups. MATERIAL AND METHODS This study was approved by the Research
and Ethics Committee of the Institute of Public
Health and Research at Universidade Federal do
Rio de Janeiro, Brazil (#05/2012). A total of 60 human caries-free premolars
extracted for orthodontic purposes were stored in
aqueous solution of thymol (0.1%) to prevent bac-
terial growth and dehydration. Teeth were selected
based on visual observation of soundness of the
buccal surfaces, absence of caries and cracks in the
coronal portion, and no previous exposure to adhe-
sive agents. The teeth roots were removed and the
crowns were embedded in self-polymerizing acryl-
ic resin with the buccal surfaces facing upwards. © 2015 Dental Press Journal of Orthodontics 79 Dental Press J Orthod. 2015 Sept-Oct;20(5):78-85 original article Efficiency of different protocols for enamel clean-up after bracket debonding: an in vitro study The same operator performed debonding and
adhesive removal without water cooling, and with
a new bur or rubber used after treating every two
teeth. The overall extent of resin removal was de-
termined by visual inspection under the light of an
operative lamp. The time required for completion of
each resin removal protocol was recorded in seconds
with a digital chronometer. tests. Pearson’s correlation test was performed to as-
sess the association between ΔRa and ΔRz and time
spent on each enamel clean-up protocol. A level of
significance of 0.05 was used for all analyses. © 2015 Dental Press Journal of Orthodontics STATISTICAL ANALYSIS FF9714 ( Jet - Beavers Dental®, Ontario, Canada), 20,000 rpm;
d DU10CA ORTHO (DhPro®, Paranaguá, PR, Brazil), 9,000 rpm;
e Renew™ Finishing System (Reliance Orthodontics® – Illinois, USA);
f Diagloss polisher (Edenta, Switzerland), 10,000 a 12,000 rpm
Groups
N
Protocols
G12L
10
12-blade tungsten carbide bur (low speed)a
G12H
10
12-blade tungsten carbide bur (high speed)b
G30L
10
30-blade tungsten carbide bur (low speed)c
GDU
10
DU10CA ORTHO Pointsd
GR
10
12-blade tungsten carbide bur (high speed)
+ Renew™ Finishing System Pointe
GD
10
Diagloss polisherf a Ref. H23R.21.012 (Brasseler®, Savannah, GA, USA), 20,000 rpm;
b Ref. H23R.31.012 (Brasseler®, Savannah, GA, USA);
c Ref. FF9714 ( Jet - Beavers Dental®, Ontario, Canada), 20,000 rpm;
d DU10CA ORTHO (DhPro®, Paranaguá, PR, Brazil), 9,000 rpm;
e Renew™ Finishing System (Reliance Orthodontics® – Illinois, USA);
f Diagloss polisher (Edenta, Switzerland), 10,000 a 12,000 rpm
Groups
N
Protocols
G12L
10
12-blade tungsten carbide bur (low speed)a
G12H
10
12-blade tungsten carbide bur (high speed)b
G30L
10
30-blade tungsten carbide bur (low speed)c
GDU
10
DU10CA ORTHO Pointsd
GR
10
12-blade tungsten carbide bur (high speed)
+ Renew™ Finishing System Pointe
GD
10
Diagloss polisherf DISCUSSION Table 2 - Mean and standard deviation (SD) for initial and final Ra and results
of paired t-test. Table 2 - Mean and standard deviation (SD) for initial and final Ra and results
of paired t-test. * Indicates statistical significance (p < 0.05). Groups
Initial Ra (µm)
Final Ra (µm)
p-value
Mean (SD)
Mean (SD)
G12L
1.60 (0.50)
1.39 (0,15)
0.289
G12H
1.99 (0.34)
1.79 (0.38)
0.187
G30L
1.96 (0.50)
1.45 (0.43)
0.003 *
GDU
1.65 (0.34)
1.45 (0.24)
0.045 *
GR
1.64 (0.32)
1.31 (0.32)
0.025 *
GD
2.04 (0.43)
1.45 (0.22)
0.001 * In this study, six protocols for removal of ad-
hesive remnant from enamel after bracket debond-
ing were assessed. The choice of burs and abrasive
points was based on the protocols most used by or-
thodontists, in other words, tungsten carbide burs
in low and high-speed handpieces,3-6 and products
launched on the market in recent years. Many studies use the parameter Ra as the only
indicator of surface smoothness. However, this uni-
versally accepted parameter has limitations when Table 3 - Mean and standard deviation (SD) for initial and final Rz and results
of paired t-test. * Indicates statistical significance (p < 0.05). Groups
Initial Rz (µm)
Final Rz (µm)
p-value
Mean (SD)
Mean (SD)
G12L
6.03 (3.04)
5.48 (0.59)
0.595
G12H
8.16 (2.16)
8.66 (1.75)
0.634
G30L
7.90 (2.33)
5.16 (1.77)
0.001*
GDU
6.26 (2.31)
5.82 (1.62)
0.404
GR
6.04 (1.50)
4.65 (1.00)
0.023*
GD
8.07 (2.47)
5.35 (1.06)
0.002*
Table 4 - Mean and standard deviation (SD) for ΔRa and ΔRz and results of
ANOVA/Tukey. Each column indicates an independent statistical analysis. Different letters indicate statistically significant difference (p < 0.05) for ANO-
VA/Tukey. Groups
ΔRa
ΔRz
Mean (SD)
Mean (SD)
G12L
- 0.20 (0.58)a
- 0.55 (3.15)AB
G12H
- 0.19 (0.43)a
0.49 (3.17)B
G30L
- 0.51 (0.39)a
- 2.74 (1.82)A
GDU
- 0.20 (0.27)a
- 0.44 (1.59)AB
GR
- 0.32 (0.38)a
- 1.39 (1.60)AB
GD
- 0.59 (0.38)a
- 2.71 (2.00)A
Table 5 - Time required for cleaning residual resin after debracketing (sec-
onds) p < 0.05. SD - Standard deviation. Different letters indicate statistically significant difference. G12L
G12H
G30L
GDU
GR
GD
Mean
(SD)
34.0
(5.73)
23.5
(5.01)
57.5
(19.9)
31.8
(4.56)
31.9
(5.85)
63.5
(13.8)
A
A
B
A
A
B
Table 6 - Pearson’s Linear Correlation Coefficient between the time required
and the variations in roughness. DISCUSSION ** p ≤ 0.01. ΔRa
ΔRz
(95% confidence interval)
(95% confidence interval)
Time
- 0.445 **
-0.475 **
- (-0.685 _ -0.143)
(- 0.627 _ -0.214) Table 3 - Mean and standard deviation (SD) for initial and final Rz and results
of paired t-test. Dental Press J Orthod. 2015 Sept-Oct;20(5):78-85
1
Table 4 - Mean and standard deviation (SD) for ΔRa and ΔRz and results of
ANOVA/Tukey. Each column indicates an independent statistical analysis. Different letters indicate statistically significant difference (p < 0.05) for ANO-
VA/Tukey. Groups
ΔRa
ΔRz
Mean (SD)
Mean (SD)
G12L
- 0.20 (0.58)a
- 0.55 (3.15)AB
G12H
- 0.19 (0.43)a
0.49 (3.17)B
G30L
- 0.51 (0.39)a
- 2.74 (1.82)A
GDU
- 0.20 (0.27)a
- 0.44 (1.59)AB
GR
- 0.32 (0.38)a
- 1.39 (1.60)AB
GD
- 0.59 (0.38)a
- 2.71 (2.00)A
Table 6 - Pearson’s Linear Correlation Coefficient between the time required
and the variations in roughness. ** p ≤ 0.01. ΔRa
ΔRz
(95% confidence interval)
(95% confidence interval)
Time
- 0.445 **
-0.475 **
- (-0.685 _ -0.143)
(- 0.627 _ -0.214) Table 4 - Mean and standard deviation (SD) for ΔRa and ΔRz and results of
ANOVA/Tukey. Table 4 - Mean and standard deviation (SD) for ΔRa and ΔRz and results of
ANOVA/Tukey. Table 3 - Mean and standard deviation (SD) for initial and final Rz and results
of paired t-test. * Indicates statistical significance (p < 0.05). Groups
Initial Rz (µm)
Final Rz (µm)
p-value
Mean (SD)
Mean (SD)
G12L
6.03 (3.04)
5.48 (0.59)
0.595
G12H
8.16 (2.16)
8.66 (1.75)
0.634
G30L
7.90 (2.33)
5.16 (1.77)
0.001*
GDU
6.26 (2.31)
5.82 (1.62)
0.404
GR
6.04 (1.50)
4.65 (1.00)
0.023*
GD
8.07 (2.47)
5.35 (1.06)
0.002* Table 5 - Time required for cleaning residual resin after debracketing (sec-
onds) p < 0.05. SD - Standard deviation. Different letters indicate statistically significant difference. G12L
G12H
G30L
GDU
GR
GD
Mean
(SD)
34.0
(5.73)
23.5
(5.01)
57.5
(19.9)
31.8
(4.56)
31.9
(5.85)
63.5
(13.8)
A
A
B
A
A
B Table 5 - Time required for cleaning residual resin after debracketing (sec-
onds) p < 0.05. SD - Standard deviation. Different letters indicate statistically significant difference. STATISTICAL ANALYSIS Results were collected and statistically analyzed
by means of SPSS version 20.0 software (Statisti-
cal Package for Social Sciences, SPSS Inc., Chi-
cago, IL, USA). Distribution of variables was as-
sessed for normality by Kolmogorov-Smirnov and
Shapiro-Wilk tests. Paired t-test was used to assess
the mean values of roughness parameters (Ra and Rz)
before and after enamel surface clean-up, and ver-
ify whether this processes altered enamel surface
roughness. Intergroup differences for ΔRa, ΔRz and
time required for cleaning the residual resin after
bracket debonding were assessed by ANOVA/Tukey The time spent for resin remnant removal is
shown in Table 5. The protocols used in Groups
G30L and GD were more time-consuming than
those used in the other groups (p < 0.05). Correlation
between time-ΔRa and time-ΔRz was negative and
moderate (Table 6). Scatter plots illustrate these re-
sults (Figs 1 and 2). Inspection in scanning electron microscopy shows
the enamel surface before bonding (Fig 3) as well as 80 Dental Press J Orthod. 2015 Sept-Oct;20(5):78-85 original article Sigilião LCF, Marquezan M, Elias CN, Ruellas AC, Sant’Anna EF Table 1 - Distribution of groups according to the protocol applied for removal
of adhesive remnant. after debonding and enamel clean-up (Fig 4). Scratch-
es produced by the 12-blade burs at low speed are
presented in Figure 4A. Deeper scratches were pro-
duced by the burs at high speed (Fig 4B). The highest
degree of surface smoothness was obtained in Group
G30L (Fig 4C) This group presented surface more
similar to the original tooth, as shown in Figure 3. In Groups GDU and GR, there was loss of periky-
mata with fine scratches caused by polishers of vary-
ing abrasiveness (Fig 4D and Fig 4E). Fine scratches,
which appeared to be well-marked and deep, caused
by the diamond particles embedded in rubber, were
also seen in Group GD (Fig 4F). a Ref. H23R.21.012 (Brasseler®, Savannah, GA, USA), 20,000 rpm;
b Ref. H23R.31.012 (Brasseler®, Savannah, GA, USA);
c Ref. DISCUSSION G12L
G12H
G30L
GDU
GR
GD
Mean
(SD)
34.0
(5.73)
23.5
(5.01)
57.5
(19.9)
31.8
(4.56)
31.9
(5.85)
63.5
(13.8)
A
A
B
A
A
B Table 5 - Time required for cleaning residual resin after debracketing (sec-
onds) p < 0.05. ** p ≤ 0.01. © 2015 Dental Press Journal of Orthodontics © 2015 Dental Press Journal of Orthodontics 81 Dental Press J Orthod. 2015 Sept-Oct;20(5):78-85 Dental Press J Orthod. 2015 Sept-Oct;20(5):78-85 Dental Press J Orthod. 2015 Sept-Oct;20(5):78-85 original ar Efficiency of different protocols for enamel clean-up after bracket debonding: an in vitro study Figure 1 - Scatter plot of variation in roughness (∆Ra) in relation to time in
all groups. Figure 2 - Scatter plot of variation in roughness (∆Rz) in relation to time in
all groups. Group
Group Figure 2 - Scatter plot of variation in roughness (∆Rz) in relation to time in
all groups. Group Group Group Figure 2 - Scatter plot of variation in roughness (∆Rz) in relation to time in
all groups. Figure 1 - Scatter plot of variation in roughness (∆Ra) in relation to time in
all groups. Figure 3 - Scanning electron microscopy (200 X magnification) of original
enamel; perikymata (P); prism end openings (arrows). Figure 4 - Scanning electron microscopy (500 X magnification) showing the
effect of enamel clean-up procedures on the surface. A) 12-blade tungsten
carbide bur (low speed) (G12L); B) 12-blade tungsten carbide bur (high speed)
(G12H); C) 30-blade tungsten carbide bur (low speed) (G30L); D) DU10CA
ORTHO polisher; E) Renew Finishing System; F) Diagloss polisher. A
C
E
B
D
F A
C
B
D A A B D C F E Figure 3 - Scanning electron microscopy (200 X magnification) of original
enamel; perikymata (P); prism end openings (arrows). Figure 4 - Scanning electron microscopy (500 X magnification) showing the
effect of enamel clean-up procedures on the surface. A) 12-blade tungsten
carbide bur (low speed) (G12L); B) 12-blade tungsten carbide bur (high speed)
(G12H); C) 30-blade tungsten carbide bur (low speed) (G30L); D) DU10CA
ORTHO polisher; E) Renew Finishing System; F) Diagloss polisher. © 2015 Dental Press Journal of Orthodontics 82 Dental Press J Orthod. 2015 Sept-Oct;20(5):78-85 al article original article Sigilião LCF, Marquezan M, Elias CN, Ruellas AC, Sant’Anna EF used alone5,7 because it does not determine the pro-
file of irregularities and makes no distinction be-
tween peaks and valleys. The association of other
parameters used in this study, such as Rz, enabled
us to study the shape of the vertical profile. condition is transient, thereby indicating that the
damage caused by this protocol is reversible, and
pulp repair occurs within about 20 days. © 2015 Dental Press Journal of Orthodontics The authors
recommend removing most of the resin under wa-
ter cooling and turning the water cooling off when
removing the last layer of resin, so that it is pos-
sible to successfully distinguish between enamel and
resin, thereby preventing further damage to enamel. Considering the results of the present study, low-
speed burs without water cooling could be used to
remove the last layer of resin, so the risk of enamel
scratches might be reduced. In this study, all resin
remnant was removed without water cooling. It is
suggested that future studies assess enamel rough-
ness and loss when following the aforementioned
recommendations. In this study, the protocols involving 12-blade
tungsten carbide burs at low and high speed pro-
duced similar results considering Ra. The Rz pa-
rameter, however, was markedly affected when the
12-blade tungsten carbide bur was used at high
speed. A ΔRz value of 0.49 was the only positive
value (indicating increase in roughness) and statis-
tically different from Groups G30L and GD. This
outcome demonstrated the increase in irregularities
with sporadic deep scratches, which were not de-
tected by ΔRa, because Ra is an indicator of mean
roughness and does not account for the presence of
an occasional peak or valley. In SEM evaluation,
the 12-blade bur produced deeper scratches at high
speed. However, no statistically significant differ-
ence was observed for both roughness parameters
(ΔRa and ΔRz) between G12L and G12H. Group GR, which involved the use of 12-blade
tungsten carbide burs at high speed followed by
Renew polisher, showed a significant (p < 0.05) de-
crease in the two roughness parameters between the
two time points, indicating the importance of gently
eliminating the last layer of resin with polishers af-
ter using burs at high speed.1,6,8 The literature shows
that the sequential use of multiple tools for polishing
is more efficient than one-step procedures2,3,17,20,23,24
in terms of reduction in surface roughness. In this
study, GR resulted in a low level of surface rough-
ness, with negative values for ΔRa and ΔRz. However, the final variation in ΔRz roughness of
GR was not statistically different from the majority
of the other groups, except for G12L (Table 4). © 2015 Dental Press Journal of Orthodontics © 2015 Dental Press Journal of Orthodontics The literature reports that the use of tungsten
carbide burs at high speed to remove resin remnant
after debonding leaves the surface similar to that of
intact enamel;2,8,13,14 however, at the cost of a sub-
stantial loss in enamel thickness (19.2 µm).2,15 Other
studies recommend the use of tungsten carbide burs
at low speed3,16-18, which create fine scratches19 with
a lower level of enamel loss (7.9 µm to 11.3 µm)2,10. In this study, enamel loss was not measured,
although this factor should be an important con-
sideration when choosing the method for resin
removal. According to Smith et al,21 the average
enamel thickness of a maxillary central incisor is ap-
proximately 0.6 mm (600 µm). Considering a single
bracket/resin removal, a loss of 10 or 20 µm might
seem harmless, but it is necessary to consider the
possibility of multiple rebondings due to bracket loss
(caused by the patient) or bonding errors (caused
by the orthodontist). Therefore, the orthodontist
should minimize enamel damage and loss. Roughness values obtained after clean-up in
Groups G30L, GDU, GR and GD were lower than
the initial roughness values. Similar results were
found in other studies,7,18,20 in which abrasive points
and 30-blade tungsten carbide burs were used to
eliminate adhesive remnant. In a previous study,
microscopic evaluation showed that the use of abra-
sive points (Optimize Discs – TDV – and Onegloss
Discs – Shofu) maintained the enamel surface of the
study groups in a similar condition to the enamel
surface of the control groups.25 The use of high-speed burs without wa-
ter cooling has been previously described by
Bicakci et al.22 They observed heating in the pulp
chamber, leading to vascular hyperemia and oc-
casional breakage of odontoblasts. However, this The time required for removing resin differed
among the six groups, mainly due to differenc-
es in the cutting power of tools used,7 which was
mainly determined by the speed of rotation,17 type
of bur, number of blades, composition, particle size, 83 Dental Press J Orthod. 2015 Sept-Oct;20(5):78-85 original arti Efficiency of different protocols for enamel clean-up after bracket debonding: an in vitro study leaving a smoother surface less prone to plaque adhe-
sion and pigmentation. and pressure applied to the handpiece.2 The latter
variable was minimized because the same operator
performed all resin removal procedures. CONCLUSIONS 1) All finishing and polishing protocols were
considered satisfactory for residual resin removal
without increasing enamel roughness. 1) All finishing and polishing protocols were
considered satisfactory for residual resin removal
without increasing enamel roughness. 2) The time spent on enamel clean-up varied
from 23.5 (12-blade tungsten carbide bur at high
speed) to 63.5 seconds (Diagloss polishers). 3) The longer the time spent on removing the remain-
ing resin, the smaller the variation in roughness level. Our
findings
corroborate
those
of
other
studies,3,14,19,20,25,26 indicating that all rotary instru-
ments cause varying changes in enamel surface. The association between the time spent and change
in roughness (ΔRa, ΔRz) showed a negative and
moderate correlation: the longer the time spent on
removing the remaining resin, the lower was the
roughness left on the enamel surface, which is in
agreement with a previous study.1 Instruments with
low cutting power perform slower resin removal, © 2015 Dental Press Journal of Orthodontics The time
required in the groups in which Diagloss polishers
(63.5 seconds) and 30-blade tungsten carbide burs
at low speed (57.5 seconds) were used, was signifi-
cantly longer than that required in the other groups
(p < 0.05). The protocol used in GD involved two
steps: use of rubber with a high gradient of dia-
mond particle concentration, which ensured resin
reduction, followed by another point for polishing. Hence, the procedure consumed more chair time. In Group G30L, the higher number of blades of the
bur used at low speed decreased its cutting power
and removed the resin layer by layer, which results
in a smoother and scratch-free surface; however, it
increased the time required for resin removal. p g
After orthodontic treatment, it is impossible to re-
store the surface of teeth to their original condition. Prophylaxis with pumice, acid etching, debonding
and aggressive resin removal procedures cause enam-
el loss.15 Rotating instruments create some degree
of enamel irregularities, and when rebonding is fre-
quently necessary, the surface is modified and the peri-
kymata pattern of young teeth is probably damaged.3
Therefore, fine scratches, such as those made when us-
ing the protocols tested in this study, appear to cause
minimum damage and must be placed in an expected
clinical perspective. It is up to the orthodontist to ap-
ply methods to minimize damage to tooth enamel.25
Thorough resin removal and polishing after debonding
is entirely dependent on the operator27 who is respon-
sible for selecting the instruments, using points with
particles with a lower degree of hardness than enamel
to minimize iatrogenic abrasions and scratches;2 for the
pressure applied to the handpiece and for eliminating
resin from the tooth surface. The fastest protocol was the use of the 12-blade
tungsten carbide bur at high speed (23.5 sec-
onds), followed by DU10CA ORTHO polisher
(31.8 seconds), and the Renew system (31.9 seconds),
which also made use of 12-blade tungsten carbide
burs at high speed, however, in a two-step procedure. Ryf et al20 assessed the Renew system, and showed
it required a considerably longer time to remove and
polish the enamel (83.6 seconds); however, the burs
were used were at low speed. The potential reasons
for this difference were the use of lower-speed hand-
pieces (under 20,000 rpm) and the use of the same
bur every 10 specimens; thus, the bur became worn
and had its cutting power diminished. Author contributions EFS: Supervised the project, guiding the experiments
and text reviewing. LCFS: Performed the experiments
and wrote the master’s thesis of which this article was
originated. MM: Performed the statistical analysis and
wrote the article. CNE: Guided shearing’s testing and
supplied the equipment and location for the experiments. ACOR: Guided microscopy analysis. © 2015 Dental Press Journal of Orthodontics 84 Dental Press J Orthod. 2015 Sept-Oct;20(5):78-85 al article original article Sigilião LCF, Marquezan M, Elias CN, Ruellas AC, Sant’Anna EF Dental Press J Orthod. 2015 Sept-Oct;20(5):78-85 REFERENCES 1. Ulusoy C. Comparison of finishing and polishing systems for residual resin
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on the enamel surface: an in vitro qualitative study. Am J Orthod Dentofacial
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techniques. Am J Orthod. 1979;75(2):121-7. 4. Hong YH, Lew KK. Quantitative and qualitative assessment of enamel surface
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with different methods. Braz J Oral Sci. 2010;9(2):81-4. 9. Bennett CG, Shen C, Waldron JM. The effects of debonding on the enamel
surface. Am J Orthod Dentofacial Orthop. 1995;108(3):284-93. 10. Montasser MA, Drummond JL. Reliability of the adhesive remnant index score
system with different magnifications. Angle Orthod. 2009;79(4):773-6. 11. Artun J, Bergland S. Clinical trials with crystal growth conditioning as an
alternative to acid-etch enamel pretreatment. Am J Orthod. 1984;85(4):333-40. 12. 4287 ANI. Geometrical Product Specifications (GPS) - Surface texture: Profile
method -Terms, definitions and surface texture parameters; 2002. 13. Rouleau BD Jr, Marshall GW Jr, Cooley RO. Enamel surface evaluations
after clinical treatment and removal of orthodontic brackets. Am J Orthod. 1982;81(5):423-6. 14. Pignatta LMB, Duarte Júnior S, Santos ECA. Evaluation of enamel surface after
bracket debonding and polishing. Dental Press J Orthod. 2012;17(4):77-84. 15. Hosein I, Sherriff M, Ireland AJ. Enamel loss during bonding, debonding, and
cleanup with use of a self-etching primer. Am J Orthod Dentofacial Orthop. 2004;126(6):717-24. 1. Ulusoy C. Comparison of finishing and polishing systems for residual resin 16. Eliades T, Kakaboura A, Eliades G, Bradley TG. Comparison of enamel colour
changes associated with orthodontic bonding using two different adhesives. Eur
J Orthod. 2001;23(1):85-90. 1. Ulusoy C. Comparison of finishing and polishing systems for residual resin
removal after debonding. J Appl Oral Sci. 2009;17(3):209-15. REFERENCES y
pi
g
p
g y
removal after debonding. J Appl Oral Sci. 2009;17(3):209-15. removal after debonding. J Appl Oral Sci. 2009;17(3):209-15. 2. Zarrinnia K, Eid NM, Kehoe MJ. The effect of different debonding techniques
on the enamel surface: an in vitro qualitative study. Am J Orthod Dentofacial
Orthop. 1995;108(3):284-93. 17. Radlanski RJ. A new carbide finishing bur for bracket debonding. J Orofac
Orthop. 2001;62(4):296-304. 18. Giampaolo ET, Machado AL, Pavarina AC, Vergani CE. Different methods of
finishing and polishing enamel. J Prosthet Dent. 2003;89(2):135-40. 3. Zachrisson BU, Arthun J. Enamel surface appearance after various debonding
techniques. Am J Orthod. 1979;75(2):121-7. 4. Hong YH, Lew KK. Quantitative and qualitative assessment of enamel surface
following five composite removal methods after bracket debonding. Eur J
Orthod. 1995;17(2):121-8. 19. Macieski K, Rocha R, Locks A, Ribeiro GU. Effects evaluation of remaining resin
removal (three modes) on enamel surface after bracket debonding. Dental Press
J Orthod. 2011;16(5):146-54. 5. Eliades T, Gioka C, Eliades G, Makou M. Enamel surface roughness following
debonding using two resin grinding methods. Eur J Orthod. 2004;26(3):333-8. 20. Ryf S, Flury S, Palaniappan S, Lussi A, van Meerbeek B, Zimmerli B. Enamel
loss and adhesive remnants following bracket removal and various clean-up
procedures in vitro. Eur J Orthod. 2012;34(1):25-32. 6. Brauchli LM, Baumgartner EM, Ball J, Wichelhaus A. Roughness of enamel
surfaces after different bonding and debonding procedures: an in vitro study. J Orofac Orthop. 2011;72(1):61-7. 21. Smith TM, Olejniczak AJ, Zermeno JP, Tafforeau P, Skinner MM, Hoffmann A,
et al. Variation in enamel thickness within the genus Homo. J Hum Evol. 2012;62(3):395-411. 7. Karan S, Kircelli BH, Tasdelen B. Enamel surface roughness after debonding. Angle Orthod. 2010;80(6):1081-8. 22. Bicakci AA, Kocoglu-Altan B, Celik-Ozenci C, Tekcan M, Babacan H, Gungor
E. Histopathologic evaluation of pulpal tissue response to various adhesive
cleanup techniques. Am J Orthod Dentofacial Orthop. 2010;138(1):12.e1-e7;
discussion 12-3. 8. Albuquerque GS, Filho MV, Lucato AS, Boeck EM, Degan V, Kuramae M. Evaluation of enamel roughness after ceramic bracket debonding and clean-up
with different methods. Braz J Oral Sci. 2010;9(2):81-4. 8. Albuquerque GS, Filho MV, Lucato AS, Boeck EM, Degan V, Kuramae M. Evaluation of enamel roughness after ceramic bracket debonding and clean-up
with different methods. Braz J Oral Sci. 2010;9(2):81-4. 23. Campbell PM. Enamel surfaces after orthodontic bracket debonding. Angle
Orthod. 1995;65(2):103-10. 9. Bennett CG, Shen C, Waldron JM. The effects of debonding on the enamel
surface. REFERENCES Am J Orthod Dentofacial Orthop. 1995;108(3):284-93. 9. Bennett CG, Shen C, Waldron JM. The effects of debonding on the enamel
surface. Am J Orthod Dentofacial Orthop. 1995;108(3):284-93. 23. Campbell PM. Enamel surfaces after orthodontic bracket debonding. Angle
Orthod. 1995;65(2):103-10. 10. Montasser MA, Drummond JL. Reliability of the adhesive remnant index score
system with different magnifications. Angle Orthod. 2009;79(4):773-6. 24. Joo HJ, Lee YK, Lee DY, Kim YJ, Lim YK. Influence of orthodontic adhesives and
clean-up procedures on the stain susceptibility of enamel after debonding. Angle
Orthod. 2011;81(2):334-40. 24. Joo HJ, Lee YK, Lee DY, Kim YJ, Lim YK. Influence of orthodontic adhesives and
clean-up procedures on the stain susceptibility of enamel after debonding. Angle
Orthod. 2011;81(2):334-40. 11. Artun J, Bergland S. Clinical trials with crystal growth conditioning as an
alternative to acid-etch enamel pretreatment. Am J Orthod. 1984;85(4):333-40. 25. De Marchi R, De Marchi LM, Terada RSS, Terada HH. Comparison between two
methods for resin removing after bracket debonding. Dental Press J Orthod. 2012;17(6):130-6. 25. De Marchi R, De Marchi LM, Terada RSS, Terada HH. Comparison between two
methods for resin removing after bracket debonding. Dental Press J Orthod. 2012;17(6):130-6. 12. 4287 ANI. Geometrical Product Specifications (GPS) - Surface texture: Profile
method -Terms, definitions and surface texture parameters; 2002. 13. Rouleau BD Jr, Marshall GW Jr, Cooley RO. Enamel surface evaluations
after clinical treatment and removal of orthodontic brackets. Am J Orthod. 1982;81(5):423-6. 26. Retief DH, Denys FR. Finishing of enamel surfaces after debonding of
orthodontic attachments. Angle Orthod. 1979;49(1):1-10. 26. Retief DH, Denys FR. Finishing of enamel surfaces after debonding of
orthodontic attachments. Angle Orthod. 1979;49(1):1-10. 27. Pont HB, Ozcan M, Bagis B, Ren Y. Loss of surface enamel after bracket
debonding: an in-vivo and ex-vivo evaluation. Am J Orthod Dentofacial Orthop. 2010;138(4):387.e1-e9; discussion 387-9. 14. Pignatta LMB, Duarte Júnior S, Santos ECA. Evaluation of enamel surface after
bracket debonding and polishing. Dental Press J Orthod. 2012;17(4):77-84. 15. Hosein I, Sherriff M, Ireland AJ. Enamel loss during bonding, debonding, and
cleanup with use of a self-etching primer. Am J Orthod Dentofacial Orthop. 2004;126(6):717-24. © 2015 Dental Press Journal of Orthodontics 85 Dental Press J Orthod. 2015 Sept-Oct;20(5):78-85
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PHYSICAL DEVELOPMENT DISORDERS OF CHILDREN WITH DIFFERENT OUTCOMES OF PERINATAL BRAIN LESIONS
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Kubanskij naučnyj medicinskij vestnik
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ЛИТЕРАТУРА 8. Федоров В. Д., Воробьев Г. И., Ривкин Л. М. Клиниче-
ская оперативная колопроктология // М.: Медицина. – 1994. –
С. 401–408. 1. Алекперов Э. Э., Коплатадзе A. M., Проценко В. М. Тактика и методы хирургического лечения острого нагноения,
эпителиального» копчикового хода // Актуальные проблемы ко-
лопроктологии : материалы Всерос. конф. (Иркутск, сентябрь
1999 г.). – Иркутск, 1999. – С. 81–82. эпителиального» копчикового хода // Актуальные проблемы ко-
лопроктологии : материалы Всерос. конф. (Иркутск, сентябрь
1999 г.). – Иркутск, 1999. – С. 81–82. 9. Azizi, R. Trends in surgical treatment of pilonidal sinus
diseases: primary closure or flap? // R.Azizi, M. Alemrajabi. World
J.Surg.2012 Jul; 36(7):1713–4. 2. Ан В.К., Ривкин B.Л. Неотложная проктология // М.; Меди-
цина. – 2003. – С.51–56. 10. Al–Jaberi, T.M. Excision and simple primary closure
of chronic pilonidal'sinus / T.M. Al–Jaberi // Eur. J. Surg., 2001; 167:
p. 133–135. 2. Ан В.К., Ривкин B.Л. Неотложная проктология // М.; Меди-
цина. – 2003. – С.51–56. Кубанский научный медицинский 3. Богданов В. Л., Татьянченко В. К. Наш опыт хирургиче-
ского лечения острого воспаления эпителиального копчиково- 3. Богданов В. Л., Татьянченко В. К. Наш опыт хирургиче-
ского лечения острого воспаления эпителиального копчиково- 3. Богданов В. Л., Татьянченко В. К. Наш опыт хирургиче-
ского лечения острого воспаления эпителиального копчиково- Поступила 23.11.2016 азота на ткани послеоперационной раны позволя-
ет рассматривать данный метод как альтернативу
длительной антибактериальной терапии. азота на ткани послеоперационной раны позволя-
ет рассматривать данный метод как альтернативу
длительной антибактериальной терапии. го хода // Материалы V научной сессии Ростовского государ-
ственного медицинского университета, посвященной 95–летию
РостГМУ. – Ростов–на–Дону, 2010. – Т. 2. – С. 624–626. 4. Галашкоян К. М., Черкасов М. Ф., Старцев Ю. М., Чер-
касов Д. М. Комплексное лечение эпителиального копчикового
хода с использованием вакуум–терапии // Альманах института
им. А.В. Вишневчкого. – 2015. – № 2. – С. 1078–1079. Таким образом, можно сделать следующие выво-
ды: 1) применение оксигенизированных лекарствен-
ных препаратов при первом этапе отсроченного хи-
рургического лечения эпителиального копчикового
хода на стадии абсцедирования является более эф-
фективным, 2) при комплексном лечении эпители-
ального копчикового хода на стадии абсцедирования
применение оксигенизированных лекарственных
препаратов, экзогенного оксида азота в сочетании с
региональной лимфатической терапией позволяет
оптимизировать результаты отсроченного радикаль-
ного оперативного лечения данной группы больных. 5. Дульцев А. А., Ривкин В. Л. Эпителиальный копчиковый
ход // М.:Медицина. – 1988. – 126 с. 6. Жданов А. И., Коротких Н. Н., Брежнев С. Г. К методи-
ке хирургического лечения эпителиального копчикового хода //
Альманах института хирургии им. А.В. Вишневского. – 2015. –
Т.2. – С. 485. 6. Жданов А. И., Коротких Н. Н., Брежнев С. Г. К методи-
ке хирургического лечения эпителиального копчикового хода // Альманах института хирургии им. А.В. Вишневского. – 2015. –
Т.2. – С. 485. 7. Карташев A. A., Чарышкин А. Л., Евтушенко Е. Г. Спо-
соб хирургического лечения больных эпителиальным копчико-
вым ходом // Хирург. – М. – 2011. – № 1. – С. 3–5. Кубанский научный медицинский вестник, № 1 (162), 2017 НАРУШЕНИЯ ФИЗИЧЕСКОГО РАЗВИТИЯ У ДЕТЕЙ С РАЗЛИЧНЫМИ
ИСХОДАМИ ПЕРИНАТАЛЬНОГО ПОРАЖЕНИЯ ГОЛОВНОГО МОЗГА
Кафедра педиатрии с курсом неонатологии ФПК и ППС ФГБОУ ВО KUBGMU Минздрава Российской
Федерации. Россия, 350063, Краснодар, ул. Седина 4; тел. 8 (918) 329-03-48; е-mail: ev2273@mail.ru В статье представлены данные о нарушениях физического развития у детей с различными исходами перина-
тального поражения головного мозга. Дети, перенесшие неонатальную реанимацию, имели различные исходы в
постнатальном развитии: неврологический дефицит – 43,7 %, минимальные мозговые дисфункции (функциональ-
ные расстройства) – 56,3 %. Нарушения физического развития выявлены у 58,2 % детей с неврологическим дефи-
цитом и у 39,4 % детей с минимальными мозговыми дисфункциями (р<0, 05). У детей с неврологическим дефицитом
определено преобладание дефицита массы тела (р<0,001), а у детей с минимальными мозговыми дисфункциями
отмечено преобладание избытка массы тела (р<0,05). Анализ физического развития проводился с использованием
метода сигмальных отклонений. Анализ психомоторного развития осуществлялся с использованием шкалы Clinical
Adaptive Test/Clinical Linguistic and Auditory Milestone Scale (CAT/CLAMS). 142
УДК 616.8-053.3-06 : 613.953 Ключевые слова: дети, физическое развитие, неврологический дефицит, минимальная мозговая дисфункция. ючевые слова: дети, физическое развитие, неврологический дефицит, минимальная мозговая дисф УДК 616.8-053.3-06 : 613. PHYSICAL DEVELOPMENT DISORDERS OF CHILDREN WITH DIFFERENT OUTCOMES OF PERINATAL
BRAIN LESIONS Pediatrics chair with a course of a neonatology of FPK and PPS FGBOU VO to KUBGMU Minzd
ussian Federation. Russia, 350063, Krasnodar, Sedina str., 4; tel. 8 (918) 329-03-48; е-mail: ev22 Pediatrics chair with a course of a neonatology of FPK and PPS FGBOU VO to KUBGMU Minzdrava of
Russian Federation. Russia, 350063, Krasnodar, Sedina str., 4; tel. 8 (918) 329-03-48; е-mail: ev2273@mail.ru
The article presents data on the physical development disorders of children with different outcomes of perinatal brain
lesions. The children, gone through neonatal reanimation, have different outcomes during postnatal development, they
are: neurological deficit – 43,7 %, minimum cerebral dysfunctions (functional disorders) – 56,3 %. Physical development
disorders were found in 58,2 % of children with neurological deficit and in 39,4 % of children with minimum cerebral The article presents data on the physical development disorders of children with different outcomes of perinatal brain
lesions. The children, gone through neonatal reanimation, have different outcomes during postnatal development, they
are: neurological deficit – 43,7 %, minimum cerebral dysfunctions (functional disorders) – 56,3 %. Physical development
disorders were found in 58,2 % of children with neurological deficit and in 39,4 % of children with minimum cerebral 142 dysfunctions (р<0, 05). The prevalence of underweight children was determined in children with neurological deficit
(р<0, 001), and the prevalence of excess weight gain was noted in children with minimum cerebral dysfunctions (р<0,
05). Analysis of physical development was carried with using of the sigma deviations method. Analysis of psychomotor
development was carried with using the Clinical Adaptive Test/Clinical Linguistic and Auditory Milestone Scale. Keywords: children, physical development, neurological deficit, minimum cerebral dysfunction. Keywords: children, physical development, neurological deficit, minimum cerebral dysfunction. Результаты исследования и их обсуждение Результаты исследования и их обсуждение
Оценка физического развития в основной и
контрольной группе проводилась у детей в воз-
расте 3, 6 месяцев и 1 года с использованием
оценочных сигмальных таблиц физического раз-
вития детей первого года жизни Краснодарско-
го края (1991 г.) [6]. В возрасте 3 и 6 месяцев у
детей, перенесших неонатальную реанимацию,
показатели длины тела, массы тела и окружности
груди находились преимущественно в диапазоне
величин ниже средних и низких величин, а пока-
затели окружности головы – в диапазоне средних
величин. В возрасте 1 года показатели массы тела
и окружности груди у детей, перенесших критиче-
ское состояние в раннем неонатальном периоде,
находились преимущественно в диапазоне вели-
чин ниже средних и низких величин, а показатели
длины тела и окружности головы – в диапазоне
средних величин. В контрольной группе все пока-
затели физического развития у большинства де-
тей находились в диапазоне средних величин. Цель исследования – изучить нарушения физи-
ческого развития у детей с различными исходами
перинатального поражения головного мозга. Введение построении шкала включает среднюю арифмети-
ческую величину (М) и отклонения от нее, измеря-
емые величиной среднего квадратического откло-
нения (б) [4, 7]. Физическое развитие является одним из основ-
ных показателей растущего организма ребенка. Каждый период развития ребенка определяется
своими темпами физического роста, возрастными
физиологическими и поведенческими реакциями
[1]. Прирост показателей физического развития
и поступательное приобретение детьми психомо-
торных навыков в соотношении с возрастом явля-
ется безусловным критерием здоровья [9]. Анализ психомоторного развития осущест-
влялся с использованием шкалы КАТ/КЛАМС
(CAT/CLAMS – The Clinical Adaptive Test/Clinical
Linguistic and Auditory Milestone Scale), позволяю-
щей раздельно оценить развитие макромоторики,
формирование навыков решения наглядных за-
дач КAT и речевых задач КЛAMС [3, 9]. Кубанский научный медицинский вестник, № 1 (162), 2017 В связи с интенсивным развитием неонаталь-
ной реанимационной службы все большее ме-
сто в структуре заболеваемости детей первых
лет жизни занимают перинатальные поражения
центральной нервной системы. Ведущее место в
структуре заболеваний, приводящих к инвалидно-
сти, занимают психические расстройства, болезни
нервной системы и органов чувств, до 60–70 %
причин детской инвалидности связаны с перина-
тальной патологией [2]. При обследовании недоношенных детей учи-
тывалась поправка на скоррегированный возраст. Статистическая обработка полученных результа-
тов проводилась на персональном компьютере с
использованием программ Microsoft Office Excel
2010, BIOSTAT. Уровень достоверности различий
между средними и относительными величинами
(р) определялся по критерию t Стьюдента [8]. Перинатальные поражения центральной нерв-
ной системы являются одной из основных причин
нарушений соматического здоровья, отклонений
физического и нервно-психического развития де-
тей, как на первом году жизни, так и в последу-
ющие периоды детства [5]. Особую актуальность
приобрели анализ физического развития и изуче-
ние определяющих его факторов у детей с пери-
натальным поражением головного мозга. Кубанский научный медицинский вестник, № 1 (162), 2017 Примечание: * р<0, 05 в сравнении с показателями детей с функциональными расстройствами;
** р<0, 01 в сравнении с показателями детей контрольной группы;
*** р<0, 001 в сравнении с показателями детей с функциональными расстройствами и контрольной группы. ** р<0, 01 в сравнении с показателями детей контрольной группы;
*** р<0, 001 в сравнении с показателями детей с функциональными расстройствами и контрольной группы. р 0, 01 в сравнении с показателями детей контрольной группы;
*** р<0, 001 в сравнении с показателями детей с функциональными расстройствами и контрольной группы. р
,
р
д
р
ру
;
*** р<0, 001 в сравнении с показателями детей с функциональными расстройствами и контро равнении с показателями детей с функциональными расстройствами и контрольной группы. при различных неврологических исходах. Резуль-
таты исследования представлены в таблице. ственно дефицитом массы тела, а у детей с ми-
нимальной мозговой дисфункцией – избытком
массы тела. В связи с этим улучшить физическое
развитие детей с перинатальным поражением го-
ловного мозга возможно при своевременной диа-
гностике и лечении неврологических нарушений,
учете особенностей вскармливания детей с проя-
вившимся неврологическим дефицитом. У детей со стойким неврологическим дефици-
том дисгармоничное развитие выявлено в 60,0 %
случаев, что достоверно чаще по сравнению с
детьми, имеющими функциональные расстрой-
ства, – 39,4 % (р<0,05) и детьми контрольной
группы – 25,0 % (р<0,01). Дефицит массы тела
определен у 49,1 % детей со стойким невроло-
гическим дефицитом, что превысило показатели
детей с функциональными расстройствами на
36,4 % и показатели детей контрольной группы
на 40,8 % (р<0,001). В контрольной группе избы-
ток массы тела выявлялся в 2 раза чаще, чем
недостаток массы тела (р>0,05). Анализируя по-
лученные данные, следует отметить, что среди
нарушений физического развития дефицит массы
тела встречался достоверно чаще по сравнению
с избытком массы у детей со стойким неврологи-
ческим дефицитом (р<0,001). Избыток массы тела
выявлялся достоверно чаще по сравнению с де-
фицитом массы тела у детей с функциональными
расстройствами (р<0,05). Материалы и методы исследования Исследование проводилось на базе ГБУЗ «Дет-
ская краевая клиническая больница» г. Краснода-
ра. В основную группу были включены 126 детей
(срок гестации от 29 до 42 недель) с перинаталь-
ным поражением головного мозга, перенесших не-
онатальную реанимацию. Контрольную группу со-
ставили 36 детей (срок гестации 37–42 недели), не
нуждавшихся в интенсивной терапии, выписанных
домой из отделения для новорожденных краевого
перинатального центра. Дисгармоничное развитие имели более поло-
вины детей основной группы. В возрасте 3 меся-
цев нарушение физического развития выявлено у
60,0 % детей, перенесших неонатальную реани-
мацию. К году у половины детей были определены
отклонения в физическом развитии. Анализ физического развития детей проводил-
ся с использованием параметрического метода
сигмальных отклонений. Для оценки физического
развития использовались сигмальные таблицы,
разработанные на основе средних статистических
показателей основных параметров физического
развития в зависимости от возраста и половой
принадлежности ребенка. При параметрическом При дальнейшем исследовании в основной
группе у 71 ребенка выявлены функциональные
расстройства,
соответствующие
проявлениям
минимальной мозговой дисфункции, у 55 детей –
стойкий неврологический дефицит, проявившийся 143 сы тела находились преимущественно в диапазоне
величин ниже средних и низких величин, показатели
длины тела – в диапазоне средних величин, что спо-
собствовало выявлению дефицита массы тела в 1,5
раза чаще по сравнению с избытком массы тела. детским церебральным параличом, симптомати-
ческой эпилепсией, нарушением психоречевого
развития на фоне структурной патологии голов-
ного мозга (кистозная энцефаломаляция, глиоз,
атрофические изменения лобных, теменных, ви-
сочных долей с расширением субарахноидально-
го пространства, межполушарной щели). Неврологический исход во многом определял
дальнейшее физическое развитие ребенка: у де-
тей с неврологическим дефицитом нарушения
физического развития проявлялись преимуще- Ретроспективно проведен анализ нарушений
физического развития у детей в возрасте 1 года Нарушения физического развития у детей исследуемых Нарушения физического развития у детей исследуемых групп в возрасте года
Признак
Основная группа (n=126)
Контрольная
группа (n=36),
абс. число ( %)
Функциональные
расстройства (n=71), абс. число ( %)
Неврологический
дефицит (n=55),
абс. число ( %)
Дефицит массы тела
9 (12,7 %)
27 (49,1 %)***
3 (8,3 %)
Избыток массы тела
19 (26,8 %)
6 (10,9 %)
6 (16,7 %)
ВСЕГО
28 (39,4 %)
33 (60,0 %)*,**
9 (25,0 %)
Примечание: * р<0, 05 в сравнении с показателями детей с функциональными расстройствами;
** р<0, 01 в сравнении с показателями детей контрольной группы;
*** р<0, 001 в сравнении с показателями детей с функциональными расстройствами и контрольной группы. я физического развития у детей исследуемых групп в возрасте года ЛИТЕРАТУРА 1. Баранов А.А., Кучма В.Р., Скоблина Н.А. Физическое раз-
витие детей и подростков на рубеже тысячелетий. – М.: НЦЗД
РАМН. – 2008. – 216 с. 2. Барашнев Ю.И. Перинатальная неврология. – М.: изда-
тельство Триада-Х. – 2001. – 638 с. 3. Виленская Г.А. Психолог у детской кроватки // Психологи-
ческое обозрение. – 2001. – № 2. – С. 5–12. 3. Виленская Г.А. Психолог у детской кроватки // Психологи-
ческое обозрение. – 2001. – № 2. – С. 5–12. 4. Воронцов И. М., Мазурин А. В. Пропедевтика детских бо-
лезней. – СПб.: Фолиант. – 2009. – 1008 с. 5. Особенности заболеваемости и физического развития
детей раннего возраста с перинатальными поражениями ЦНС
в зависимости от уровня нервно–психического развития / О.М. Филькина, Л.А. Пыхтина, Т.Г. Шанина и др. // Паллиативная ме-
дицина и реабилитация. – 2010. – № 3. – С.19–22. Оценка психомоторного развития по шка-
ле КАТ/КЛАМС проводилась у детей в возрасте
1 года. В результате исследования у детей с не-
врологическим дефицитом выявлено отставание в
моторном, познавательном и речевом развитии, то
есть снижение показателей по всем линиям разви-
тия. У детей с функциональными расстройствами
преимущественно отмечалось отставание в рече-
вом развитии. В контрольной группе по всем ли-
ниям развития значения показателей превышали
80,0 %, что соответствовало нормальному уровню
развития детей. 6. Оценочные таблицы физического развития детей первого
года жизни Краснодарского края: методические рекомендации для
педиатров / Под ред. Л. А. Никулина. – Краснодар. – 1991. – 140 с. 7. Юрьев В.В., Симаходский А.С., Воронович Н.Н., Хомич
М.М. Рост и развитие ребенка: краткий справочник. – СПб:
ГИМА, 2000. – 197с. 7. Юрьев В.В., Симаходский А.С., Воронович Н.Н., Хомич
М.М. Рост и развитие ребенка: краткий справочник. – СПб:
ГИМА, 2000. – 197с. 8. Реброва О.Ю. Статистический анализ медицинских дан-
ных. Применение пакета программ Statistica. – М.: Медиасфе-
ра. – 2006. – 312с. 8. Реброва О.Ю. Статистический анализ медицинских дан-
ных. Применение пакета программ Statistica. – М.: Медиасфе-
ра. – 2006. – 312с. 9. Сахарова Е.С., Кешишян Е.С., Алямовская Г.А. Особенности
психомоторного развития глубоконедоношенных детей // Вестник
современной клинической медицины. – 2013. – № 6. – С.84–90. 9. Сахарова Е.С., Кешишян Е.С., Алямовская Г.А. Особенности
психомоторного развития глубоконедоношенных детей // Вестник
современной клинической медицины. – 2013. – № 6. – С.84–90. Таким образом, у детей, перенесших неонаталь-
ную реанимацию, в возрасте 1 года показатели мас- 144 Поступила 22.12.2016
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Erratum for Mirhendi et al., The first case of onychomycosis in a koala (Phascolarctos cinereus) due to atypical isolates of Microsporum gypseum, a diagnostic challenge
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Current medical mycology
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ERRATUM Volume 2, no. 2, 45-50, 2016, DOI: 10.18869/acadpub.cmm.2.2.2 [1]. The original version of this article contained
incorrect dose of terbinafine. This erratum adds the correct dose as shown below. Terbinafine dosage should change from 250 mg/kg to 250 mg/day. How to cite this paper
Mirhendi H, Nishiyama Y, Rezaei-Matehkolaei A, Satoh K, Makimura K. The first case of onychomycosis in a koala (Phascolarctos
cinereus) due to atypical isolates of Microsporum gypseum: a diagnostic challenge. Curr Med Mycol. 2018; 4(1): 35. DOI:
10.18502/cmm.4.1.34 1. Mirhendi H, Nishiyama Y, Rezaei-Matehkolaei A, Satoh K, Makimura K. The first case of onychomycosis in a koala
(Phascolarctos cinereus) due to atypical isolates of Microsporum gypseum: a diagnostic challenge. Curr Med Mycol. 2016; 2(2):
45-50. DOI: 10.18869/acadpub.cmm.2.2.2 Mirhendi H1, 2*, Nishiyama Y3, Rezaei-Matehkolaei A4, Satoh K5, Makimura K5 4 Department of Medical Mycology, School of Medicine, Health Research Institute, Infectious and Tropical Diseases Research Cent er, Ahvaz
Jundishapur University of Medical Sciences, Ahvaz, Iran
5 Current Medical Mycology Current Medical Mycology 2018, 4(1): 35 How to cite this paper
Mirhendi H, Nishiyama Y, Rezaei-Matehkolaei A, Satoh K, Makimura K. The first case of onychomycosis in a koala (Phascolarctos
cinereus) due to atypical isolates of Microsporum gypseum: a diagnostic challenge. Curr Med Mycol. 2018; 4(1): 35. DOI:
10.18502/cmm.4.1.34 5 General Medical Education and Research Center, Teikyo University, Tokyo, Japan 4 Department of Medical Mycology, School of Medicine, Health Research Institute, Infectious and Tropical Diseases Research Cent er, Ahvaz
Jundishapur University of Medical Sciences, Ahvaz, Iran
5 Erratum for Mirhendi et al., the first case of onychomycosis in
a koala (Phascolarctos cinereus) due to atypical isolates of
Microsporum gypseum, a diagnostic challenge Mirhendi H1, 2*, Nishiyama Y3, Rezaei-Matehkolaei A4, Satoh K5, Makimura K5 References References
1. Mirhendi H, Nishiyama Y, Rezaei-Matehkolaei A, Satoh K, Makimura K. The first case of onychomycosis in a koala
(Phascolarctos cinereus) due to atypical isolates of Microsporum gypseum: a diagnostic challenge. Curr Med Mycol. 2016; 2(2):
45-50. DOI: 10.18869/acadpub.cmm.2.2.2 Copyright© 2018, Published by Mazandaran University of Medical Sciences on behalf of Iranian Society of Medical Mycology and Invasive Fungi Research Center. This is
an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International (CC BY) License (http://creativecommons.org/) which permits
unrestricted use, distribution and reproduction in any medium, provided appropriate credit to the original author(s) and the source, provide a link to the Creative Commons
license, and indicate if changes were made.
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Autologous c-Met/PD-L1-specific CAR T-cells
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Definitions
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Open Peer Review on Qeios Open Peer Review on Qeios Qeios ID: P0CTGL · https://doi.org/10.32388/P0CTGL Qeios · Definition, February 2, 2020 Autologous c-Met/PD-L1-specific CAR T-
cells National Cancer Institute National Cancer Institute Source Source
National Cancer Institute. Autologous c-Met/PD-L1-specific CAR T-cells. NCI Thesaurus. Code C162481. A preparation of autologous T-lymphocytes that have been transduced with a lentiviral
vector encoding a chimeric antigen receptor (CAR) specific for human hepatocyte growth
factor receptor (HGFR or c-Met) and the immunosuppressive ligand, programmed cell
death-1 ligand 1 (PD-L1; cluster of differentiation 274; CD274), with potential
antineoplastic activities. Upon infusion, the autologous c-Met/PD-L1-specific CAR T-cells
bind to and induce selective toxicity in c-Met- and PD-L1-expressing tumor cells. cMET, a
receptor tyrosine kinase that is overexpressed or mutated in many tumor cell types,
plays a key role in cancer cell growth, survival, angiogenesis, invasion, and metastasis. PD-L1 is also overexpressed by many human cancer cell types and plays a key role in the
downregulation of the immune system and tumor evasion from host immunity. 1/1
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Are some species ‘robust’ to exploitation? Explaining persistence in deceptive relationships
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Evolutionary ecology
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Amy L Brunton-Martin
albruntonmartin@gmail.com Evolutionary Ecology (2022) 36:321–339
https://doi.org/10.1007/s10682-022-10174-9 Evolutionary Ecology (2022) 36:321–339
https://doi.org/10.1007/s10682-022-10174-9 IDEAS & PERSPECTIVES Are some species ‘robust’ to exploitation? Explaining
persistence in deceptive relationships Amy L Brunton-Martin1
· James C O’Hanlon2
· Anne C Gaskett1 Received: 11 August 2021 / Accepted: 12 March 2022 / Published online: 10 April 2022
© The Author(s) 2022 1
Te Kura Mātauranga Koiora, Waipapa Taumata Rau, Aotearoa; School of Biological Sciences,
The University of Auckland, Auckland Central, Auckland, New Zealand Keywords Sexual deception · Antagonistic coevolution · Persistence 2
School of Environmental and Rural Science, The University of New England, Armidale,
NSW, Australia 1
Te Kura Mātauranga Koiora, Waipapa Taumata Rau, Aotearoa; School of Biological Sciences,
The University of Auckland, Auckland Central, Auckland, New Zealand
2
School of Environmental and Rural Science, The University of New England, Armidale,
NSW, Australia Introduction A species’ evolutionary trajectory can change due to interactions with other species. Coevo
lution, or reciprocal change between species (Bronstein 2001; Price 2003; Soler 2013; Hui
et al. 2015), can drive selection for traits that maintain or increase an individual’s fitness
(Brockhurst and Koskella 2013; Hembry et al. 2014). Coevolution involves a range of inter
actions, including mutualisms. However, exploitative relationships, where exploiters secure
fitness benefits (e.g. food or improved reproduction) at the cost of the other party, are also a
common outcome of coevolution (Mokkonen and Lindstedt 2016). The evolutionary maintenance of mutualistic relationships is relatively straightforward
– if both parties benefit, there is strong selection for mutualisms or factors that reinforce
mutualisms (Bronstein 2009, 2015). Indeed, the most compelling evidence for coevolu
tion or co-speciation arises from research on mutualistic relationships (Vienne et al. 2013). Contrarily, the relationships that impose fitness costs on only one partner are harder to
explain (Kokko and Brooks 2003). Theory predicts that, if costs are sufficient, deceptive
relationships should break down, due to the extinction, decline, or evolved resistance of
the exploited partner (Gibson et al. 2010; Ricklefs 2010; Hesse and Buckling 2016; Vitale
and Best 2019). Extant deceptive relationships may well be a temporary snapshot in evolu
tionary time. However, exploitative relationships can persist in nature, due to a fascinating
variety of mechanisms. Orchids provide an overlooked opportunity for studying exploitation and mechanisms of
persistence. Although many orchids do offer pollinators a reward such as nectar, rewardless
ness and deception are very common (Shrestha et al. 2020). Deceptive orchids exploit their
pollinators’ foraging and reproductive behaviours: they do not offer any reward, and can
even harm their pollinator (Wong and Schiestl 2002; Brunton Martin et al. 2020). Sexual
deception can be an extreme form of pollination, in which flowers lure a male pollinator
with duplicitous sex signals. Fooled males mate with the flower and unintentionally collect
or deliver pollinia (Schiestl 2005). In some cases, males will ejaculate and waste sperm on
the orchid (Blanco and Barboza 2005; Gaskett et al. 2008; Brunton Martin et al. 2020). As
well as missed mating opportunities, new data shows that sperm wastage is costly (Wong
and Schiestl 2002; de Jager and Ellis 2014), as males can become sperm depleted, at least
temporarily (Brunton Martin et al. 2020). Abstract Animals and plants trick others in an extraordinary diversity of ways to gain fitness ben
efits. Mimicry and deception can, for example, lure prey, reduce the costs of parental
care or aid in pollination–in ways that impose fitness costs on the exploited party. The
evolutionary maintenance of such asymmetric relationships often relies on these costs be
ing mitigated through counter-adaptations, low encounter rates, or indirect fitness benefits. However, these mechanisms do not always explain the evolutionary persistence of some
classic deceptive interactions. Sexually deceptive pollination (in which plants trick male pollinators into mating with
their flowers) has evolved multiple times independently, mainly in the southern hemi
sphere and especially in Australasia and Central and South America. This trickery imposes
considerable costs on the males: they miss out on mating opportunities, and in some cases,
waste their limited sperm on the flower. These relationships appear stable, yet in some
cases there is little evidence suggesting that their persistence relies on counter-adaptations,
low encounter rates, or indirect fitness benefits. So, how might these relationships persist? Here, we introduce and explore an additional hypothesis from systems biology: that
some species are robust to exploitation. Robustness arises from a species’ innate traits and
means they are robust against costs of exploitation. This allows species to persist where a
population without those traits would not, making them ideal candidates for exploitation. We propose that this mechanism may help inform new research approaches and provide
insight into how exploited species might persist. 1 3 1 3 Evolutionary Ecology (2022) 36:321–339 322 Existing hypotheses for the maintenance of exploitation Exploitation occurs across an extensive range of ecological interactions and taxa, and costs
vary. For instance, deception can result in death for the exploited party: to hunt spiders,
assassin bugs imitate the vibrations of trapped insects (Wignall and Taylor 2011); pray
ing mantises mimic flowers to lure pollinators as prey (O’Hanlon et al. 2014). Exploited
individuals may also lose young - brood-parasitic cuckoos have eggs and chicks that mimic
those of their hosts, and parasitic chicks will often kill their nest-mates (Feeney et al. 2014)
– or lose the potential to have offspring: sexually deceptive orchids use mimicry and signal
biases to fool male insects into mating with, and pollinating their flowers (Gaskett 2011;
Gaskett et al. 2017), sometimes eliciting ejaculation. On the other hand, costs of exploita
tion may be negligible, as when quorum sensing bacteria signal others to produce ‘common
goods’ without producing it themselves (Diggle et al. 2007; Czárán and Hoekstra 2009;
Katzianer et al. 2015). When there are costs, exploitative relationships might be maintained via antagonistic
coevolution, or an arms-race, as occurs between cuckoos and their hosts (Feeney 2017). In
other taxa, the maintenance of exploitative interactions is via indirect fitness benefits accru
ing to the exploited partner, for instance in praying mantids where sexual cannibalism also
enhances paternity (Barry et al. 2008; Sardell et al. 2012). Finally, low incidence or encoun
ter rates can mean exploitation imposes only weak selection on the exploited partner (Kokko Table 1 Summary of studies that have investigated evidence for negative frequency dependent selection in
deceptive orchid genera
Deception
Species
Polymorphism
Finding
Conclusion
Study
Food
deceptive
Dactylorhiza
sambucina
Colour
Male and female reproductive
success of plants declined with
frequency of colour morph in
the population. Advantage
for rare
colour
morph. Gigord
et al. 2001
No correlation between male
and female reproductive success
and colour morph frequency. Red morphs received more
second visits, yellow more first
visits. Pollinia removal and fruit
set increased with frequency of
yellow morph. No advan
tage for
rare colour
morph. Pel
legrino
et al. 2005
The more dominant colour
morph (yellow) had the most
pollinia export and deposition. No advan
tage for
rare colour
morph. Groiß
et al. 2017
Sexually
deceptive
Lepanthes
rupestris
Colour
Reproductive success did not
differ between colour morphs. No advan
tage for
rare colour
morph. Introduction Despite these costs, sexual deception persists and
is reasonably common: 20% of deceptive genera present sexual deception (Jersáková et
al. 2006). Although recent work challenges the long-held idea that one-third of orchids are
deceptive, rewardlessness, and therefore sexual deception, are probably relatively common
in the family Orchidaceae (Shrestha et al. 2020), despite the known costs to pollinators. Here, we explore how relationships persist when there are high costs, focusing on decep
tive orchids. We assess the evidence for low encounter rates, indirect benefits, or coevolu
tionary arms-races. In addition, we use an extreme example of sexual deception that triggers
pollinator ejaculation to propose an additional hypothesis that applies an idea from the field
of systems biology, (Kitano 2004; Whitacre 2012)robustness, that could help explain the
persistence of such deceptive relationships. We propose that future work exploring persis
tence might test for these traits in other exploitative interactions. 1 3 1 3 1 Evolutionary Ecology (2022) 36:321–339 323 Low encounter rates and frequency dependent selection One of the most intuitive mechanisms for the persistence of costly relationships is simply
the idea that low encounter rates allow populations to persist because incidences of exploi
tation or conflict are very rare (Kokko and Rankin 2006; Kokko et al. 2008). The less an
exploited group encounters the exploiter, the less of an impact that exploiter has on the pop
ulation – reducing the chance for learning or counteradaptation and allowing the deceptive
relationship to persist. In deceptive orchids, low encounter rates may be a product of decep
tive orchid density and pollinator learning resulting in negative frequency-dependent selec
tion. However, few studies test the presence of negative-frequency dependent selection, and
there is mixed evidence of any benefit of being rare (Table 1). In one food deceptive orchid,
Dactylorhiza sambucina, rare colour morphs had a reproductive advantage (Gigord et al. 2001) demonstrating evidence for negative-frequency dependent selection – but this is the
only known example, and has not yet been successfully replicated in the same species Pel
legrino et al. 2005; Groiß et al. 2017; reviewed in Sapir et al. 2021). In several other studies,
this was also not the case either in terms of colour polymorphisms (Tremblay and Ackerman
2007; reviewed in Juillet and Scopece 2010); or scent polymorphisms (Braunschmid and
Dötterl 2020). In sexually deceptive orchids, low encounter rates could arise because pollinators quickly
learn to avoid deceptive orchids, a mechanism that may even be accelerated when orchids
are highly abundant. For example, a few studies suggest short-term pollinator avoidance
in response to sexual deception (Wong and Schiestl 2002; Schiestl 2005; Paulus 2006; Jer
sáková et al. 2006; Whitehead and Peakall 2013; Weinstein et al. 2016). However, avoid
ance of orchids may not necessarily be a learned behaviour in response to orchids. If we
consider the pre-existing behaviour of these sexually deceptive orchids’ pollinators (which
are typically solitary parasitoid wasps or bees; Gaskett 2011), a male wasp may typically
avoid a site where he has mated previously to reduce the chance of remating with the same
female. The typical mating system of solitary parasitoid wasps involves females releasing
sex pheromones to attract a male and mating just once during her lifetime (monandry). Males of these species are therefore under intense scramble competition to find and mate
with a signalling female, but they will mate with several females in their life (polygyny;
Quicke 2014). Existing hypotheses for the maintenance of exploitation Trem
blay
and
Acker
man
2007
Cypripedium
calceolus
Scent
Rarer scent morphs did not
significantly differ in probability
of fruit set. No advan
tage for
rare scent
morph. Braun
schmid
and
Dötterl
2020 1 3 324 Evolutionary Ecology (2022) 36:321–339 and Rankin 2006; Kokko et al. 2008). This is often suggested as an explanation for why
pollinators do not learn to avoid rewardless or deceptive orchids (Pérez-Hérnandes et al. 2011). Alternatively, low encounter rates might also be maintained by negative frequency-
dependent selection, which may arise from pollinator learning or avoidance (Schiestl 2005). Here, we briefly discuss each of these mechanisms in the context of orchid deception: Indirect fitness benefits Exploitation can persist when indirect fitness benefits compensate for the costs to the
exploited party (Sardell et al. 2012; Dimitriu et al. 2016). For example, indirect benefits are
obtained by cannibalised male praying mantids, because although sexual cannibalism kills
males, it provides them with paternity benefits. Cannibalistic females substantially improve
their body condition by consuming males before, during or immediately after mating (Barry
et al. 2008). Becoming lunch, of course, comes at a cost to the courting male. Neverthe
less, males indirectly benefit through increased fertilisation success and improved offspring
survival (Welke and Schneider 2012), although not at all if they are eaten before mating.fi No studies have found that deceptive orchids offer any indirect benefits to any polli
nators. One (very tenuous) possibility is that pollinators could benefit from brief periods
of shelter in sexually deceptive orchids with trap mechanisms, e.g. Pterostylis. Hence, it
is unlikely that sexual deception persists due to this mechanism – there are certainly no
rewards they offer that would outweigh the costs of reproductive loss. Low encounter rates and frequency dependent selection Thus, there is likely to be strong selection on males to avoid responding
to mated females, depending on their learning rate. When a solitary male parasitoid has
found and mated with a virgin female, he usually will not return to that site – even if a new
virgin female is placed there (Goh and Morse 2010; Quicke 2014). Although, in some sexu
ally deceptive systems experienced males can learn to recognise that deceptive flowers are
mimics, reducing their mating behaviour (de Jager and Ellis 2014). Interestingly, male bees
deceived by Ophrys orchids can learn to avoid the scent of one flower, but variation in scent
means that the male will go back to different flowers on the same or different plants (Ayasse
et al. 2007). In other rewardless systems (e.g. food deception), pollinators will learn to avoid 1 3 3 1 Evolutionary Ecology (2022) 36:321–339 325 all plants in a dense patch of deceivers, but they are less likely to avoid a deceptive orchid
when among similar, rewarding plants (Johnson et al. 2003) – thus, low density allows for
the persistence of the relationship. all plants in a dense patch of deceivers, but they are less likely to avoid a deceptive orchid
when among similar, rewarding plants (Johnson et al. 2003) – thus, low density allows for
the persistence of the relationship. Does sexual deception co-evolve? Although there are few studies of coevolution between sexually deceptive orchids and their
pollinators, it is widely accepted that orchid species adapt to their pollinator preferences
(box 1). Floral scent mimicry has received the most research attention and it is clear that
sexual deception relies primarily on orchids’ precise chemical mimicry of the species-spe
cific sex pheromones of the female of the pollinating species (Bohman et al. 2016; Peakall
et al. 2020; Hayashi et al. 2021), particularly for long-range attraction. Pollinators can also
exert selection on orchid colour and morphology: sexually deceptive orchids that adapt to
match the morphologies of their pollinators can achieve more efficient pollination (Newman
et al. 2015). While there are no explicitly documented cases of coevolution between sexually decep
tive orchids and their pollinators, it is clear that deceptive orchids can evolve in response
to their pollinators and are labile in terms of their morphology, scent, and colour. Do their
pollinators coevolve in response to these changes? Coevolution is documented between
rewarding flowers and their pollinators (Ricklefs 2010; Bili et al. 2016; Vamosi et al. 2006;
Arditti et al. 2012) and between rewarding orchids and their pollinators (Boberg et al. 2014;
Anderson and Johnson 2007). However, there is presently no evidence that pollinators
evolve in response to deceptive pollination. Furthermore, phylogenies suggest that coevolu
tion is unlikely because of the different branch lengths (time since splitting) of pollinator
and orchid groups (Mant et al. 2002). Regardless, there are very few explorations of how
coevolution may function in deceptive plant-pollinator relationships (Wong and Schiestl
2002). In order to best explore this possibility, it is vital that the fitness costs of sexual
deception are established. Coevolutionary arms-race Another potential mechanism for the maintenance of sexual deception is through a coevo
lutionary arms-race. Under this mechanism, deception by orchids could select for pollina
tor traits that improve the exploited species’ fitness, e.g., increased ability to differentiate
between orchids and real females. In turn, these defences select for counter-adaptations in
the deceiver, e.g., more accurate mimicry. Cuckoo brood parasites and their hosts are a clas
sic example of an arms-race. Exploitation selects for host recognition of parasitic eggs and
mobbing of parasites (Kilner and Langmore 2011; Feeney et al. 2014). In turn, this selects
for cuckoo cryptic plumage and behaviour, and eggs and chicks with features that match
those of their hosts (Marchetti 2000; Spottiswoode and Stevens 2011; Feeney 2017). Resistance or tolerance can be key mechanisms reducing costs of arms-races (Svens
son and Råberg 2010). In brood parasitism, resistance minimises the number of exploita
tions, e.g. hosts that mob and deter cuckoos. Resistance can drive deceptive relationships to
extinction (Vienne et al. 2013), whereas tolerance reduces the fitness impact of exploitation. For instance, Eurasian magpie (Pica pica) tolerate brood parasitism by rearing their young
alongside cuckoo chicks, and increasing their own clutch size (Soler et al. 2001). Tolerance
may not result in an arms-race because it does not inhibit exploiter fitness (Svensson and
Råberg 2010; Fornoni 2011). Importantly, a coevolutionary arms-race is unlikely to allow
maintenance of deception as it often results in the exploited species becoming extinct or
escaping exploitation (some theoretical models suggest prolonged coexistence; Huang et al. 2017; Hui et al. 2018; Schenk et al. 2020). 3 1 Evolutionary Ecology (2022) 36:321–339 326 Box 1: Evidence for pollinator selection on orchid traits Deceived orchid pollinators can exert selection on both orchid morphology and scent. For
instance, Peakall et al. (2010) found that changes in the floral scent of Australian sexually
deceptive Chiloglottis orchids attracts new pollinator species, leading to floral isolation and
speciation. Similarly, morphological manipulation of Chiloglottis species shows that pol
linators reduce copulation duration (and therefore pollen transfer) when pollinating flowers
with abnormal callus-tip distances or shortened labella (de Jager and Peakall 2016, 2019).f In european Ophrys orchids, pollinators are less effective at pollinating when they inter
act with flowers that have altered lips (the third petal of the orchid that serves as a landing
platform for pollinators) and therefore inadequate gripping points on the flower (Rakosy et
al. 2017). Sexually deceptive orchids may also be under selection to be highly detectable for pol
linators (Rakosy et al. 2012; Kelly and Gaskett 2014; Gaskett et al. 2017) or have colours
that match those of female pollinators (Gaskett and Herberstein 2009). Are costs of sexual deception sufficient for coevolution? The costs sexually deceptive orchids impose on their pollinators, particularly in terms of
sperm wastage, have only recently been explored or discovered (Blanco and Barboza 2005;
Brunton Martin et al. 2020; Cohen et al. 2021). If orchid interference is a substantial cost 3 1 Evolutionary Ecology (2022) 36:321–339 327 to pollinators, we might expect coevolutionary responses in the pollinator’s reproductive
behaviour or anatomy– such as an arms’ race like that observed between cuckoos and their
hosts.f to pollinators, we might expect coevolutionary responses in the pollinator’s reproductive
behaviour or anatomy– such as an arms’ race like that observed between cuckoos and their
hosts.f Sexual deception can affect male pollinators by causing missed mating opportunities
with real females and wasted ejaculate Wong et al. 2004; de Jager and Ellis 2014; Wong
and Schiestl 2002; Brunton Martin et al. 2020). Females of the pollinator species are also
likely to suffer costs. They must compete with orchids for mates in both space and time
because their mating season coincides with orchid flowering season, and the orchids only
occur where wasp populations are present (Gaskett and Herberstein 2006; Brunton-Martin
et al. 2021). Sperm production can be energetically costly: male cockroaches, Nauphoeta cinerea,
demonstrate a nutrient trade-off between sperm production and pre-copulatory attractive
ness (Bunning et al. 2015); male and female seed beetles, Callosobruchus maculatus, invest
equally reproductive effort, losing body mass – and males who mate more than once show
reduced ejaculate size (Savalli and Fox 1999; Wagner and Bakare 2017). In flour beetles,
Tribolium castaneum, males with higher mating success have longer sperm, which was
shown to be costly to produce as protein-restricted males had shorter, less successful sperm
(Godwin et al. 2017). This was also found in Drosophila melanogaster: long sperm males
had lower mating success, suggesting a trade-off between pre-copulatory sexual selection
and post-copulatory advantages of long sperm (Zajitschek et al. 2019). Parasitoid wasps,
who are the primary pollinators of sexually deceptive orchids (Gaskett 2011) often have
limited sperm production in their lifetime (Boivin et al. 2005; Damiens and Boivin 2006;
Boulton et al. 2015). Parasitoid females generally cannot detect whether males have sperm,
so if they mate with a sperm-depleted male, they may never receive a full complement of
sperm, or remain pseudo-virgin for their lifetime (Abe 2019). Are costs of sexual deception sufficient for coevolution? This change in sperm allocation behaviour
might be a strategic response to the perception of abundant females (and orchids) in the
males’ habitat (Parker and Pizzari 2010), or lower quality females (i.e. orchids; Reinhold
et al. 2002). For both early studies, it is unclear whether these behavioural changes arise
through plasticity or counter-adaptation. It would be interesting to determine whether there
are any morphological changes in deceived populations of pollinators. For instance, since
pheromone mimicry is the main source of attraction in orchids (Ayasse et al. 2011; Bohman
et al. 2019), future research might focus on differences in the chemosensory structures of
pollinators. These might indicate counteradaptations that would allow pollinators to better
distinguish orchids from real females and allow assessment of whether this correlates with
reduced encounter rates. Are costs of sexual deception sufficient for coevolution? If males do replenish sperm,
they typically produce fewer spermatozoa at a lower rate and with a higher proportion of
infertile sperm with every mating event (Wedell et al. 2002; Pizzari et al. 2008; Vega-Trejo
et al. 2019). To date, there is only one study estimating sperm production in an orchid pol
linator: males of Lissopimpla excelsa were found to at least become temporarily sperm
depleted (Brunton Martin et al. 2020). The procedural challenges of detecting spermato
phores on orchids (Bressac et al. 2008) may have led to an underestimation of how common
ejaculation is. However, recent work is discovering the presence of spermatophores in other
deceptive interactions: Cohen et al. (2021) recently reported that beetle sperm was found
on the labellum of the sexually deceptive orchid, Disa forficaria, and Blanco and Barboza
(2005) report a putative spermatophore on flowers of the sexually deceptive orchid, Lep
anthes glicensteinii. Building on this, it would be interesting to determine pollinator sperm
capacity (and whether sperm deposits are present) in other sexually deceptive systems. If
ejaculation on orchids is common, then this cost may be sufficient to alter pollinators’ fit
ness, even if temporary (see Brunton-Martin et al. 2021b); if not, the interaction’s costs may
well be negligible. At present, there are just a few tantalising suggestions that these types of costs could be
sufficient to drive change in a sexually deceived pollinator species. One study has suggested
that the presence of sexually deceptive orchids interrupt the normal mating behaviour of
pollinators, and in response, selection may act on females’ learned avoidance of deceptive
orchids, to improve their chances of reproduction (Wong et al. 2004). We note, however,
that this might occur because female parasitoids perceive a high density of females, and are 1 3 Evolutionary Ecology (2022) 36:321–339 328 avoiding competition for hosts (Mathiron et al. 2019). Another study, comparing male pol
linators of a single species from sites with and without natural populations of orchids, found
that males in sympatry with sexually deceptive orchids had smaller ejaculates but a similar
sperm volume (Brunton Martin et al. 2020). This change might be a counteradaptation in
response to sperm loss, but confirming this will rely on establishing both proximate and
ultimate costs (see De Mazancourt et al. 2005). Fig. 1 In pursuit of pollination,
a Cryptostylis subulata orchid
tricks its ichneumonid wasp
pollinator, Lissopimpla excelsa,
into ejaculating and wasting his
sperm. Sperm loss and missed
mating opportunities could
impose great costs on deceived
pollinators’ populations – how
does this relationship persist?
Image courtesy of C. Young Fig. 1 In pursuit of pollination,
a Cryptostylis subulata orchid
tricks its ichneumonid wasp
pollinator, Lissopimpla excelsa,
into ejaculating and wasting his
sperm. Sperm loss and missed
mating opportunities could
impose great costs on deceived
pollinators’ populations – how
does this relationship persist?
Image courtesy of C. Young Applying existing hypotheses to Cryptostylis-Lissopimpla Low encounter rates may maintain the interaction between Cryptostylis and its sperm-
depleted pollinator - individual-based modelling of a haplodiploid pollinator population
indicates that the less males search and find deceptive orchids, the higher the pollinator
population’s persistence (see: Brunton-Martin et al. 2021b). However, this mechanism does
not fully explain the persistence and success of this relationship: Cryptostylis’ high pollina
tion rates suggest frequent interactions with the pollinator, counter to what we might expect
if encounter rates were low. Regardless of the mechanism behind this phenomenon, it is
crucial to assess the lifetime fitness costs imposed on pollinators, and pollinator population
sizes in areas with orchids, to determine the true impact of deceptive orchids and whether
their success is simply owed to pollinator abundance. Indirect fitness benefits are unlikely,
as deceptive orchids never offer rewards to their pollinators – although this has not been
explicitly tested for Cryptostlyis orchids. The costs of sperm limitation may be sufficient
to promote an arms-race between orchid and pollinator, but this has not yet been explicitly
tested. Case study: Cryptostylis orchids The Australasian sexually deceptive Cryptostylis orchids are exceptional deceivers, exploit
ing males of the solitary parasitoid wasp species Lissopimpla excelsa (Family: Ichneumoni
dae, subfamily: Pimplinae, Fig. 1). These orchids frequently trick males into mating with,
and ejaculating on, the flower during pollination (Gaskett et al. 2008; Brunton Martin et
al. 2020). Parasitoid species can become permanently or temporarily sperm depleted after
several mating events with females (Olsson et al. 1997; Boivin et al. 2005; Damiens and
Boivin 2006; de Jager and Ellis 2014; Boulton et al. 2015), and male L. excelsa may become Fig. 1 In pursuit of pollination,
a Cryptostylis subulata orchid
tricks its ichneumonid wasp
pollinator, Lissopimpla excelsa,
into ejaculating and wasting his
sperm. Sperm loss and missed
mating opportunities could
impose great costs on deceived
pollinators’ populations – how
does this relationship persist? Image courtesy of C. Young 3 1 Evolutionary Ecology (2022) 36:321–339 329 at least temporarily sperm depleted (Brunton Martin et al. 2020) when mating with orchids. Cryptostylis orchids may therefore interfere in pollinator reproduction by imposing sperm
limitation on females. This could be by depleting males of sperm or preventing males from
encountering real females. Despite this, orchids achieve exceptionally high pollination rates
for sexually deceptive orchids (Cryptostylis: 70% (Gaskett 2011; Schiestl et al. 2004) com
pared to an average of 13–39% for sexually deceptive orchids in Australia (Brundrett 2019)
excluding Cryptostlyis orchids). Haplodiploidy and persistence The three mechanisms – low encounter rates, indirect fitness benefits and counteradapta
tions – do not seem to have sufficient evidence to support the idea that they, alone, might
explain the persistence of the interaction between Cryptostylis orchids and their pollina
tor. Indirect fitness benefits are unlikely, the evidence for low encounter rates is meagre
(Table 1); and the presence of an arms-race has not yet been thoroughly explored. Indeed,
many gaps remain in our understanding of this system – and each of these three hypoth
eses warrant explicit testing in the context of sexual deception. However, we propose the
application of another hypothesis (robustness) derived from work in systems biology. This
hypothesis does not preclude or require the other three mechanisms, but is simply another
way in which we might understand exploited species’ persistence. Sexually deceptive orchids are almost exclusively pollinated by solitary hymenopterans
(Gaskett et al. 2008), but sexual deception with sperm wastage in beetles and fungus gnats
has recently been reported (Phillips et al. 2014; Cohen et al. 2021). All Hymenoptera (wasps,
bees and ants) are haplodiploid and have one ancient, arrhenotokous origin (Normark 2006),
that pre-dates the evolution of sexual deception. Sexual deception likely arose between 16.1
and 4.6 MYA (corresponding with divergence of the genus, Ophrys (Breitkopf et al. 2015),
whereas haplodiploid Ichneumonidae arose ~ 228MYA ((Peters et al. 2017). Diploidy has
never evolved in the hymenopterans, and the most prevalent form of haplodiploidy in the 1 3 Evolutionary Ecology (2022) 36:321–339 330 Hymenoptera is arrhenotokous haplodiploidy, where diploid females arise from fertilised
eggs, and haploid males from unfertilised eggs (Heimpel & de Boer, 2008). Hymenoptera is arrhenotokous haplodiploidy, where diploid females arise from fertilised
eggs, and haploid males from unfertilised eggs (Heimpel & de Boer, 2008). The preponderance of haplodiploid pollinators for deceptive orchids is striking when
comparing the relative proportions of haplodiploid and diploid pollinators across different
orchid pollination strategies (Fig. 2). Using the Atlas of Orchid Pollination (Van der Cingel
2001) for an overview of pollinators and pollination strategies, we found an association
between sex-determination systems in pollinators and orchid pollination strategy. Reward
ing orchid species and food deceptive orchid pollinators are approximately equally split
between diploids and haplodiploids, whereas sexually deceptive orchid species have 90%
haplodiploid pollinators (Fig. 2). Orchids with other deceptive pollination systems such as
brood site deception and sensory traps are also predominantly pollinated by non-haplodip
loid pollinators. Fig. 2 Relative proportions of
haplodiploid and not haplodip
loid pollinators across different
known pollination strategies
for 755 species of Orchidaceae
(excluding autogamous orchids;
data from Van der Cingel 2001).
Pollinators included Hymenop
tera (haplodiploid), and Diptera,
Coleoptera, Lepidoptera and
birds (all not haplodiploid). Pol
linator type (haplodiploid or not)
appears to relate to pollination
strategy. Pollination strategies
that were not rewarding, but
otherwise unclear were described
as ‘sensory traps’ Haplodiploidy and persistence We note that the deception of hymenopteran pollinators (an entirely hap
lodiploid order) appears to be the ancestral state in at least the subtribe Orchidinae (which
includes the large European sexually deceptive genus Ophrys; Inda et al. 2012), and we
predict it may be ancestral for the sister tribe Diurideae from the same subfamily (Orchi
doideae), which includes all 9 of the Australian hymenopteran-pollinated sexually decep
tive genera. Yet, the discovery of other sexually deceived orchid pollinators (e.g. beetles
and fungus gnats; (Reiter et al. 2019; Cohen et al. 2021; Hayashi et al. 2021) that are not 1 3
Fig. 2 Relative proportions of
haplodiploid and not haplodip
loid pollinators across different
known pollination strategies
for 755 species of Orchidaceae
(excluding autogamous orchids;
data from Van der Cingel 2001). Pollinators included Hymenop
tera (haplodiploid), and Diptera,
Coleoptera, Lepidoptera and
birds (all not haplodiploid). Pol
linator type (haplodiploid or not)
appears to relate to pollination
strategy. Pollination strategies
that were not rewarding, but
otherwise unclear were described
as ‘sensory traps’ 1 3 1 Evolutionary Ecology (2022) 36:321–339 331 haplodiploid. Hence, more work identifying the pollinators of sexually deceptive orchids
will be useful to confirm whether haplodiploid pollinators are, indeed, as common as they
seem, and thus if haplodiploidy is a critical factor in the evolutionary maintenance of sexual
deception. haplodiploid. Hence, more work identifying the pollinators of sexually deceptive orchids
will be useful to confirm whether haplodiploid pollinators are, indeed, as common as they
seem, and thus if haplodiploidy is a critical factor in the evolutionary maintenance of sexual
deception. However, in the context of Cryptostylis orchids, haplodiploidy may ensure their pollina
tors are robust to potential costs of sperm limitation elicited by sexual deception. Haplo
diploid females can reproduce without sperm (albeit, all their offspring are male; Godfray
1990) and can facultatively adjust the sex ratio of their offspring when they do (Burton-
Chellew et al. 2008; Booksmythe et al. 2017). Therefore, female mating failure due to sperm
loss or missed mating opportunities could drive an increase in sons from generation to gen
eration, causing a male-biased sex ratio. In turn, this male-biased sex ratio would reduce the
costs of deception to the pollinator population and benefit the orchids. There would be new,
naïve males for orchids to exploit, but also enough males in the population to keep some
females fertilised and producing daughters. Haplodiploidy and persistence In this way, the population might persist over
time. This contrasts with sexually deceptive orchids that target a diploid pollinator species,
which cannot reproduce without matings and sperm. Using mathematical modelling, we tested whether haplodiploidy renders pollinators
robust to sexual deception (Brunton-Martin et al. 2021a). We found that haplodiploidy does
indeed act as a mitigating mechanism: deceived haplodiploid populations are better able to
withstand costs associated with sexual deception (in terms of persistence and maintaining
fitness) than an otherwise identical diploid population. Their persistence partially resulted
from the haplodiploid pollinator population reaching a stable, male-biased sex ratio. Using
surveys of museum and citizen science records, we were able to test this aspect of the
model: finding that pollinator populations that live in sympatry with Cryptostylis orchids
were more male-biased than populations that do not live with Cryptostylis orchids (Brunton-
Martin et al. 2021b). Future work should also establish the sex-ratio of pollinator popula
tions in the field. In this way, haplodiploidy allows pollinators to be robust against sexual deception, and
can be readily exploited with a lower risk of extinction. We propose that this phenomenon
may exist in other exploitative systems: a life-history trait may influence whether a given
species or group is prone to exploitation, henceforth, we refer to this new hypothesis as
‘robustness’ (and traits that confer ‘robustness’ as ‘robust traits’). Defining robustness The idea of robustness has been explored in systems biology. Specifically, this is where
biological systems are hypothesised to have an intrinsic ability to maintain functions when
exposed to perturbations (Kitano 2004; Whitacre 2012). In the context of sexual deception,
we propose that this intrinsic ability arises from a trait that exists prior to the exploitative
relationship. The definition of robustness is perhaps better understood by contrasting it
with the existing concepts of resistance and tolerance (Fig. 3; Svensson and Råberg 2010). Robustness, like tolerance, is distinct from resistance in that it neither acts to lower exploiter
fitness, nor does it reduce the direct costs of exploitation. However, robustness is distinct 3 Evolutionary Ecology (2022) 36:321–339 332 Fig. 3 Contrasting different mechanisms for persistance: resistance, exaptation, tolerance against ‘robust
ness’. Citations: Resistance – 1de Jager and Ellis 2014; Exaptation 2Schiestl and Cozzolino 2008, chemical
compound example from Bohman et al. 2019; Tolerance –3Tiffin 2000; Robustness – 4(Kitano 2004; Whita
cre 2012) Fig. 3 Contrasting different mechanisms for persistance: resistance, exaptation, tolerance against ‘robust
ness’. Citations: Resistance – 1de Jager and Ellis 2014; Exaptation 2Schiestl and Cozzolino 2008, chemical
compound example from Bohman et al. 2019; Tolerance –3Tiffin 2000; Robustness – 4(Kitano 2004; Whita
cre 2012) from both strategies because it does not arise as a defensive strategy in response to an
antagonist. Instead, rather than evolving as a defence strategy, robust traits are pre-existing
in the deceived species (Vitale and Best 2019). Pre-existing traits are observed in another similar evolutionary scenario: exaptation (i.e. co-opting an existing trait for a new defence function; Fig. 3, Gould and Vrba 1982). Unlike
an exaptation, however, a robust trait is still utilised for its original function, but also simul
taneously improves a populations’ persistence in the face of exploitation. We propose that robustness does not exclude the evolution of adaptations and counter
adaptations, but may weaken the costs that drive them. Robust traits may operate in concert
with one of the other hypotheses for the maintenance of deceptive systems, low encounter
rates, but seems unlikely to be compatible with the indirect benefits hypothesis, because
robustness does not involve any benefit to the exploited species. 1 3 Towards a general mechanism This concept of robustness could be useful for exploring persistence of an exploited species
in other systems and may be particularly useful in exceptional instances of extreme exploi
tation. Intriguingly, one other orchid that triggers pollinator ejaculation may have a different
robust trait preventing pollinator extinction. Lepanthes orchids provoke ejaculation, but their
pollinator is diploid fungus gnats (Diptera; Blanco and Barboza 2005), and fungus gnats
may well be a much more common sexually deceived pollinator than currently documented
(Reiter et al. 2020; Hayashi et al. 2021). However, fungus gnats can also have a male biased
sex ratio, not via an internal mechanism like haplodiploid insects, instead fungus gnat sex
ratios become more male-biased at cool temperatures (Nigro et al. 2007; Pandey and Tripa
thi 2008; Farsani et al. 2013). Lepanthes orchids bloom in colder weather (April – June) and
grow in cold climates, and so fungus gnat populations might well have a male-biased sex
ratio. This may confer robustness to these pollinators but remains to be tested empirically. Robustness may also be conveyed, for instance, via differences in developmental modes of
cuckoo hosts (altricial, or early development state at birth, versus precocial, or late develop
ment state at birth) may play an important role in understanding the long-term stability of
obligate interspecific brood parasitism (Yom-Tov and Geffen 2006). Three of the four orders
of species that exhibit obligate interspecific brood parasitism (Cuculiformes, Piciformes,
and Passeriformes) are altricial, while only one order (Anseriformes) is precocial. There is a
greater duration of parental care in altricial species that might allow parasitised individuals
time to reduce the costs of deception (Sheriff et al. 2018), and yet the appearance of altricial
young may allow parasitic chicks to remain inconspicuous for longer (Scheiber et al. 2017). Additionally, because precocial species have longer incubation times and invest more in
eggs, the cost of rejecting an egg may be greater for them, whereas an altricial species may
reject an egg and re-lay (Augustine et al. 2019) with lower costs. Better understanding these
types of relationships are important, particularly when we consider that these systems are
often species-specific and thus fragile to climate change and other anthropogenic effects. Robustness, combined with the mechanisms mentioned previously for the maintenance
of costly relationships, is hopefully a useful avenue of research in behavioural and evolu
tionary ecology. Testing for robust traits Testing for the presence of robust traits in an exploitative relationship is difficult, as it will
vary from trait to trait. We propose that the framework used in the Cryptostylis case-study
may prove useful when exploring this phenomenon. Firstly, mathematical modelling and
simulations are a useful tool for capturing traits that might impact persistence (Kitano 2002;
Daniels et al. 2008; Whitacre 2012). For instance, modelling the exploited population with
and without the proposed trait will produce testable outcomes. While robust traits are fixed
in the species, theoretical modelling that removes the trait and holds all else equal allows for
clarity on how, exactly, a trait might influence persistence. For a trait to be robust, we would
expect the exploited population with the trait to have better persistence and fitness than the
same (theoretical) population without the trait. Then, quantitative research to assess whether the predicted outcomes indeed occur in
nature, and perhaps comparisons with sister species that do and do not have a fixed robust
trait. Furthermore, meta-analyses that utilise existing knowledge and explore the prepon
derance of putative robust traits in antagonistic relationships (parasitism or predation, for
example) and the impact on exploiters (in terms of mortality rates, infection rates or repro
duction rates) might reveal the importance of robustness as a mechanism. 1 3 1 Evolutionary Ecology (2022) 36:321–339 333 Towards a general mechanism This mechanism may help shape our understanding of the coexistence of
exploitative species and their hosts. Acknowledgements We would like to acknowledge Callum Young, Ryan deRegnier and Dianne Brunton for
their helpful comments on the manuscript. Author contribution ABM, ACG and JCO formulated and developed the ideas for the manuscript, ABM
wrote the manuscript, ACG and JCO edited drafts of the manuscript, ACG provided funding. Funding Work funded by Marsden Fund Fast-Start Grant. Declarations Conflict of interest The authors have no conflicts of interest to declare. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which 3 334 Evolutionary Ecology (2022) 36:321–339 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
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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
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regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright
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in Drosophila melanogaster. J Evol Biol 32:1300–1309. https://doi.org/10.1101/652248 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
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Right ventricular function in dilated cardiomyopathy and ischemic heart disease: assessment with non-invasive imaging
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Neth Heart J (2015) 23:232–240
DOI 10.1007/s12471-015-0673-x Neth Heart J (2015) 23:232–240
DOI 10.1007/s12471-015-0673-x Original Article - Design Study Article Abstract Background Dilated cardiomyopathy and ischaemic heart
disease can both lead to right ventricular (RV) dysfunction. Direct comparisons of the two entities regarding RV size
and function using state-of-the-art imaging techniques have
not yet been performed. We aimed to determine RV func-
tion and volume in dilated cardiomyopathy and ischaemic
heart disease in relation to left ventricular (LV) systolic and
diastolic function and systolic pulmonary artery pressure. Conclusions In patients with dilated cardiomyopathy and
ischaemic heart disease, RV function is determined by
LV systolic and diastolic function, the underlying cause
of LV dysfunction, and pulmonary artery pressure. It was
demonstrated that RV function is more impaired in dilated
cardiomyopathy. Methods and results A well-characterised group (cardiac
magnetic resonance imaging, echocardiography, coronary
angiography and endomyocardial biopsy) of 46 patients with
dilated cardiomyopathy was compared with LV ejection
fraction (EF)-matched patients (n = 23) with ischaemic heart
disease. Volumes and EF were determined with magnetic
resonance imaging, diastolic LV function and pulmonary
artery pressure with echocardiography. Right ventricular function in dilated cardiomyopathy and
ischemic heart disease: assessment with non-invasive imaging S. Schalla · C. Jaarsma · S.C. Bekkers · J. Waltenberger · R. Dennert ·
H.J. Crijns · J. Wildberger · S. Heymans · H-P. Brunner-La Rocca Published online: 6 March 2015
© The Author(s) 2015. This article is published with open access at Springerlink.com transmitral Doppler flow velocity as measure of LV filling
pressure (r = − 0.52, p < 0.001) and tricuspid regurgita-
tion flow velocity as measure of pulmonary artery pres-
sure (r = − 0.38, p = 0.001). RVEF was significantly worse
in patients with dilated cardiomyopathy compared with
ischaemic heart disease: median 48 % (interquartile range
(IQR) 37–55 %) versus 56 % (IQR 48–63 %), p < 0.05. transmitral Doppler flow velocity as measure of LV filling
pressure (r = − 0.52, p < 0.001) and tricuspid regurgita-
tion flow velocity as measure of pulmonary artery pres-
sure (r = − 0.38, p = 0.001). RVEF was significantly worse
in patients with dilated cardiomyopathy compared with
ischaemic heart disease: median 48 % (interquartile range
(IQR) 37–55 %) versus 56 % (IQR 48–63 %), p < 0.05. S. Schalla () · C. Jaarsma · S.C. Bekkers · R. Dennert ·
H.J. Crijns · S. Heymans · H.-P. Brunner-La Rocca
Department of Cardiology, Maastricht University Medical Center,
P. Debyelaan 25,
P.O. Box 5800, 6202 AZ Maastricht, The Netherlands
e-mail: s.schalla@mumc.nl Echocardiography Echocardiography was used to determine LV diastolic func-
tion, LA volume and peak tricuspid regurgitation (TI) Dop-
pler velocity. Transthoracic echocardiograms were performed
using dedicated systems (Sonos 5500 with S3 or IE33 with
S5-1 transducers, Philips Medical Systems, Andover, MA,
USA). Echocardiographic investigations were performed in
the standard views, according to the recommendations of the
American Society of Echocardiography [7]. LV diastolic func-
tion was assessed by measuring mitral (ratio of peak early and
peak atrial transmitral Doppler flow velocity, E/A) and pulmo-
nary vein flow velocities (ratio of peak systolic and diastolic
pulmonary vein Doppler flow velocity, S/D), tissue Doppler
flow velocities from the basal septal and lateral wall to cal-
culate the ratio of early transmitral inflow and myocardial
tissue velocity (E/e’) and left atrial (LA) volumes. Since we
did not include patients with normal LV function, we used the
E/A ratio as an easily obtainable measure of LV filling pres-
sure. Peak tricuspid regurgitation (TI) Doppler flow velocity
was used as a measure of pulmonary artery systolic pressure
(PAP). Images were digitally stored and analysed off-line by
an investigator blinded to CMR results and clinical data. Study subjects in the IHD group were included from our
infarct database matched for LVEF as measured by CMR. Thus, 23 patients with impaired systolic LV function due to
chronic non-inferior myocardial infarction (infarction in left
anterior descending (LAD) or left circumflex (LCX) coronary
artery territory to avoid inclusion of patients with RV infarc-
tion) and without significant valvular disease were included. The study was approved by the local ethics committee. Study population Study population CMR images were analysed by an investigator blinded
to clinical and echocardiographic data. Endocardial and
epicardial LV and endocardial RV contours were manually
traced in end-diastolic and end-systolic phases on short-axis
slices to determine LV and RV end-diastolic volume (EDV),
end-systolic volume (ESV), ejection fraction (EF) and LV
end-diastolic mass. Areas of late enhancement were visually
assessed to confirm the presence and extent of infarction in
the LAD or LCX territory as the reason for impaired LVEF
in the IHD group. Two groups of patients were included in the study: patients
with DCM from Maastricht University Medical Center and
patients with IHD from our infarct database matched for
LVEF. Study subjects in the DCM group comprised 46 consecu-
tive patients with DCM referred to our heart failure program. All underwent a standard diagnostic evaluation including
electrocardiogram (ECG), coronary angiography, endomyo-
cardial biopsy, echocardiography and cardiac magnetic res-
onance imaging (CMR). LV and RV volumes and systolic
function were determined by CMR. LV diastolic function
and RV systolic pressure were assessed with echocardiog-
raphy as described later in the text. Patients with a history
of myocardial infarction, history of cardiotoxic agents, sig-
nificant coronary artery stenosis on coronary angiography,
valvular heart disease on echocardiography as well as other
known causes of impaired systolic function such as inflam-
mation or infiltrative disorders on endomyocardial biopsy
were excluded from the study. Patients with permanent
pacemakers, rhythm other than sinus and significant chronic
renal failure ( ≥ stage 3 kidney disease) were also excluded. Magnetic resonance imaging Patients were examined with a 1.5-T scanner (Gyroscan
Intera,Philips Medical Systems, Best, the Netherlands). ECG-gatedcine images were acquired for functional analy-
sis using a steady-state free precession sequence (slice thick-
ness 6 mm, gap 4 mm, repetition time/echo time 3.8/1.9 ms,
flip angle 50°, field of view 350 mm, matrix 256 × 256,
22–25 phases per cardiac cycle). A breath-hold multislice
T1-weighted three-dimensional inversion-recovery gradi-
ent-echo sequence (acquired/reconstructed slice thickness
12/6 mm, average repetition time/echo time 3.9/2.4 ms,
multi-shot [50 profiles/shot] segmented partial echo read-
out, flip angle 15°, field of view 400 mm, matrix 256 × 256)
to evaluate the presence of myocardial infarction of the left
and right ventricle was used to acquire images 10 min after
intravenous administration of 0.2 mmol/kg body weight
gadolinium diethylenetriaminepentaacetic acid (Magnevist, Introduction Right ventricular (RV) dysfunction and dilatation are corre-
lated to limited exercise capacity and poor outcome [1–4],
but often neglected in the clinical setting [5, 6]. Dilated
cardiomyopathy (DCM) and ischaemic heart disease (IHD)
can both lead to RV dysfunction. Direct comparisons of
the two entities with respect to RV size and function using
state-of-the-art imaging techniques have not yet been per-
formed. Such a comparison may help to better understand
the underlying pathophysiology of RV dysfunction. There-
fore, we determined RV function and volume in relation
to left ventricular (LV) systolic and diastolic function, and
pulmonary artery pressure in patients with DCM to assess
the main mechanisms of RV dysfunction. In addition, after
matching for LV ejection fraction (LVEF), patients with
IHD due to infarction of the left coronary artery were also
examined. After multivariable linear regression, four factors inde-
pendently influenced RVEF (R2 = 0.51, p < 0.001): LVEF
(r = 0.54, p < 0.001), ratio of peak early and peak atrial S. Schalla () · C. Jaarsma · S.C. Bekkers · R. Dennert ·
H.J. Crijns · S. Heymans · H.-P. Brunner-La Rocca
Department of Cardiology, Maastricht University Medical Center,
P. Debyelaan 25,
P.O. Box 5800, 6202 AZ Maastricht, The Netherlands
e-mail: s.schalla@mumc.nl 1 Neth Heart J (2015) 23:232–240 233 Methods Bayer, Germany). Inversion times were adjusted to null nor-
mal myocardium (200–280 ms). Bayer, Germany). Inversion times were adjusted to null nor-
mal myocardium (200–280 ms). Statistical analysis Variables are expressed as percentage, mean ± standard
deviation or median (interquartile range (IQR)) as appro-
priate. The Kolmogorov–Smirnov test was applied to test
whether continuous variables were normally distributed. Group comparisons were performed by using the Pearson
χ2 test, Student’s t-test or Mann–Whitney U test, as appro-
priate. For correlations, Pearson’s r was used. Finally, inde-
pendent variables influencing RVEF were sought by using
the multivariable linear regression model with a backward
procedure (inclusion p < 0.05, exclusion p < 0.1). DCM dilated cardiomyopathy, IHD ischaemic heart disease, NYHA New York Heart Association class, COPD chronic obstructive pulmonary
disease, HF heart failure, BP blood pressure, LBBB left bundle branch block, RBBB right bundle branch block, ACE angiotensin converting
enzyme, AT II angiotensin II, RV right ventricular, LV left ventricular, EDV end-diastolic volume, ESV end-systolic volume, SV stroke volume,
EF ejection fraction, PASP pulmonary artery systolic pressure, RA right atrial, LA left atrial, E peak early transmitral Doppler flow velocity, E/A
ratio of peak early and peak atrial transmitral Doppler flow velocity, dt E-top deceleration time of peak early transmitral Doppler flow signal,
S/D ratio of peak systolic and diastolic pulmonary vein Doppler flow velocity, E/e’ IVS ratio of peak early transmitral Doppler flow velocity
and peak early diastolic tissue Doppler flow velocity from the basal septal left ventricular wall, E/e’ ratio of peak early transmitral Doppler
flow velocity and peak early diastolic tissue Doppler flow velocity from the basal lateral left ventricular wall, TI peak tricuspid regurgitation
Doppler flow velocity Differences between patients with DCM and IHD The RVEF was more impaired and RV size larger in
patients with DCM in comparison with patients with IHD
(Figs. 3 and 4) despite similar LV systolic and diastolic
dysfunction. There was an isolated increase in E/e’ of the
septum in DCM patients, but an opposite trend laterally
and no further differences were seen in diastolic parameters
between the groups, suggesting no difference in diastolic
LV function. LV EDV and ESV were not different between
the groups. All CMR and echocardiography parameters are
summarised in Table 1. CMR image analysis was possible in all patients. In three
patients, however, image quality on echocardiography was
not sufficient to obtain reliable pulmonary vein flow Dop-
pler signals. In two of them, image quality was also not suf-
ficient to obtain tissue Doppler signals, but the E/A ratio
could be determined in all patients. A total of 21 patients with IHD had infarcts in the
LAD territory and 2 patients in the LAD and LCX terri-
tory (Fig. 1). Infarction of the RV free wall was recognised
in only one patient of the IHD group: a small area of late
enhancement of the RV apex was visible on CMR images. This patient had a transmural infarction in the LAD territory
showing late enhancement from the anterior wall to the apex
of the LV continuing to the RV apex (Fig. 2). None of the Statistical analysis 1 3 1 3 3 234 Neth Heart J (2015) 23:232–240 Table 1 Clinical, cardiac magnetic resonance imaging and echocardiographic characteristics of the patients with dilated cardiomyopa
and ischaemic heart disease (IHD)
Characteristic
DCM (n = 46)
IHD (n = 23)
p Value
Age, years
49 ± 14
59 ± 16
< 0.01
Male, n (%)
27 (59 %)
18 (78 %)
0.18
Body surface area, m2
1.92 ± 0.25
1.97 ± 0.19
0.28
Dyspnoea
0.48
NYHA 1/2, n (%)
38 (83 %)
21 (91 %)
NYHA 3/4, n (%)
8 (17 %)
2 (9 %)
Duration HF, months
4 (1–18)
3 (0–19)
0.98
Diabetes mellitus
0.62
Type 1, n (%)
1 (2 %)
0 (0 %)
Type 2, n (%)
4 (9 %)
1 (4 %)
COPD, n (%)
6 (13 %)
2 (9 %)
0.71
Hypertension, n (%)
9 (18 %)
8 (35 %)
0.24
Systolic BP
125 ± 20
123 ± 19
0.80
Diastolic BP
75 ± 12
74 ± 10
0.75
Heart rate, beats/min
77 ± 14
73 ± 13
0.32
PR duration, ms
150 ± 57
157 ± 60
0.66
QRS duration, ms
111 ± 31
92 ± 43
0.10
LBBB, n (%)
16 (35 %)
4 (17 %)
0.17
RBBB, n (%)
0 (0 %)
2 (9 %)
0.11
Creatinine, µmol/l
92 ± 27
95 ± 45
0.74
Beta-blocker
36 (78 %)
18 (78 %)
0.34
ACE-inhibitor or AT-II-receptor blocker
40 (87 %)
21 (91 %)
0.20
Diuretic
32 (70 %)
9 (39 %)
0.03
Aldosterone antagonist
12 (26 %)
4 (17 %)
0.28
Calcium channel blocker
0 (0 %)
1 (4 %)
0.13
RV EDV, ml/m2
78 (65–92)
71 (63–78)
0.03
RV ESV, ml/m2
41 (35–51)
32 (24–39)
0.03
RV SV, ml/m2
35 (28–43)
38 (29–43)
0.98
RVEF, %
48 (37–55)
56 (48–63)
0.05
LV EDV, ml/m2
120 (96–158)
131 (101–165)
0.79
LV ESV, ml/m2
82 (64–117)
85 (66–128)
0.63
LV SV, ml/m2
39 (29–46)
38 (34–43)
0.75
LVEF, %
31 (22–40)
34 (18–39)
0.77
LV mass, g/m2
75 (62–84)
68 (62–86)
0.86
RA volume, ml/m2
20 (16–32)
22 (17–26)
0.43
LA volume, ml/m2
32 (26–53)
37 (28–46)
0.86
E max velocity, cm/s
71 (55–82)
68 (53–98)
0.67
E/A
1.00 (0.70–1.40)
0.96 (0.63–1.78)
0.99
dt E-top ms
170 (130–205)
160 (130–220)
0.68
S/D
1.04 (0.81–1.33)
1.00 (0.62–1.45)
0.79
E/e’ IVS
10.0 (8.0–13.0)
6.5 (4.9–7.5)
< 0.001
E/e’
7.7 (5.4–9.7)
8.4 (6.6–11.5)
0.31
TI peak velocity, m/s
2.13 (1.86–2.44)
2.42 (2.02–3.03)
0.04
Values represent mean ± standard deviation, median (interquartile range) or n, numbers of patients (%)
DCM dilated cardiomyopathy, IHD ischaemic heart disease, NYHA New York Heart Association class, COPD chronic obstructive
disease, HF heart failure, BP blood pressure, LBBB left bundle branch block, RBBB right bundle branch block, ACE angiotensin
enzyme, AT II angiotensin II, RV right ventricular, LV left ventricular, EDV end-diastolic volume, ESV end-systolic volume, SV stro
EF ejection fraction, PASP pulmonary artery systolic pressure, RA right atrial, LA left atrial, E peak early transmitral Doppler flow v
ratio of peak early and peak atrial transmitral Doppler flow velocity, dt E-top deceleration time of peak early transmitral Doppler
S/D ratio of peak systolic and diastolic pulmonary vein Doppler flow velocity, E/e’ IVS ratio of peak early transmitral Doppler f
and peak early diastolic tissue Doppler flow velocity from the basal septal left ventricular wall, E/e’ ratio of peak early transmit
flow velocity and peak early diastolic tissue Doppler flow velocity from the basal lateral left ventricular wall, TI peak tricuspid re
Doppler flow velocity Table 1 Clinical, cardiac magnetic resonance imaging and echocardiographic characteristics of the patients with dilated cardiomyopathy (DCM)
and ischaemic heart disease (IHD) etic resonance imaging and echocardiographic characteristics of the patients with dilated cardiomyopathy (DCM)
D) Values represent mean ± standard deviation, median (interquartile range) or n, numbers of patients (% DCM dilated cardiomyopathy, IHD ischaemic heart disease, NYHA New York Heart Association class, COPD chronic obstructive pulmonary
disease, HF heart failure, BP blood pressure, LBBB left bundle branch block, RBBB right bundle branch block, ACE angiotensin converting
enzyme, AT II angiotensin II, RV right ventricular, LV left ventricular, EDV end-diastolic volume, ESV end-systolic volume, SV stroke volume,
EF ejection fraction, PASP pulmonary artery systolic pressure, RA right atrial, LA left atrial, E peak early transmitral Doppler flow velocity, E/A
ratio of peak early and peak atrial transmitral Doppler flow velocity, dt E-top deceleration time of peak early transmitral Doppler flow signal,
S/D ratio of peak systolic and diastolic pulmonary vein Doppler flow velocity, E/e’ IVS ratio of peak early transmitral Doppler flow velocity
and peak early diastolic tissue Doppler flow velocity from the basal septal left ventricular wall, E/e’ ratio of peak early transmitral Doppler
flow velocity and peak early diastolic tissue Doppler flow velocity from the basal lateral left ventricular wall, TI peak tricuspid regurgitation
Doppler flow velocity 1 3 3 Neth Heart J (2015) 23:232–240 235 patients with DCM had focal fibrosis of the RV free wall
myocardium on CMR late enhancement images. Fig. 1 Late gadolinium enhance-
ment (a, b) and cine (four-cham-
ber view in end-diastole (c) and
end-systole (d)) magnetic reso-
nance images of a patient with a
large myocardial infarction (left
ventricular ejection fraction 15 %)
of the left anterior descending
coronary artery (right ventricular
ejection fraction 56 %) RV volume and function The clinical characteristics of the DCM and IHD patients
are summarised in Table 1. Patients in the IHD group were
older and less often treated with diuretics. All other param-
eters were not significantly different between the groups. Parameters influencing RV function the presence of DCM, systolic and diastolic
function of the LV and elevation of the PAP. RV function and volumes. RV systolic function was influ-
enced by different factors including the underlying disease
process, i.e. the presence of DCM, systolic and diastolic
function of the LV and elevation of the PAP. tion negatively correlated with RV function. Also, the TI
velocity as measure of PAP was negatively correlated with
RVEF. Finally, age and heart rate showed significant corre-
lations with RVEF. RVEF was higher in women compared
with men (55 ± 10 versus 44 ± 13 %, p = 0.001). Other clini-
cal parameters had no influence on RVEF (data not shown). Overall, there were no significant differences in these cor-
relations between patients with DCM and IHD (data not
shown). In ST-segment elevation myocardial infarction (STEMI)
patients with cardiogenic shock, RV dysfunction had been
identified as a prognostic parameter [8]. Between patients
with and without RV dysfunction, no significant differences
in infarct-related artery and infarct location were observed. RV dysfunction in patients in whom the right coronary artery
(RCA) was not the infarct-related artery was also present. This suggests that not only direct RV infarction causes RV
dysfunction but also mechanisms such as reduced blood
supply of the septum, low RV preload due to low output
and diminished contribution of LV contraction to RV systole
[8]. RV function may depend on LV septal contractile con-
tribution transmitted through systolic ventricular interaction
[9–11] and the septum itself may contribute to the systolic
function of both ventricles [12]. RV function was experi-
mentally as much impaired with septal ligation as with
RCA ligation [13]. Our results, however, suggest that other
factors such as elevated LV filling pressure and PAP are of
additional importance to explain reduced RV function. Multivariable analysis revealed that the RV function (i.e. RVEF) was correlated with LVEF, DCM compared with
IHD, E/A ratio as measure of LV filling pressure, and TI
velocity as measure of PAP as depicted in Table 3. Although
the relationship with TI velocity was of borderline signifi-
cance, it was in addition to a measure of LV filling pressure,
e.g. a possible active process in addition to passive elevation
of PAP due to elevated LV filling pressures. Parameters influencing RV function As depicted in Table 2, the RVEF was significantly cor-
related with LVEF and the volumes of all four cardiac
chambers. Furthermore, parameters of LV diastolic func- 1 3
Fig. 1 Late gadolinium enhance-
ment (a, b) and cine (four-cham-
ber view in end-diastole (c) and
end-systole (d)) magnetic reso-
nance images of a patient with a
large myocardial infarction (left
ventricular ejection fraction 15 %)
of the left anterior descending
coronary artery (right ventricular
ejection fraction 56 %) 1 3 3 236 Neth Heart J (2015) 23:232–240 RV function and volumes. RV systolic function was influ-
enced by different factors including the underlying disease
process, i.e. the presence of DCM, systolic and diastolic
function of the LV and elevation of the PAP. In ST-segment elevation myocardial infarction (STEMI)
patients with cardiogenic shock, RV dysfunction had been
tion negatively correlated with RV function. Also, the TI
velocity as measure of PAP was negatively correlated with
RVEF. Finally, age and heart rate showed significant corre-
lations with RVEF. RVEF was higher in women compared
with men (55 ± 10 versus 44 ± 13 %, p = 0.001). Other clini-
cal parameters had no influence on RVEF (data not shown). Fig. 2 Late gadolinium enhance-
ment (four-chamber view (a), 2
chamber view (b)) and cine (four-
chamber view in end-diastole (c)
and end-systole (d)) magnetic
resonance images of a patient
with a large myocardial infarction
(left ventricular ejection fraction
22 %) of the left anterior descend-
ing coronary artery, continuing
from the left ventricular apex to
the right ventricle (right ventricu-
lar ejection fraction 57 %) (white
arrow) A corresponding akinetic
region of the right ventricular
apex can be depicted on the
end-systolic cine image (D, black
arrow) Fig. 2 Late gadolinium enhance-
ment (four-chamber view (a), 2
chamber view (b)) and cine (four-
chamber view in end-diastole (c)
and end-systole (d)) magnetic
resonance images of a patient
with a large myocardial infarction
(left ventricular ejection fraction
22 %) of the left anterior descend-
ing coronary artery, continuing
from the left ventricular apex to
the right ventricle (right ventricu-
lar ejection fraction 57 %) (white
arrow) A corresponding akinetic
region of the right ventricular
apex can be depicted on the
end-systolic cine image (D, black
arrow) RV function and volumes. RV systolic function was influ-
enced by different factors including the underlying disease
process, i.e. Discussion In the current study, the non-invasive imaging techniques
CMR and echocardiography were applied in patients with
DCM and IHD without RV free wall infarction, to evaluate 1 3 237 Neth Heart J (2015) 23:232–240 c Fig. 3 Late gadolinium enhance-
ment (a, b) and cine (four-
chamber view in end-diastole (c)
and end-systole (d)) magnetic
resonance images of a patient
with idiopathic dilated cardiomy-
opathy (left ventricular ejection
fraction 24 %, right ventricular
ejection fraction 45 %) The left
ventricular systolic function of
this patient is slightly better than
that of the patients with ischaemic
heart disease from Figs. 1 and 2
while the right ventricular func-
tion is more impaired. Areas of
late enhancement are not present risk for necrosis, although the resulting final RV infarction
size was small. A possible explanation for the discrepancy
between this and our findings is that only a small portion of
our patients had transmural infarcts of the whole septum. risk for necrosis, although the resulting final RV infarction
size was small. A possible explanation for the discrepancy
between this and our findings is that only a small portion of
our patients had transmural infarcts of the whole septum. RV infarction detected by late enhancement CMR and
RV systolic function were outcome predictors in a general
population of STEMI patients [14]. In inferior STEMI, RV
systolic function was related to the presence and extent of
RV infarction, while in anterior STEMI it was related to LV
systolic dysfunction. When imaging more early after infarc-
tion, RV infarction was not related to prognosis [15]. This
discrepancy could be explained by the fact that RV function
may recover to a greater extent than LV function, depend-
ing on recovery of septal function [9]. Thus, global RV per-
formance recovered within days after infarction, regardless
of artery patency [16]. Moreover, global RV performance
improved greatly even in chronic RCA occlusion, despite
persistent severe RV free wall dysfunction [17]. To exclude
the influence of direct RV free wall infarct on RV function,
we included only patients with infarcts of the left coronary
artery territory (LAD and LCX infarct). To our knowledge,
this has not been applied in previous comparative studies of
patients with DCM and IHD. Discussion 4 Late gadolinium enhance-
ment (a, b) and cine (four-
chamber view in end-diastole (c)
and end-systole (d)) magnetic
resonance images of a patient
with idiopathic dilated cardio-
myopathy with more pronounced
dilation of the right ventricle (left
ventricular ejection fraction 36 %,
end-diastolic volume 211 ml;
right ventricular ejection frac-
tion 23 %, end-diastolic volume
269 ml) Although elevated left
ventricular filling pressure and TI
velocity were present, the degree
of right ventricular dilation is out
of proportion, possibly suggesting
an active unknown process affect-
ing the right ventricle more than
the left ventricle Table 2 Correlations of clinical, cardiac magnetic resonance imaging
(CMR) and echocardiographic characteristics with right ventricular
ejection fraction as measured with CMR Table 2 Correlations of clinical, cardiac magnetic resonance imaging
(CMR) and echocardiographic characteristics with right ventricular
ejection fraction as measured with CMR Table 3 Regression analysis testing the association between right ven-
tricular ejection fraction and various potential predictors in the entire
study population (dilated cardiomyopathy and ischaemic heart disease) (CMR) and echocardiographic characteristics with right ventricular
ejection fraction as measured with CMR
Variable
Correlation
p Value
Age
0.29
0.02
Systolic BP
0.13
0.28
HR
− 0.46
< 0.001
LVEF
0.54
< 0.001
LV EDV
− 0.29
0.02
RV EDV
− 0.50
< 0.001
LA volume
− 0.41
< 0.001
RA volume
− 0.37
< 0.001
E/A ratio
− 0.52
< 0.001
S/D ratio
0.48
< 0.001
TI velocity
− 0.38
0.001
BP blood pressure, HR heart rate, LV left ventricular, RV right
ventricular, EF ejection fraction, EDV end-diastolic volume, LA
left atrial, RA right atrial, E/A ratio of peak early and peak atrial
transmitral Doppler flow velocity, S/D ratio of peak systolic and
diastolic pulmonary vein Doppler flow velocity, TI peak tricuspid
regurgitation Doppler flow velocity y p p
(
y p
y
)
Variable
Regression coefficient
p Value
LVEF (per %)
0.50
< 0.0001
DCM versus IHD
9.41
0.0005
E/A ratio (per unit)
− 3.60
0.02
TI velocity (per m/s)
− 4.84
0.06
(R2 = 0.51)
DCM dilated cardiomyopathy, IHD ischaemic heart disease,
LVEF left ventricular ejection fraction, E/A ratio of peak early and
peak atrial transmitral Doppler flow velocity, TI peak tricuspid
regurgitation Doppler flow velocity y p p
(
y p
y
)
Variable
Regression coefficient
p Value
LVEF (per %)
0.50
< 0.0001
DCM versus IHD
9.41
0.0005
E/A ratio (per unit)
− 3.60
0.02
TI velocity (per m/s)
− 4.84
0.06
(R2 = 0.51) DCM dilated cardiomyopathy, IHD ischaemic heart disease,
LVEF left ventricular ejection fraction, E/A ratio of peak early and
peak atrial transmitral Doppler flow velocity, TI peak tricuspid
regurgitation Doppler flow velocity DCM dilated cardiomyopathy, IHD ischaemic heart disease,
LVEF left ventricular ejection fraction, E/A ratio of peak early and
peak atrial transmitral Doppler flow velocity, TI peak tricuspid
regurgitation Doppler flow velocity DCM dilated cardiomyopathy, IHD ischaemic heart disease,
LVEF left ventricular ejection fraction, E/A ratio of peak early and
peak atrial transmitral Doppler flow velocity, TI peak tricuspid
regurgitation Doppler flow velocity RV dysfunction. Discussion We observed a small exten-
sion of infarct from the left to the RV myocardium in one
patient only, which is in some contrast to a recent CMR
study in 20 patients with reperfused proximal LAD occlu-
sion where a small RV infarct was observed in 40 % of the
patients [18]. They found a relatively large area of the RV at p
p
The underlying process also determines RV function. Patients with DCM experienced larger RV volumes and more
severe impairment of RV function. In contrast to previous
suggestions [19, 20], assessing systolic RV function alone is
not sufficient to distinguish between DCM and IHD in indi-
vidual patients. Few studies have directly compared the RV
function in patients with DCM and IHD. Our findings are
supported by some earlier studies where patients with DCM
showed more severe RV dysfunction than patients with
IHD using radionuclide angiography, thermodilution and
invasive RV angiography [19–21]. However, these findings
have not been uniform. By applying tissue Doppler imag-
ing, the RV dysfunction was more pronounced in patients
with IHD than DCM in an echocardiographic study [22]. Since only two patients had inferior wall infarction based
on ECG criteria, the authors concluded that this worse RV
function had not been due to more infarcted RV myocar-
dium. However, patients were not sufficiently matched with
respect to other factors potentially influencing RV function. Thus, patients with IHD exhibited more severe LV diastolic 1 3 3 3 238 Neth Heart J (2015) 23:232–240 Fig. References Nat Rev Cardiol. 2010;7:551–63. 7. Gottdiener JS, Bednarz J, Devereux R, et al. American Society of
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lar ejection fraction to exercise capacity in chronic left ventricular
failure. Am J Cardiol. 1984;54:596–9. The current study has some limitations. Patients in the
IHD group were older and less often treated with diuretics. Older age had a negative influence on RV function. How-
ever, although the patients in the IHD group were older, they
showed a less impaired RV function than the patients from
the DCM group. Thus, not matching for age did not result
in a relevant bias. Treatment with diuretics can lower LV
filling pressure and PAP and might have had an influence on
the lower TI peak velocity measured in patients with DCM
[28]. In contrast to IHD, DCM usually comprises a hetero-
geneous group of diseases in many studies. However, with
a comprehensive diagnostic routine including endomyocar-
dial biopsy, imaging and blood tests, certain diseases such
as acute myocarditis, infiltrative and storage diseases were
excluded from our study to make the DCM study popula-
tion as uniform as possible. Regional RV wall motion as
measured by, e.g. speckle tracking was not assessed. Early
stages of dyssynchronicity between the right and left ventri-
cle were therefore not detected. Other potential mechanisms
were also not addressed, e.g. interventricular interaction and
effects of changes of geometry. Finally, this was a diagnos-
tic study. However, the study revealed different potential
mechanisms of RV dysfunction that may be therapeutically
influenced. It is important to evaluate RV function, espe-
cially in DCM, and it may be necessary to treat elevated
filling pressure and PAP more aggressively or with new
therapeutic strategies such as phosphodiesterase inhibitors
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state of the art. Discussion Moreover, assessment of RV dimensions
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limitations. In the present study, we therefore used the cur-
rent reference standard CMR to assess RV volumes and EF
[6, 23, 24].i RV dysfunction. Moreover, assessment of RV dimensions
and systolic function by echocardiography has important
limitations. In the present study, we therefore used the cur-
rent reference standard CMR to assess RV volumes and EF
[6, 23, 24].i Elevated LV filling pressure, which leads to passive ele-
vation of PAP, was one of the factors related to RV dysfunc-
tion in the current study. Since the prognosis of patients with
impaired RV function is worse in comparison with patients
with impaired systolic LV function only [25, 26], assess-
ment of RV function and possibly more aggressive treatment dysfunction and higher PAP. Such a difference in PAP and
diastolic function was not present in our patient groups, but
PAP and diastolic function were independently related to 1 3 3 Neth Heart J (2015) 23:232–240 239 of elevated LV filling pressure as suggested by Stevenson
et al. [27] might be important, irrespective of the underly-
ing cause. An additional active component of elevated pul-
monary artery pressure, i.e. out-of-proportion pulmonary
hypertension leading to fixed pulmonary hypertension, may
also have contributed to worsening of RV function since
elevated PAP was related to RV dysfunction independently
of LV filling pressure. Open Access This article is distributed under the terms of the Creative
Commons Attribution License which permits any use, distribution, and
reproduction in any medium, provided the original author(s) and the
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pressed right ventricular ejection fraction in acute inferior infarc-
tion. Br Heart J. 1977;39:1319–23. Conflict of interest None declared. This study was approved by the Medical Ethics Committee of the
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therapy tailored for severe congestive heart failure in patients
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Improved genome of<i>Agrobacterium radiobacter</i>type strain provides new taxonomic insight into<i>Agrobacterium</i>genomospecies 4
|
PeerJ
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cc-by
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Improved genome of Agrobacterium
radiobacter type strain provides new
taxonomic insight into Agrobacterium
genomospecies 4 Han Ming Gan1,2,3, Melvin V.L. Lee3 and Michael A. Savka4 Han Ming Gan1,2,3, Melvin V.L. Lee3 and Michael A. Savka4
1 Deakin Genomics Centre, Deakin University, Geelong, VIC, Australia
2 Centre for Integrative Ecology, School of Life and Environmental Sciences, Deakin University,
Geelong, VIC, Australia Deakin Genomics Centre, Deakin University, Geelong, VIC, Australia
2 Centre for Integrative Ecology, School of Life and Environmental Sciences, Deakin University,
Geelong, VIC, Australia
3 3 School of Science, Monash University Malaysia, Petaling Jaya, Selangor, Malaysia
4 College of Science, The Thomas H. Gosnell School of Life Sciences, Rochester Institute of
Technology, Rochester, NY, USA How to cite this article Gan HM, Lee MVL, Savka MA. 2019. Improved genome of Agrobacterium radiobacter type strain provides new
taxonomic insight into Agrobacterium genomospecies 4. PeerJ 7:e6366 DOI 10.7717/peerj.6366 ABSTRACT The reported Agrobacterium radiobacter DSM 30174T genome is highly fragmented,
hindering robust comparative genomics and genome-based taxonomic analysis. We re-sequenced the Agrobacterium radiobacter type strain, generating a
dramatically improved genome with high contiguity. In addition, we sequenced the
genome of Agrobacterium tumefaciens B6T, enabling for the first time, a proper
comparative genomics of these contentious Agrobacterium species. We provide
concrete evidence that the previously reported Agrobacterium radiobacter type strain
genome (Accession Number: ASXY01) is contaminated which explains its
abnormally large genome size and fragmented assembly. We propose that
Agrobacterium tumefaciens be reclassified as Agrobacterium radiobacter subsp. tumefaciens and that Agrobacterium radiobacter retains it species status with the
proposed name of Agrobacterium radiobacter subsp. radiobacter. This proposal
is based, first on the high pairwise genome-scale average nucleotide identity
supporting the amalgamation of both Agrobacterium radiobacter and Agrobacterium
tumefaciens into a single species. Second, maximum likelihood tree construction
based on the concatenated alignment of shared genes (core genes) among
related strains indicates that Agrobacterium radiobacter NCPPB3001 is sufficiently
divergent from Agrobacterium tumefaciens to propose two independent sub-clades. Third, Agrobacterium tumefaciens demonstrates the genomic potential to
synthesize the L configuration of fucose in its lipid polysaccharide, fostering its ability
to colonize plant cells more effectively than Agrobacterium radiobacter. Submitted 23 October 2018
Accepted 20 December 2018
Published 8 February 2019
Corresponding author
Michael A. Savka, massbi@rit.edu
Academic editor
Mikhail Gelfand
Additional Information and
Declarations can be found on
page 15
DOI 10.7717/peerj.6366
Copyright
2019 Gan et al. Di t ib t d
d Subjects Agricultural Science, Genomics, Microbiology, Plant Science, Taxonomy
Keywords Type strain, Average nucleotide identity, Phylogenomics, Agrobacterium radiobacter,
Agrobacterium tumefaciens, Lipopolysaccharide, Agrobacterium, Ti plasmid INTRODUCTION Distributed under
Creative Commons CC-BY 4.0 The taxonomy and phylogeny of the genus Agrobacterium has proven to be complex and
controversial. Bacteria of the genus Agrobacterium have been grouped into six species based on the disease phenotype associated, in part, with the resident disease-inducing
plasmid. Among those six species are Agrobacterium tumefaciens causing crown gall
on dicotyledonous plants, stone fruit and nut trees and Agrobacterium radiobacter that is
not known to cause plant diseases of any kind (Bouzar & Jones, 2001; Conn, 1942;
Kerr & Panagopoulos, 1977; Panagopoulos, Psallidas & Alivizatos, 1978; Riker et al., 1930;
Starr & Weiss, 1943; Süle, 1978). An alternative classification approach grouped
Agrobacterium organisms into three biovars based on physiological and biochemical
properties without consideration of disease phenotype (Keane, Kerr & New, 1970; Kerr &
Panagopoulos, 1977; Panagopoulos, Psallidas & Alivizatos, 1978). The species
and biovar classification schemes do not coincide well, in a large part, because of the
disease-inducing plasmids, tumor-inducing (pTi) and hairy root-inducing (pRi), are
readily transmissible plasmids (Young et al., 2001). Many widely used approaches for bacterial species definition include composition of
peptidoglycan, base composition of DNA, fatty acid and 16S rDNA sequence
(Stackebrandt et al., 2002) in addition to newer methods based on the whole-genome
analysis (Coutinho et al., 2016; Jain et al., 2018), horizontal gene transfer analysis (Bobay &
Ochman, 2017) or the core genome analysis (Moldovan & Gelfand, 2018) which is
used in the present study. The genus Agrobacterium is a prime example with many
proposals and oppositions regarding the amalgamation of Agrobacterium and Rhizobium
over the last three or four decades (Farrand, Van Berkum & Oger, 2003; Gaunt et al., 2001;
Young et al., 2001, 2003). However, more recent studies appear to favor the
preservation of the genus Agrobacterium backed by strong genetic and genomic
evidence (Gan & Savka, 2018; Ramírez-Bahena et al., 2014). Within the genus
Agrobacterium, the taxonomic status of Agrobacterium radiobacter and Agrobacterium
tumefaciens remains contentious (Sawada et al., 1993; Young, 2008; Young, Pennycook &
Watson, 2006). Agrobacterium radiobacter (originally proposed as Bacillus radiobacter)
is a non-pathogenic soil bacterium associated with nitrogen utilization isolated
more than a century ago in 1902 (Beijerinck & Van Delden, 1902; Conn, 1942). On the
other hand, Agrobacterium tumefaciens (previously Bacterium tumefaciens) is a plant
pathogen capable of inducing tumorigenesis (Smith & Townsend, 1907). Gan et al. (2019), PeerJ, DOI 10.7717/peerj.6366 INTRODUCTION However,
the descriptive assignment for Agrobacterium tumefaciens was later found to be
contributed by a set of genes located on the large Ti plasmid that can be lost (Gordon &
Christie, 2014). In other words, the curing of Ti plasmid in Agrobacterium tumefaciens
will change its identity to the non-pathogenic species, Agrobacterium radiobacter. Furthermore, comparative molecular analysis based on single-copy housekeeping genes also
supports the close relatedness of Agrobacterium radiobacter and Agrobacterium
tumefaciens, blurring the taxonomic boundaries between these species (Mousavi et al., 2015;
Shams et al., 2013). As taxa are reclassified into different populations that do not conform to
the characteristics of the original description, the given names lose their significant and
descriptive importance. Consistent with the Judicial Commission according to the Rules of
the International Code of Nomenclature of Bacteria, Tindall (2014) concluded that the
combination of Agrobacterium radiobacter has priority over the combination Agrobacterium
tumefaciens when the two are treated as members of the same species since Agrobacterium Gan et al. (2019), PeerJ, DOI 10.7717/peerj.6366 2/23 radiobacter was the first proposed and described in 1902 whereas Agrobacterium tumefaciens
was first proposed and described in 1907) (Tindall, 2014). However, given that
Agrobacterium tumefaciens has been more widely studied than Agrobacterium radiobacter
due to its strong relevance to agriculture (Bourras, Rouxel & Meyer, 2015), it remains unclear
but interesting to see if the broader scientific community will obey this rule by
adopting the recommended species name change in future studies. To our knowledge, a detailed comparative genomics analysis of Agrobacterium
radiobacter and Agrobacterium tumefaciens type strains has not been reported despite their
genome availability (Zhang et al., 2014). The high genomic relatedness of both type strains
was briefly mentioned by Kim & Gan (2017) through whole genome alignment and
pairwise nucleotide identity calculation from homologous regions. However, evidence is
now mounting that the Agrobacterium radiobacter DSM 30147T reported by Zhang et al. (2014) is contaminated, warranting immediate investigation (Jeong, Pan & Park,
2016). The assembled genome is nearly 7 megabases, the largest among Agrobacterium
currently sequenced at that time with up to 6,853 predicted protein-coding genes
contained in over 600 contigs. At sequencing depth of nearly 200, its genome assembly
is unusually fragmented even for a challenging microbial genome (Utturkar et al.,
2017). MATERIALS AND METHODS DNA extraction and whole genome sequencing INTRODUCTION Furthermore, the phylogenomic placement of Agrobacterium radiobacter DSM
30147T based on this genome assembly has been questionable as evidenced by its
basal position and substantially longer branch length relative to other members of the
species (Gan & Savka, 2018). The overly fragmented nature of this assembly also precludes
fruitful comparative genomics focusing on gene synteny analysis. More importantly,
analysis done on a contaminated assembly but with the assumption that it is not,
will likely lead to incorrect biological interpretations (Allnutt et al., 2018). In this study, we sequenced the whole genome of Agrobacterium radiobacter using a
type strain that was sourced from the National Collection of Plant Pathogenic
Bacteria (NCPPB). We produced a contiguous genome assembly exhibiting genomic
statistics that are more similar to other assembled Agrobacterium genomes. We show here,
through comparative genomics and phylogenetics, that the previously assembled
Agrobacterium radiobacter DSM 30147T genome contains substantial genomic
representation from another Agrobacterium sp. isolated and sequenced by the same lab,
consistent with our initial suspicion of strain contamination. Using the newly assembled
genome for subsequent comparative analysis, we provide genomic evidence that
Agrobacterium radiobacter DSM 30147T and Agrobacterium tumefaciens B6T are the
same species. However, strain DSM 30147T should not be considered as a merely
non-tumorigenic strain of Agrobacterium tumefaciens as substantial genomic variation
exists between these two type strains notably in the nucleotide sugar metabolism pathway
that may contribute to their ecological niche differentiation. MATERIALS AND METHODS Gan et al. (2019), PeerJ, DOI 10.7717/peerj.6366 DNA extraction and whole genome sequencing Approximately 10 bacterial colonies were scrapped using a sterile P200 pipette tip from a
3-day-old nutrient agar culture and resuspended in lysis buffer with proteinase K Gan et al. (2019), PeerJ, DOI 10.7717/peerj.6366 (Sokolov, 2000) followed by incubation at 56
C for 3 h. DNA purification was performed
as previously described. The extracted DNA was normalized to 0.2 ng/mL and prepared
using the Nextera XT library preparation kit (Illumina, San Diego, CA, USA)
according to the manufacturer’s instructions. The library was sequenced on an Illumina
MiSeq desktop sequencer located at the Monash University Malaysia Genomics Facility
(2 250 bp run configuration) that routinely sequences mostly decapod crustacean
mitogenomes (Gan, Tan & Austin, 2016a; Gan et al., 2016b; Tan et al., 2015)
and occasionally microbial genomes (Gan et al., 2014, 2015; Wong et al., 2014) without
prior history of processing any member from the Agrobacterium genomospecies 4. Phylogenetic analysis Reconstruction of the Agrobacterium phylogeny used PhyloPhlAN (Segata et al., 2013). PhyloPhlAN is a bioinformatic pipeline that identifies conserved proteins (400 markers)
from microbial genomes and uses them to construct a high-resolution phylogeny
using maximum likelihood inference approach (Price, Dehal & Arkin, 2010). For single
gene tree construction, protein sequences were aligned with mafft v7.3 (Katoh & Standley,
2013) using the the most accurate setting (–localpair –maxiterate 1000) followed by
phylogenetic tree construction via IqTree v1.65 with optimized model (Kalyaanamoorthy
et al., 2017; Nguyen et al., 2014). Visualization and annotation of phylogenetic trees was
performed with Figtree v1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/). Protein clustering Gene prediction used Prodigal v2.6 (Hyatt et al., 2010). Clustering of the predicted
coding sequence was performed with CD-HIT-EST using the settings “-C 0.95, -T 0.8”
(Li & Godzik, 2006). Identification of unique and shared clusters were done using
basic unix commands, for example, csplit, grep, sort and uniq. The specific commands
used and files generated during clustering can be found in the Zenodo database
(https://doi.org/10.5281/zenodo.1489356). De novo assembly and genome completeness assessment Raw paired-end reads were adapter-trimmed using Trimmomatic v0.36 (Bolger, Lohse &
Usadel, 2014) followed by error-correction and de novo assembly using Spades
Assembler v3.9 (Bankevich et al., 2012) (See Data S1 for specific trimming and assembly
settings). Genome completeness was assessed with BUSCOv3 (Rhizobiales database)
(Waterhouse et al., 2017). Gan et al. (2019), PeerJ, DOI 10.7717/peerj.6366 An improved Agrobacterium radiobacter type strain genome Raw sequencing data and whole genome assembly for strains B6 and NCPPB3001 reported
in this study are linked to the NCBI Bioproject IDs PRJNA300485 and PRJNA300611,
respectively. The newly assembled genome of Agrobacterium radiobacter type strain that
was sourced from the NCPPB is approximately 30% smaller than the first reported
Agrobacterium radiobacter DSM 30147T genome with 96% less contigs (22 vs 612), 20-fold
longer N50 (480 vs 23 kb) and assembled length that is much more similar to other
Agrobacterium spp. (Table 1). In addition, it is near-complete with 685 out of 686 BUSCO
Rhizobiale single-copy genes detected as either partial or complete with minimal
evidence of contamination as indicated by the near absence of duplicated single-copy gene
(<0.1%). On the contrary, the current DSM 30147 genome is missing 25.1% of the single
copy gene with up to 34.8% duplication rate. At the time of this manuscript writing,
another genome of Agrobacterium radiobacter type strain that was sourced from another
culture collection centre, for example, the Belgian Coordinated Collections of
Microorganisms has been deposited in the NCBI wgs database (Agrobacterium radiobacter
LMG140T; Table 1) with assembly statistics that are highly similar to the type strain
genome reported in this study. Detection and visualization of Ti plasmid Genome sequences of each member of the genomospecies 4 except for the problematic
DSM 37014T strain were used as the query for blastN search (e-value 1e-100) against the
octopine-type Ti plasmid (Altschul et al., 1990). The result of the similarity search
was subsequently visualized in Blast Ring Image Generator v0.95 (Alikhan et al., 2011). Pan-genome construction and phylogenomics Whole genome sequences were reannotated with Prokka v1.1 using the default setting
(Seemann, 2014). The Prokka-generated gff files were used as the input for Roary
v3.12.0 to calculate the pan-genome (Page et al., 2015). Maximum likelihood tree
construction of the core-genome alignment and tree visualization used FastTree2 v2.1.10
(-nt -gtr) (Price, Dehal & Arkin, 2010) and FigTree v 1.4.3, respectively. Input and
output files associated with the Roary analysis have been deposited in the Zenodo database
(https://doi.org/10.5281/zenodo.1489356). Gan et al. (2019), PeerJ, DOI 10.7717/peerj.6366 Genome annotation and KEGG pathway reconstruction Whole genome sequences of Agrobacterium tumefaciens B6T and Agrobacterium
radiobacter NCPPB 3001T were submitted to the online server GhostKoala (Kanehisa,
Sato & Morishima, 2016b) for annotation and the annotated genomes were subsequently
used to reconstruct KEGG pathways (Kanehisa et al., 2016a) in the same webserver. Identification of proteins with TIGRFAM signatures of interest (Haft, Selengut &
White, 2003) used HMMsearch v3.1b2 with the option “–cut_tc” activated to filter for only
protein hits passing the TIGRFAM trusted cutoff values (Johnson, Eddy &
Portugaly, 2010). Gan et al. (2019), PeerJ, DOI 10.7717/peerj.6366 The inflated genome size of Agrobacterium radiobacter DSM 30147(T)
is due to technical errors genome-scale based on whole proteome clustering of Agrobacterium radiobacter DSM
30147T/NCPPB 3001T (Previous study, GCF_000421945; This study, GCF_001541305),
A. sp. TS43 (unpublished, GCF_001526605) and Agrobacterium tumefaciens B6
(GCF_001541315), we observed a high number of proteins that were exclusively shared
between Zhang et al. Agrobacterium radiobacter DSM 30147 and A. sp. TS43 belonging
to genomospecies 7 (Fig. 1B). Coincidentally, despite not sharing the same
Bioproject ID, the whole genomes of strains DSM 30147T and TS43 were sequenced by
the Zhang et al., and submitted to NCBI on the same date, 30 May 2013, hinting strain
contamination during sample processing in the lab. The inflated genome size of Agrobacterium radiobacter DSM 30147(T)
is due to technical errors Instead of sharing a recent common ancestor as would be expected for a recently
duplicated gene, the duplicated single copy genes coding for seryl-tRNA synthetase in
Agrobacterium radiobacter DSM 30147T were placed in two distinct clusters with one
affiliated to genomospecies 4 and the other affiliated to genomospecies 7 (Fig. 1A). Such an
unexpected clustering pattern raises the suspicion of genome assembly from two or
more non-clonal bacterial strains. In addition, by performing comparison at the Gan et al. (2019), PeerJ, DOI 10.7717/peerj.6366 Table 1 Genome statistics of publicly available Agrobacterium genomospecies 4 whole genome sequences. Assembly
accession
Strain
Isolation source
Country
Size
GC%
# Contig
GCF_900045375
B6
Apple Gall (Iowa)
USA
5.8
59.07
4
GCF_001541315*
B6
Apple Gall (Iowa)
USA
5.6
59.32
52
GCF_001692245
B140/95
Peach/Almond Rootstock
USA
5.7
59.23
45
GCF_002179795
LMG 215
Humulus lupulus gall
(USA)
USA
5.4
59.48
33
GCF_000233975
CCNWGS0286
R. pseudoacacia nodules
China
5.2
59.53
49
GCF_900011755
Kerr 14 = LMG 15 = CFBP 5761
Soil around Prunus dulcis
Australia
5.9
59.04
5
GCF_002591665
186
English Walnut gall
California
5.7
59.42
22
GCF_002008215
LMG 140 = NCPPB 3001 = CFBP
5522= DSM 30147
Saprobic soil
Germany
5.5
59.34
22
GCF_000421945
LMG 140 = NCPPB 3001 = CFBP
5522 = DSM 30147
Saprobic soil
Germany
7.17
59.86
612
GCF_001541305*
LMG 140 = NCPPB 3001 = CFBP
5522 = DSM 30147
Saprobic soil
Germany
5.5
59.36
22
GCF_900012605
CFBP 5621
Lotus corniculata, root
tissue commensal
France
5.4
59.32
3
GCF_003031125
LAD9 (CGMCC No. 2962)
Landfill leachate treatment
system
China
5.9
59.13
49
GCF_000384555
224MFTsu31
Rhizosphere of L. luteus in
Hungary, formerly
R. lupini H13-3
USA
4.8
59.73
21
GCF_900188475
719_389
Rhizosphere and
endosphere of
Arabidopsis thaliana. USA
4.9
59.73
18
GCF_000384555
UNC420CL41Cvi
Plant associated
USA
5
59.69
18
Note:
* Reported in this study genome-scale based on whole proteome clustering of Agrobacterium radiobacter DSM
30147T/NCPPB 3001T (Previous study, GCF_000421945; This study, GCF_001541305),
A. sp. TS43 (unpublished, GCF_001526605) and Agrobacterium tumefaciens B6
(GCF_001541315), we observed a high number of proteins that were exclusively shared
between Zhang et al. Agrobacterium radiobacter DSM 30147 and A. sp. TS43 belonging
to genomospecies 7 (Fig. 1B). Coincidentally, despite not sharing the same
Bioproject ID, the whole genomes of strains DSM 30147T and TS43 were sequenced by
the Zhang et al., and submitted to NCBI on the same date, 30 May 2013, hinting strain
contamination during sample processing in the lab. Gan et al. (2019), PeerJ, DOI 10.7717/peerj.6366 Gan et al. (2019), PeerJ, DOI 10.7717/peerj.6366 Genome-scale average nucleotide identity calculation supports the
amalgamation of Agrobacterium radiobacter and Agrobacterium
tumefaciens into a single genomospecies (2019), PeerJ, DOI 10.7717/peerj.6366 7/23 As expected, pairwise ANI of less than 92% was observed when they were compared with
strains from genomospecies 7 (strains RV3 and Zutra 3/1). A 100% pairwise ANI was
observed between Agrobacterium radiobacter type strains that were sourced from
NCPPB and LMG. In addition, non-type strains B140/95 and CFBP5621 also exhibit
a strikingly high pairwise ANI (>99%) to the type strains of Agrobacterium tumefaciens
and Agrobacterium radiobacter, respectively, leading to the formation of sub-clusters
within genomospecies 4 (Fig. 2). Figure 2 A heatmap showing the hierarchical clustering of Agrobacterium strains based on genomic
distance. Values in boxes indicate pairwise average nucleotide identity. Horizontal colored bar below the
heatmap indicate the genomospecies assigned to each genome (G7, genomospecies 7; G4, genomospecies 4). Boxed labels indicate genomes sequenced in this study. Full-size
DOI: 10.7717/peerj.6366/fig-2 Figure 2 A heatmap showing the hierarchical clustering of Agrobacterium strains based on genomic
distance. Values in boxes indicate pairwise average nucleotide identity. Horizontal colored bar below the
heatmap indicate the genomospecies assigned to each genome (G7, genomospecies 7; G4, genomospecies 4). Boxed labels indicate genomes sequenced in this study. Full-size
DOI: 10.7717/peerj.6366/fig-2 As expected, pairwise ANI of less than 92% was observed when they were compared with
strains from genomospecies 7 (strains RV3 and Zutra 3/1). A 100% pairwise ANI was
observed between Agrobacterium radiobacter type strains that were sourced from
NCPPB and LMG. In addition, non-type strains B140/95 and CFBP5621 also exhibit
a strikingly high pairwise ANI (>99%) to the type strains of Agrobacterium tumefaciens
and Agrobacterium radiobacter, respectively, leading to the formation of sub-clusters
within genomospecies 4 (Fig. 2). Genome-scale average nucleotide identity calculation supports the
amalgamation of Agrobacterium radiobacter and Agrobacterium
tumefaciens into a single genomospecies Genome-scale average nucleotide identity calculation supports the
amalgamation of Agrobacterium radiobacter and Agrobacterium
tumefaciens into a single genomospecies Single gene tree shows that Agrobacterium radiobacter NCPPB 3001T and Agrobacterium
tumefaciens B6T belong to the genomospecies 4 clade (Fig. 1A), corroborating with the
PhyloPhlAN phylogenomic tree that was constructed based on the alignment of Gan et al. (2019), PeerJ, DOI 10.7717/peerj.6366 Figure
1 Phylogenetic and genomic evidence indicating contamin
A. radiobacter DSM 30147T genome. (A) Maximum likelihood phylogenet
thetases from Agrobacterium genomospecies 4 and 7. Codes after the tildes
corresponding homologs. Node labels indicate ultra-fast bootstrap support
indicates number of substitutions per site. Duplicated homologs in the proble
30147 genome were colored red. (B) Venn diagram of the core proteome
strains from genomospecies 4. Numbers in the overlapping regions indica
sequences (CDS) that shared by two or more groups at 95% nucleotide iden
Full-size
DO Figure
1 Phylogenetic and genomic evidence indicating contamination
in the published
A. radiobacter DSM 30147T genome. (A) Maximum likelihood phylogenetic tree of seryl-tRNA syn-
thetases from Agrobacterium genomospecies 4 and 7. Codes after the tildes are contigs containing the
corresponding homologs. Node labels indicate ultra-fast bootstrap support value and branch length
indicates number of substitutions per site. Duplicated homologs in the problematic A. radiobacter DSM
30147 genome were colored red. (B) Venn diagram of the core proteome of selected Agrobacterium
strains from genomospecies 4. Numbers in the overlapping regions indicate the number of coding
sequences (CDS) that shared by two or more groups at 95% nucleotide identity cutoff. ll
fi 400 universal single-copy proteins (Fig. S1). The pairwise average nucleotide identity
(ANI) among strains within this clade is consistently more than 95% further supporting
their affiliation to the same genomospecies (Fig. 2) (Coutinho et al., 2016; Jain et al., 2018). 400 universal single-copy proteins (Fig. S1). The pairwise average nucleotide identity
(ANI) among strains within this clade is consistently more than 95% further supporting
their affiliation to the same genomospecies (Fig. 2) (Coutinho et al., 2016; Jain et al., 2018). 400 universal single-copy proteins (Fig. S1). The pairwise average nucleotide identity
(ANI) among strains within this clade is consistently more than 95% further supporting
their affiliation to the same genomospecies (Fig. 2) (Coutinho et al., 2016; Jain et al., 2018). Gan et al. Gan et al. (2019), PeerJ, DOI 10.7717/peerj.6366 Is Agrobacterium radiobacter a non-tumorigenic strain of
Agrobacterium tumefaciens? A majority of the currently sequenced strains from genomospecies 4 are non-tumorigenic
as evidenced by the near complete lack of genomic region with significant nucleotide
similarity to the octopine-type Ti reference plasmid (Fig. 3). Of the 14 genomes analyzed,
only strains B6T and B140/95 exhibit a complete coverage of the Ti plasmid with
near 100% sequence identity while strain 186 shows hits mainly to the essential gene
clusters of a Ti plasmid such as the vir gene cluster (black rings and gene labels in Fig. 3) at
a substantially lower sequence identity (50% < x < 90%) (Fig. 3), suggesting that it
may be harboring a dissimilar variant of Ti plasmid, for example, different opine type. In addition, although lacking hits to the virulence gene of the Ti plasmid, the tra and trb
clusters involved in plasmid conjugal transfer are present in strains Kerr 14,
CCNWGS0286 and UNC420CL41Cvi. Despite belonging to the same genomospecies, Gan et al. (2019), PeerJ, DOI 10.7717/peerj.6366 core genome alignment and phylogenomic analysis indicates that Agrobacterium
radiobacter NCPPB3001T is sufficiently divergent from Agrobacterium tumefaciens B6T
leading to their separation into two distinct sub-clusters (Fig. 4A). This is also resonated by
Figure 3 Prevalence and sequence conservation of the octopine-type Ti plasmid among
Agrobacterium genomospecies 4. Each genome (labelled 1–15) is represented by a colored ring sha-
ded based on nucleotide percentage similarity to the reference Ti plasmid (min. 50%; max. 100%). The outermost ring highlights the gene regions involved in tumorigenesis (vir, iaa and ipt) and plasmid
conjugation (trb and tra). Asterisks indicate genomes sequenced in this study. Full-size
DOI: 10.7717/peerj.6366/fig-3 Figure 3 Prevalence and sequence conservation of the octopine-type Ti plasmid among
Agrobacterium genomospecies 4. Each genome (labelled 1–15) is represented by a colored ring sha-
ded based on nucleotide percentage similarity to the reference Ti plasmid (min. 50%; max. 100%). The outermost ring highlights the gene regions involved in tumorigenesis (vir, iaa and ipt) and plasmid
conjugation (trb and tra). Asterisks indicate genomes sequenced in this study. Full-size
DOI: 10.7717/peerj.6366/fig-3 Figure 3 Prevalence and sequence conservation of the octopine-type Ti plasmid among
Agrobacterium genomospecies 4. Each genome (labelled 1–15) is represented by a colored ring sha-
ded based on nucleotide percentage similarity to the reference Ti plasmid (min. 50%; max. 100%). The outermost ring highlights the gene regions involved in tumorigenesis (vir, iaa and ipt) and plasmid
conjugation (trb and tra). Gan et al. (2019), PeerJ, DOI 10.7717/peerj.6366 Gan et al. (2019), PeerJ, DOI 10.7717/peerj.6366 Is Agrobacterium radiobacter a non-tumorigenic strain of
Agrobacterium tumefaciens? Asterisks indicate genomes sequenced in this study. Full-size
DOI: 10.7717/peerj.6366/fig-3 core genome alignment and phylogenomic analysis indicates that Agrobacterium
radiobacter NCPPB3001T is sufficiently divergent from Agrobacterium tumefaciens B6T
leading to their separation into two distinct sub-clusters (Fig. 4A). This is also resonated by core genome alignment and phylogenomic analysis indicates that Agrobacterium
radiobacter NCPPB3001T is sufficiently divergent from Agrobacterium tumefaciens B6T
leading to their separation into two distinct sub-clusters (Fig. 4A). This is also resonated by Gan et al. (2019), PeerJ, DOI 10.7717/peerj.6366 Figure 4 Genomic divergence among genomospecies 4 strains. (A) Unrooted maximum likelihood
tree constructed based on the core genome alignment. Branch length and node labels indicate number of
substitutions per site and FastTree2 SH-like support values, respectively. Putative subclades were colored
blue, red and purple (B) Distribution of accessory (non-core) gene clusters among strains determined
with Roary and plotted with the perl script roary2svg.pl (https://github.com/sanger-pathogens/Roary/
blob/master/contrib/roary2svg/roary2svg.pl). A total of 7,906 accessory gene clusters were identified
by Roary and the number of accessory genes presence in each genome are shown in the most right
column. Vertical gray lines/bars along the plot indicate presence of accessory gene. Asterisks indicate
genomes sequenced in this study. Full-size
DOI: 10.7717/peerj.6366/fig-4 Figure 4 Genomic divergence among genomospecies 4 strains. (A) Unrooted maximum likelihood
tree constructed based on the core genome alignment. Branch length and node labels indicate number of
substitutions per site and FastTree2 SH-like support values, respectively. Putative subclades were colored
blue, red and purple (B) Distribution of accessory (non-core) gene clusters among strains determined
with Roary and plotted with the perl script roary2svg.pl (https://github.com/sanger-pathogens/Roary/
blob/master/contrib/roary2svg/roary2svg.pl). A total of 7,906 accessory gene clusters were identified
by Roary and the number of accessory genes presence in each genome are shown in the most right
column. Vertical gray lines/bars along the plot indicate presence of accessory gene. Asterisks indicate
genomes sequenced in this study. Full-size
DOI: 10.7717/peerj.6366/fig-4 their different sub-cluster placement in the pairwise ANI heatplot (Fig. 2). Furthermore,
strains from both subclades could be broadly differentiated by the set of core accessory
genes that they harbor (Fig. 4B). Therefore, even though Agrobacterium radiobacter does
not harbor a Ti plasmid, it cannot be considered as a non-tumorigenic strain of
Agrobacterium tumefaciens given multiple lines of evidence indicating its substantial
genomic divergence from Agrobacterium tumefaciens. Gan et al. Gan et al. (2019), PeerJ, DOI 10.7717/peerj.6366 Is Agrobacterium radiobacter a non-tumorigenic strain of
Agrobacterium tumefaciens? (2019), PeerJ, DOI 10.7717/peerj.6366 Figure 5 KEGG pathway of nucleotide sugar metabolism associated with Agrobacterium
lipopolysaccharide synthesis. (A & B) genomic potential of A. tumefaciens B6 and A. radiobacter
DSM 30147, respectively, in the biosynthesis of dTDP-L-rhamnose. (C & D) genomic potential of
A. tumefaciens B6 and A. radiobacter DSM 30147, respectively, in the biosynthesis of GDP-L-Fucose. Numbers in boxes indicate Enzyme Commission numbers. White and green boxes indicate
absence and presence of the corresponding enzymes, respectively, based on GhostKoala annotation
(Kanehisa, Sato & Morishima, 2016b). Full-size
DOI: 10.7717/peerj.6366/fig-5 Figure 5 KEGG pathway of nucleotide sugar metabolism associated with Agrobacterium Figure 5 KEGG pathway of nucleotide sugar metabolism associated with Agrobacterium
lipopolysaccharide synthesis. (A & B) genomic potential of A. tumefaciens B6 and A. radiobacter
DSM 30147, respectively, in the biosynthesis of dTDP-L-rhamnose. (C & D) genomic potential of
A. tumefaciens B6 and A. radiobacter DSM 30147, respectively, in the biosynthesis of GDP-L-Fucose. Numbers in boxes indicate Enzyme Commission numbers. White and green boxes indicate
absence and presence of the corresponding enzymes, respectively, based on GhostKoala annotation
(Kanehisa, Sato & Morishima, 2016b). Full-size
DOI: 10.7717/peerj.6366/fig-5 Agrobacterium genomospecies 4 strains differ in their genomic
potential for nucleotide sugar metabolism
Individual comparison of the reconstructed KEGG pathways in Agrobacterium
tumefaciens (Fig. 5A) and Agrobacterium radiobacter (Fig. 5B) revealed stark contrast in
the anabolism of dTDP-L-rhamnose which is commonly found in the O-antigen of
717/peerj.6366
11/23 Agrobacterium genomospecies 4 strains differ in their genomic
potential for nucleotide sugar metabolism Individual comparison of the reconstructed KEGG pathways in Agrobacterium
tumefaciens (Fig. 5A) and Agrobacterium radiobacter (Fig. 5B) revealed stark contrast in
the anabolism of dTDP-L-rhamnose which is commonly found in the O-antigen of Gan et al. (2019), PeerJ, DOI 10.7717/peerj.6366 Table 2 Identification of Agrobacterium proteins with TIGRFAM domains involved in the biosynthesis of nucleotide sugar. Is Agrobacterium radiobacter a non-tumorigenic strain of
Agrobacterium tumefaciens? Assembly ID
Strain
TIGR01479
(EC 5.4.2.8)
TIGR01472
(EC 4.2.1.47)
TIGR01207
(EC 2.7.7.24)
TIGR01181
(EC 4.2.1.46)
TIGR01221
(EC 5.1.3.13)
TIGR01214
(EC 1.1.1.133)
1st hit
2nd hit
GCF_900045375
B6
690.2
566.6
589.5
GCF_001541315
B6
690.2
566.6
589.5
GCF_001692245
B140/95
690.2
566.6
589.5
GCF_900011755
Kerr14
691.3
690.2
428.6*
GCF_001541305
NCPPB3001
690.2
494.6
488.5
215.4
331.5
GCF_002008215
LMG140
690.2
494.6
488.5
215.4
331.5
GCF_900012605
CFBP5621
689.3
494.6
489.5
215.4
331.5
GCF_002591665
186
689.3
494.6
488.5
215.4
331.8
GCF_003031125
LAD9
688.5
494.4
487.9
215.4
329.9
GCF_000233975
CCNWGS
644.8
494.6
487.5
215.4
331.8
GCF_002179795
LMG215
690.2
GCF_000384555
224MFTsu31
644.8
GCF_000482285
UNC420CL41Cvi
644.8
GCF_900188475
719_389
687.5
Notes:
Numbers indicate bit scores calculated based on protein alignment to the model with higher scores indicating stronger and more significant hits. * Formed a separate protein cluster from the rest of genomospecies 4 GDP-mannose-4,6-dehydratase orthologs (<70% pairwise protein identity). Table 2 Identification of Agrobacterium proteins with TIGRFAM domains involved in the biosynth ntification of Agrobacterium proteins with TIGRFAM domains involved in the biosynthesis of nucleotide Notes:
Numbers indicate bit scores calculated based on protein alignment to the model with higher scores indicating stronger and more significant hits. * Formed a separate protein cluster from the rest of genomospecies 4 GDP-mannose-4,6-dehydratase orthologs (<70% pairwise protein identity). Notes:
Numbers indicate bit scores calculated based on protein alignment to the model with higher scores indicating stronger and more significant hits. * Formed a separate protein cluster from the rest of genomospecies 4 GDP-mannose-4,6-dehydratase orthologs (<70% pairwise protein identity). lipopolysaccharide (LPS) in gram-negative bacteria. Surprisingly, the entire enzyme set
required for the generation of dTDP-L-rhamnose from D-glucose-phosphate (Table 2) is
absent in Agrobacterium tumefaciens B6, suggesting that this common nucleotide sugar
may be absent from the LPS O-antigen of strain B6. A manual inspection of the accessory
genes uniquely shared by Agrobacterium tumefaciens strains B6 and B140/95 identified a
homolog cluster containing GDP-L-fucose synthase (EC 1.1.1.271) that is involved in the
enzymatic production of GDP-L-fucose from GDP-4-dehydro-6-deoxy-D-mannose and
NADH (Table 2; Fig. 5C). As expected, the genes coding for this enzyme and GDP-
mannose 4,6-dehydratase involved in the conversion of GDP-alpha-D-mannose to
GDP-4-dehydro-6-deoxy-D-mannose, are absent in the Agrobacterium radiobacter
NCPPB3001 genome (Fig. 5D). Intriguingly, HMMsearch scan revealed the presence of
two protein hits to the TIGR01479 HMM profile in Agrobacterium tumefaciens B6
that corresponds to D-mannose 1,6-phosphomutase (EC 5.4.2.8) required for the synthesis
of D-mannose 6-phosphate. Gan et al. (2019), PeerJ, DOI 10.7717/peerj.6366 Is Agrobacterium radiobacter a non-tumorigenic strain of
Agrobacterium tumefaciens? In addition to strain B6, its close relative, strain B140/95,
and a more distantly related strain Kerr14 also harbor two copies of this gene. However,
one of the D-mannose 1,6-phosphomutases in strain Kerr14 is more divergent with a
lower TIGRFAM HMM sequence score (Table 2). Furthermore, it exhibits less than
70% protein identity to the Agrobacterium tumefaciens B6 and B140/95 homologs,
forming a private protein cluster in the pan-genome (data not shown). lipopolysaccharide (LPS) in gram-negative bacteria. Surprisingly, the entire enzyme set
required for the generation of dTDP-L-rhamnose from D-glucose-phosphate (Table 2) is
absent in Agrobacterium tumefaciens B6, suggesting that this common nucleotide sugar
may be absent from the LPS O-antigen of strain B6. A manual inspection of the accessory
genes uniquely shared by Agrobacterium tumefaciens strains B6 and B140/95 identified a
homolog cluster containing GDP-L-fucose synthase (EC 1.1.1.271) that is involved in the
enzymatic production of GDP-L-fucose from GDP-4-dehydro-6-deoxy-D-mannose and
NADH (Table 2; Fig. 5C). As expected, the genes coding for this enzyme and GDP-
mannose 4,6-dehydratase involved in the conversion of GDP-alpha-D-mannose to
GDP-4-dehydro-6-deoxy-D-mannose, are absent in the Agrobacterium radiobacter
NCPPB3001 genome (Fig. 5D). Intriguingly, HMMsearch scan revealed the presence of
two protein hits to the TIGR01479 HMM profile in Agrobacterium tumefaciens B6
that corresponds to D-mannose 1,6-phosphomutase (EC 5.4.2.8) required for the synthesis
of D-mannose 6-phosphate. In addition to strain B6, its close relative, strain B140/95,
and a more distantly related strain Kerr14 also harbor two copies of this gene. However,
one of the D-mannose 1,6-phosphomutases in strain Kerr14 is more divergent with a
lower TIGRFAM HMM sequence score (Table 2). Furthermore, it exhibits less than
70% protein identity to the Agrobacterium tumefaciens B6 and B140/95 homologs,
forming a private protein cluster in the pan-genome (data not shown). DISCUSSION The distinct separation of Agrobacterium genomospecies 4 and 7 at 95% ANI cutoff
corroborates with the previously established “genomic yardstick” for species
differentiation (Konstantinidis & Tiedje, 2005; Richter & Rosselló-Móra, 2009). Using this
percentage cutoff, the ANI approach has been successfully used to provide a near
“black-and-white” pattern of species separation in even some of the most diverse bacterial
genera such as Pseudomonas, Arcobacter and Stenotrophomonas (Pérez-Cataluña et al.,
2018; Tran, Savka & Gan, 2017; Vinuesa, Ochoa-Sánchez & Contreras-Moreira, 2018). Given the increasing evidence highlighting the robustness and reliability of the ANI
approach in species delineation, the pairwise ANI between Agrobacterium tumefaciens and
Agrobacterium radiobacter type strains that is at least 2.5% higher than the 95% cutoff
value is rigorous evidence that they belong to the same genomospecies, effectively
serving as the final nail in the coffin for the decade-long debate on their taxonomic status. The amalgamation of Agrobacterium radiobacter and Agrobacterium tumefaciens into
a single species have been repeatedly suggested in the past few years but was complicated
by the special status of Agrobacterium tumefaciens as the type species of the genus
Agrobacterium despite the priority that Agrobacterium radiobacter has over Agrobacterium
tumefaciens as it was isolated and described 3 years before Agrobacterium tumefaciens
(Young et al., 2001, 2003). Despite sharing numerous morphological and biochemical
features, differences in genomic features such as pairwise ANI, phylogenomic clustering
and core accessory gene contents do exist among members in Agrobacterium
genomospecies 4 that can facilitate the identification of genotypic and phenotypic variants
to accurately delimit sub-species relationships in the future (Brenner, Staley & Krieg, 2000;
Jezbera et al., 2011; Meier-Kolthoff et al., 2014; Tan et al., 2013). To date the LPS for both type strains have been determined (De Castro et al., 2002,
2004). In stark contrast to Agrobacterium radiobacter, the Agrobacterium tumefaciens
LPS consists of D-arabinose and L-fucose that have yet been reported to date in another
members of the genus Agrobacterium (De Castro et al., 2002). The presence of the
L configuration of fucose is considered to be rare even among plant pathogenic bacteria but
may be associated with the ability of Agrobacterium tumefaciens to colonize or bind to
wounded plant cell (Lippincott, Whatley & Lippincott, 1977; Whatley et al., 1976;
Whatley & Spiess, 1977). DISCUSSION We re-sequenced the genome of Agrobacterium radiobacter type strain using strain
directly obtained from NCPPB. The assembled Agrobacterium radiobacter genome Gan et al. (2019), PeerJ, DOI 10.7717/peerj.6366 reported in this study exhibits assembly statistics that are consistent with a high-quality
draft genome such as high genome completeness and contiguity, near-zero contamination/
duplication and comparable genome size to other closely related strains (Gan, Lee &
Savka, 2018; Parks et al., 2015). Furthermore, given the improved contiguity and dramatic
reduction in the number of contigs of this newly assembled draft genome, we recommend
using this genome in place of the previously published draft genome for future
Agrobacterium comparative studies. g
p
The distinct separation of Agrobacterium genomospecies 4 and 7 at 95% ANI cutoff
corroborates with the previously established “genomic yardstick” for species
differentiation (Konstantinidis & Tiedje, 2005; Richter & Rosselló-Móra, 2009). Using this
percentage cutoff, the ANI approach has been successfully used to provide a near
“black-and-white” pattern of species separation in even some of the most diverse bacterial
genera such as Pseudomonas, Arcobacter and Stenotrophomonas (Pérez-Cataluña et al.,
2018; Tran, Savka & Gan, 2017; Vinuesa, Ochoa-Sánchez & Contreras-Moreira, 2018). Given the increasing evidence highlighting the robustness and reliability of the ANI
approach in species delineation, the pairwise ANI between Agrobacterium tumefaciens and
Agrobacterium radiobacter type strains that is at least 2.5% higher than the 95% cutoff
value is rigorous evidence that they belong to the same genomospecies, effectively
serving as the final nail in the coffin for the decade-long debate on their taxonomic status. The amalgamation of Agrobacterium radiobacter and Agrobacterium tumefaciens into
a single species have been repeatedly suggested in the past few years but was complicated
by the special status of Agrobacterium tumefaciens as the type species of the genus
Agrobacterium despite the priority that Agrobacterium radiobacter has over Agrobacterium
tumefaciens as it was isolated and described 3 years before Agrobacterium tumefaciens
(Young et al., 2001, 2003). Despite sharing numerous morphological and biochemical
features, differences in genomic features such as pairwise ANI, phylogenomic clustering
and core accessory gene contents do exist among members in Agrobacterium
genomospecies 4 that can facilitate the identification of genotypic and phenotypic variants
to accurately delimit sub-species relationships in the future (Brenner, Staley & Krieg, 2000;
Jezbera et al., 2011; Meier-Kolthoff et al., 2014; Tan et al., 2013). Gan et al. (2019), PeerJ, DOI 10.7717/peerj.6366 DISCUSSION It has been previously shown that the LPS of Agrobacterium
tumefaciens but not Agrobacterium radiobacter can bind to the plant cells thus providing
protection against subsequent infection by pathogenic strains (Whatley et al., 1976). The presence and absence of nucleotide sugars in the O-chain constituent of LPS in both
type strains corroborates with their observed genomic potential in the nucleotide sugar
metabolism pathway thus underscoring the utility of comparative genomics in facilitating Gan et al. (2019), PeerJ, DOI 10.7717/peerj.6366 the prediction of microbial host range and ecological niche (Klosterman et al., 2011). For
example, the absence of L-rhamnose and L-fucose in the LPS of Agrobacterium
tumefaciens B6 and Agrobacterium radiobacter DSM30147, respectively, is consistent with
the lack of genes coding for enzymes involved with the particular nucleotide sugar
metabolism. Generation of Agrobacterium tumefaciens B6 LPS mutant via targeted gene
deletion (Kaczmarczyk, Vorholt & Francez-Charlot, 2012) or the classical but
more laborious transposon mutagenesis approach followed by characterization of the LPS
mutant host-range and phytopathogenicity will be instructive (Gan et al., 2011;
Reuhs et al., 2005). Our current genomic sampling indicates that the Ti plasmid appears to be restricted to
the Agrobacterium tumefaciens subclade. The maintenance of the Ti plasmid is
metabolically taxing given its large size (Barker et al., 1983; Glick, 1995). Even if the
Ti plasmid was conjugally transfer, for example, to Agrobacterium radiobacter, the inability
of Agrobacterium radiobacter to colonize plant host as evidenced by its LPS incompatibility
will not confer an advantage to the new plasmid host in a natural environment
(Thomashow et al., 1980). Furthermore, in the absence of high density Acyl-homoserine
lactone (AHL) signals which is required to trigger Ti plasmid conjugation (Fuqua &
Winans, 1994; Pappas, 2008; Zhang, Wang & Zhang, 2002), the newly acquired Ti plasmid
in Agrobacterium radiobacter may be cured in its natural soil habitat after a few
generations. Although the spontaneous transfer of the Ti plasmid from tumorigenic
Agrobacterium tumefaciens to Agrobacterium radiobacter K84 has been reported
previously, strain K84 was re-classified based on a recent core gene analysis to Rhizobium
rhizogenes K84 (Velázquez et al., 2010; Vicedo et al., 1996), reiterating the pervasive
taxonomic inconsistency within the genus Agrobacterium that may have confound
previous biological interpretations (De Ley et al., 1966; Lindström et al., 1995; Young,
2008). Gan et al. (2019), PeerJ, DOI 10.7717/peerj.6366 Competing Interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. Funding Michael A. Savka and Han Ming Gan received support from the College of Science and the
Thomas H. Gosnell School of Life Sciences at Rochester Institute of Technology. The funders had no role in study design, data collection and analysis, decision to publish,
or preparation of the manuscript. DISCUSSION Given that a large majority of Agrobacterium genetics was performed during the
pre-NGS era (Gan & Savka, 2018), it remains unknown as to how many Agrobacterium
tumefaciens and Agrobacterium radiobacter strains have been molecularly misclassified
due to their high genomic relatedness. The inability to accurately identify plasmid and chromosomal-derived contigs among
the draft genomes means that some of the core accessory genes among tumorigenic strains
may be plasmid-derived and should be treated with caution as the low-copy-number
Ti-plasmid is prone to curing in the absence of AHL signals. Despite the value of complete
genome assembly in enabling the accurate partitioning of plasmid and chromosomal
genomic region (Arredondo-Alonso et al., 2017), the representation of complete
Agrobacterium genomes in current database is still very low as a majority of the genomes
were assembled from short Illumina reads that cannot effectively span repetitive region
(Wibberg et al., 2011; Wood et al., 2001). Furthermore, most Agrobacterium strains
harbor multiple large plasmids that further complicate short-read-only assembly
graph (Kado & Liu, 1981; Lowe et al., 2009; Shao et al., 2018). Given the currently available
genomic resources for Agrobacterium, defining subspecies within the Agrobacterium
genomospecies 4 based on the identification of lineage-specific gene set (Moldovan &
Gelfand, 2018) will be challenging. However, we anticipate that the advent of Gan et al. (2019), PeerJ, DOI 10.7717/peerj.6366 high throughput long-read sequencing that can span large repetitive region in recent years
is likely going to overcome this limitation allowing a more accurate depiction of microbial
pangenome (Gan et al., 2012; Gan, Lee & Austin, 2017; Schmid et al., 2018a, 2018b). Future hybrid genome assemblies (Illumina and Nanopore/PacBio reads) of members
from genomospecies 4 with comprehensive metadata and reliable phenotypic information,
will be instructive. CONCLUSIONS Despite belonging to the same genomospecies, Agrobacterium tumefaciens and
Agrobacterium radiobacter are by no means clonal at the chromosomal level and instead
demonstrate sufficient genomic characters that qualify their separation into two
sub-species. In addition, the difference in the LPS profile among two type strains will
have implications to host specificity leading to geographical separation. In the spirit
of preserving the naming of both species but at the same time respecting the taxonomic
jurisdiction for strain priority, we propose Agrobacterium tumefaciens to be reclassified as
Agrobacterium radiobacter subsp. tumefaciens and for Agrobacterium radiobacter to
retains its species status with the proposed name of Agrobacterium radiobacter
subsp. radiobacter. Grant Disclosures The following grant information was disclosed by the authors:
College of Science. School of Life Sciences at Rochester Institute of Technology. School of Life Sciences at Rochester Institute of Technology. Gan et al. (2019), PeerJ, DOI 10.7717/peerj.6366 Data Availability The following information was supplied regarding data availability: LMVK00000000.1, ASM154131v1: https://www.ncbi.nlm.nih.gov/assembly/GCF_
001541315.1; LMVJ00000000.1, ASM154130v1: https://www.ncbi.nlm.nih.gov/assembly/GCF_
001541305.1; LMVJ00000000.1, ASM154130v1: https://www.ncbi.nlm.nih.gov/assembly/GCF_
001541305.1; Code and data are available at Han Ming Gan. (2018). Dataset for “Improved genome of
Agrobacterium radiobacter type strain provides new taxonomic insight into Agrobacterium
genomospecies 4” [Data set]. Zenodo. DOI 10.5281/zenodo.1489356. Code and data are available at Han Ming Gan. (2018). Dataset for “Improved genome of
Agrobacterium radiobacter type strain provides new taxonomic insight into Agrobacterium
genomospecies 4” [Data set]. Zenodo. DOI 10.5281/zenodo.1489356. Author Contributions Han Ming Gan conceived and designed the experiments, performed the experiments,
analyzed the data, contributed reagents/materials/analysis tools, prepared figures and/or
tables, authored or reviewed drafts of the paper, approved the final draft. Melvin V.L. Lee performed the experiments, analyzed the data, contributed
reagents/materials/analysis tools, prepared figures and/or tables, approved the final
draft. Michael A. Savka conceived and designed the experiments, analyzed the data, authored
or reviewed drafts of the paper, approved the final draft. Gan et al. (2019), PeerJ, DOI 10.7717/peerj.6366 DNA Deposition The following information was supplied regarding the deposition of DNA sequences: Raw sequencing data and whole genome assembly for strains B6 and NCPPB3001
reported in this study are linked to the NCBI Bioproject IDs PRJNA300485 and
PRJNA300611, respectively. Supplemental Information Supplemental information for this article can be found online at http://dx.doi.org/10.7717/
peerj.6366#supplemental-information. Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ Supplemental information for this article can be found online at http://dx.doi.org/10.7717/
peerj.6366#supplemental-information. Supplemental information for this article can be found online at http://dx.doi.org/10.7717/
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Correspondence: Charles Nduhiu Wamucii (charles.wamucii@wur.nl) Received: 15 March 2021 – Discussion started: 29 March 2021
Revised: 13 July 2021 – Accepted: 22 September 2021 – Published: 3 November 2021 Received: 15 March 2021 – Discussion started: 29 March 2021 Received: 15 March 2021 – Discussion started: 29 March 2021
Revised: 13 July 2021 – Accepted: 22 September 2021 – Published: 3 November 2021 Revised: 13 July 2021 – Accepted: 22 September 2021 – Published: 3 November 2021 Revised: 13 July 2021 – Accepted: 22 September 2021 – Published: 3 November 202 Abstract. East African forested mountain regions are vital
in generating and supplying water resources to adjacent arid
and semi-arid lowlands. However, these ecosystems are un-
der pressure from both climate and land use changes. This
study aimed to analyze the effects of climate and land use
changes on water yield using the Budyko framework as a
first-order conceptual framework assuming steady-state for
pristine/protected forested areas. For nine selected forested
water towers in East Africa, the amount and distribution of
water resources and their decadal changes were analyzed. Results show that most areas inside and outside the wa-
ter towers are under pressure from human influences. Wa-
ter yield was more sensitive to climate changes compared
to land use changes within the selected East African water
towers themselves. However, for the surrounding lowlands,
the effects of land use changes had greater impacts on water
yield. We conclude that the East African water towers have
seen a strong shift towards wetter conditions, especially in
the period of 2011–2019, while, at the same time, the po-
tential evapotranspiration is gradually increasing. Given that
most of the water towers were identified as non-resilient to
these changes, future water yield is likely to also experience
more extreme variations. Hydrol. Earth Syst. Sci., 25, 5641–5665, 2021
https://doi.org/10.5194/hess-25-5641-2021
© Author(s) 2021. This work is distributed under
the Creative Commons Attribution 4.0 License. Hydrol. Earth Syst. Sci., 25, 5641–5665, 2021
https://doi.org/10.5194/hess-25-5641-2021
© Author(s) 2021. This work is distributed under
the Creative Commons Attribution 4.0 License. Published by Copernicus Publications on behalf of the European Geosciences Union. Land use and climate change effects on water yield from East
African forested water towers Charles Nduhiu Wamucii1, Pieter R. van Oel2, Arend Ligtenberg3, John Mwangi Gathenya4, and Adriaan J. Teuling1 Charles Nduhiu Wamucii1, Pieter R. van Oel2, Arend Ligtenberg3, John Mwangi Gathenya4, and Adriaan J. Teuling1
1Hydrology and Quantitative Water Management Group Wageningen University & Research harles Nduhiu Wamucii1, Pieter R. van Oel2, Arend Ligtenberg3, John Mwangi Gathenya4, a cii1, Pieter R. van Oel2, Arend Ligtenberg3, John Mwangi Gathenya4, and Adriaan J. Teuling 1Hydrology and Quantitative Water Management Group, Wageningen University & Research,
6700 AA Wageningen, the Netherlands
2 Hydrology and Quantitative Water Management Group, Wageningen University & Research,
700 AA Wageningen the Netherlands g
g
2Water Resources Management Group, Wageningen University & Research, 6700 AA Wageningen, the Netherlands
3Laboratory of Geo-information Science and Remote Sensing, Environmental Sciences, Wageningen University & Research,
6708 PB Wageningen, the Netherlands 4Soil, Water and Environmental Engineering Department, School of Biosystems and Environmental Engineering,
Jomo Kenyatta University of Agriculture and Technology, P.O. Box 62000 – 00200 Nairobi, Kenya Correspondence: Charles Nduhiu Wamucii (charles.wamucii@wur.nl) C. N. Wamucii et al.: Land use and climate change effects on water yield 5642 Understanding historical climate and human-induced land
use changes and their impacts on streamflow can explain
some of the hydrological events experienced in the adjacent
lowlands. This can help inform the role of forested water
towers in observed extremities in the lowlands, such as floods
and hydrological droughts. To our knowledge, there are no
studies that have focused on the East African forested wa-
ter towers and their ability to generate streamflow under a
changing climate and land use. At the regional scale, stud-
ies have either focused on studying forest trends such as de-
forestation (Aleman et al., 2018) or the effects of land use
changes on climate (Otieno and Anyah, 2012). At the river
basin scale, studies have focused on hydrological responses
(Hyandye et al., 2018; Mango et al., 2011; Gabiri et al.,
2020). the generation of streamflow to the drier lowland areas –
an important aspect for arid and semi-arid areas in the East
African region. EAC et al. (2016) defined the key water tow-
ers based on major rivers in East Africa and delineated the
following three major montane forest ecosystems: the Alber-
tine Rift, the Kenyan highlands, and the Ethiopian Highlands. Although research on water towers has focused mainly on
glaciated mountain chains (Immerzeel et al., 2020) where the
temperature is a key factor in determining water flows from
glaciated mountain chains, there is growing awareness that
forested mountains can provide similar services (Viviroli and
Weingartner, 2004). Mountainous areas in Africa cover ap-
proximately 20 % of Africa’s surface area and maintain sig-
nificantly more precipitation than adjacent lowlands (UNEP,
2014; EAC et al., 2016). Mountain forests capture, store, pu-
rify, and release water to lowland areas (UNEP, 2014). The
East African region is one of the most mountainous areas of
Africa, with several peaks above 4500 m, and hosts the three
highest mountains on the continent, namely Mt. Kilimanjaro
(5895 m), Mount Kenya (5119 m), and the Rwenzori Moun-
tains (5109 m; UNEP, 2014). Many of the water towers are of
volcanic origin which can be linked to the extensive plate tec-
tonics of the East African rift system (Dawson, 2008; Scoon,
2020). Guzha et al. (2018), in their review, emphasized the impor-
tance of forests in streamflow generation in the East African
region, with forest degradation leading to increased stream
discharges and surface runoff. Muthoni et al. C. N. Wamucii et al.: Land use and climate change effects on water yield However, the challenges of mod-
eling approaches, e.g., SWAT, is that the underlying pro-
cesses must be explicit, and they require complex and mul-
tiple data inputs and time-consuming calibration and valida-
tion (Zhang et al., 2012). The application of modeling ap-
proaches is, therefore, limited to small watersheds where de-
tailed streamflow observations are available or in watersheds
that are well monitored with extensive, long-term available
data on vegetation, soil, topography, land use, hydrology,
and climate (Wei and Zhang, 2011). Conceptual approaches, Various approaches have been used for studying the ef-
fects of climate and land use changes on streamflow. Jiang
et al. (2015) categorized such methods into the following
two: (a) deterministic rainfall–runoff models and (b) statis-
tical methods. Ma et al. (2014) combined the two categories
by running rainfall statistics and recorded land use change
patterns in reverse order in calibrated process-based mod-
els. Dey and Mishra (2017) reviewed the existing approaches
and categorized these approaches into the following four cat-
egories; (i) experimental approach, e.g., paired catchment
method (Bosch and Hewlett, 1982), (ii) hydrological mod-
eling, e.g., SWAT (Tech, 2019), (iii) conceptual approaches,
e.g., Budyko approach (Budyko, 1974), and (iv) analytical
approaches, e.g., climate elasticity method (Schaake, 1990). The water towers of East Africa are under pressure from
human intensification and climate change (WWF, 2005; Ge-
brehiwot et al., 2014). According to the Intergovernmental
Panel for Climate Change (IPCC) Fifth Assessment Report,
the average annual temperature for Africa has risen by at
least 0.5 ◦C during the last 100 years, and this is predicted to
increase by approximately 3.2 ◦C by 2080. This will dramat-
ically diminish glaciers in East African water towers, whose
surface area has already decreased by 80 % since the 1990s
(EAC et al., 2016), affecting runoff and water resources
downstream. The East African montane forest zones con-
tinue to be lost to agriculture and other anthropogenic uses. This is mainly attributed to a high and increasing popula-
tion density, which is a major driving force of environmental
change in the mountainous areas (UNEP, 2014). Generally, the different approaches can be grouped into
modeling (or distributed models) and conceptual approaches
(Marhaento et al., 2017; Mianabadi et al., 2020). The advan-
tage of modeling approaches is that the results are more reli-
able (Booij et al., 2019). 1
Introduction Many mountainous areas act as water towers by generating
and supplying runoff and streamflow to adjacent lowlands
that would otherwise be much drier. Beyond the supply of
water, the elevated water towers maintain high actual evap-
otranspiration, hence playing a key role in regional rainfall
recycling (WWF, 2005). Water towers have been defined in
various ways. Viviroli et al. (2007) categorized water towers
into four types, namely essential, supportive, occasional, and
limited water towers, based on their contribution to down-
stream discharge. Dewi et al. (2017) introduced the follow-
ing quantification criterion for defining water towers as an
area that satisfies two conditions: (a) an aridity–humidity in-
dex (i.e., a ratio of precipitation to potential evapotranspira-
tion) of above 0.65 and (b) where the aridity–humidity in-
dex relative to the elevation is greater than 2.77. Immerzeel
et al. (2020) introduced a global Water tower Index (WTI),
which ranks water towers in terms of their water-supplying
role and the downstream dependence of ecosystems and so-
ciety. The United Nations Environment Programme (UNEP,
2010) defined water towers as elevated areas of land (gener-
ally at least 200 m above the surrounding area) that receive at
least 750 mm of rainfall and 250 mm of runoff per year and
are significant water sources for populations beyond their im-
mediate delineated boundaries. What is evident in all definitions is that high elevation
and high precipitation are key components that determine ublished by Copernicus Publications on behalf of the European Geosciences Union. C. N. Wamucii et al.: Land use and climate change effects on water yield (2019) fo-
cused on spatiotemporal trends and variability in precipita-
tion within eastern and southern Africa. However, there is
limited information on the partitioning of the available pre-
cipitation into water yield and evapotranspiration from the
forested water towers of the East African region. Given the
semi-arid conditions in the lowlands of the East African re-
gion, there is a need to study the sensitivity of forested water
towers to changes in climate and land use. Montane forest ecosystems in the East African region
are classified as water towers due to their high elevations
and high humidity, thus generating water yield for adjacent
lowland areas (UNEP, 2010). There is a high dependency
on surface water in the East African region (Jacobs et al.,
2018), but rainfall distribution is insufficient in most parts of
the region, with several areas experiencing frequent severe
droughts (Nicholson, 2017). El Tom (1972) tested the relia-
bility of rainfall and showed that, in the dry areas, the rainfall
is highly variable and nearly independent of the mean an-
nual value, affecting rainfed agriculture in the region. Fluc-
tuations in rainfall are evident in both seasonal and decadal
time series mainly in the semi-arid zones (Hulme, 1990). The
forested water towers in the region are, therefore, important
sources of water that sustain environmental and human water
demands in the dry lowland areas. towers to changes in climate and land use. Various approaches have been used for studying the ef-
fects of climate and land use changes on streamflow. Jiang
et al. (2015) categorized such methods into the following
two: (a) deterministic rainfall–runoff models and (b) statis-
tical methods. Ma et al. (2014) combined the two categories
by running rainfall statistics and recorded land use change
patterns in reverse order in calibrated process-based mod-
els. Dey and Mishra (2017) reviewed the existing approaches
and categorized these approaches into the following four cat-
egories; (i) experimental approach, e.g., paired catchment
method (Bosch and Hewlett, 1982), (ii) hydrological mod-
eling, e.g., SWAT (Tech, 2019), (iii) conceptual approaches,
e.g., Budyko approach (Budyko, 1974), and (iv) analytical
approaches, e.g., climate elasticity method (Schaake, 1990). Generally, the different approaches can be grouped into
modeling (or distributed models) and conceptual approaches
(Marhaento et al., 2017; Mianabadi et al., 2020). The advan-
tage of modeling approaches is that the results are more reli-
able (Booij et al., 2019). N. Wamucii et al.: Land use and climate change effects on water yield A steady state is reached when the total input (i.e.,
precipitation) equals the total output (i.e., evapotranspiration
and water yield; Han et al., 2020) and changes in soil water
storage are zero (Donohue et al., 2007). Hence, a simple wa-
ter balance equation assuming steady-state conditions can be
written as follows: In this paper, we aim to use the Budyko framework to an-
alyze the hydroclimatic changes over the past few decades,
focusing on the selected forested water towers of East Africa. The selection of water towers was based on aridity index
(AI), high elevation, and continuous forest block. The se-
lected water towers have AI ≥0.65 (i.e., humid) and are lo-
cated in high elevated areas under a continuous forest block
from the foot slope contour to the peak. The montane forests
are the three major forest ecosystems in East Africa, as de-
fined and delineated by (UNEP, 2010; EAC et al., 2016), and
they include the Albertine Rift, the Kenyan highlands, and
the Ethiopian Highlands. They were defined and delineated
based on major rivers in the region. All the selected water
towers in this study fall into the three forest ecosystems. The Budyko framework was used to answer the following
research question: what are the effects of climate and land
use changes on water yield for the selected forested water
towers? P = ET + Q,
(1) (1) P = ET + Q, where P is precipitation, ET is actual evapotranspiration, and
Q is water yield. The steady-state assumption of the Budyko curve has been
challenged in several studies (Van der Velde et al., 2014;
Donohue et al., 2007; Mianabadi et al., 2020; Du et al.,
2016). Most of these studies have, however, challenged the
steady-state assumption for studies focused on catchments in
the lower slopes at finer temporal or spatial scales and the
unreliability of the Budyko framework to predict future wa-
ter cycles. In the lower slopes, the hydrological processes are
under the influence of anthropogenic changes, e.g., urbaniza-
tion, water abstraction, agricultural intensification, deforesta-
tion, etc. To our knowledge, the steady-state assumption has
not been challenged in studies focusing on the upper slopes
of the elevated forested mountains, where we assume steady-
state assumptions would probably hold due to pristine con-
ditions. Du et al. N. Wamucii et al.: Land use and climate change effects on water yield C. N. Wamucii et al.: Land use and climate change effects on water yield yield (Mianabadi et al., 2020; Teng et al., 2012; Zeng et al.,
2020). The framework is particularly useful in evaluating the
sensitivity of water yield to changes in climate and catch-
ment characteristics (Mianabadi et al., 2020; Liu et al., 2013;
Sankarasubramanian et al., 2001; Sun et al., 2014; Yang et
al., 2014; Zhang et al., 2004; Roderick and Farquhar, 2011;
Creed et al., 2014; Jiang et al., 2015; Xu et al., 2013; Mwangi
et al., 2016). In spite of the criticism on the validity of the
Budyko model outlined before, it is being applied widely and
successfully at scales ranging from coarse global grid resolu-
tion to smaller basins of less than 10 km2 (Zhang et al., 2004;
Redhead et al., 2016; Teuling et al., 2019). such as Budyko frameworks, require fewer data, hence mak-
ing them flexible in their application from small to large
study areas, and they generally give logical primary results
(Marhaento et al., 2017; Booij et al., 2019). These primary
results can be very crucial for data-limited regions such as
East Africa and can form the basis for detailed hydrologi-
cal studies (Teng et al., 2012). In this study, we selected the
Budyko framework, assuming a steady state, to analyze the
impact of land use and climate changes on water yield for the
selected forested water towers of East Africa. such as Budyko frameworks, require fewer data, hence mak-
ing them flexible in their application from small to large
study areas, and they generally give logical primary results
(Marhaento et al., 2017; Booij et al., 2019). These primary
results can be very crucial for data-limited regions such as
East Africa and can form the basis for detailed hydrologi-
cal studies (Teng et al., 2012). In this study, we selected the
Budyko framework, assuming a steady state, to analyze the
impact of land use and climate changes on water yield for the
selected forested water towers of East Africa. The Budyko framework considers both water and energy
constraints in hydrological processes over a long-term pe-
riod. The framework has been applied to quantify or separate
the impacts of climate change and human activities on runoff
(Jiang et al., 2015; Xu et al., 2013; Roderick and Farquhar,
2011). N. Wamucii et al.: Land use and climate change effects on water yield (2016) demonstrated that the basins in the
upper slopes met steady-state conditions, while this was not
the case for downstream basins due to human interference
and water inflows from the upstream basins. In their study,
Van der Velde et al. (2014) demonstrated that the climate and
land use changes do not outpace the ability of the forests to
adapt their water use and energy use strategies to the prevail-
ing conditions. Han et al. (2020) studied 1057 global unim-
paired catchments and showed that over 70 % of the catch-
ments attain a steady-state within 10 years, with co-evolution
between climate and vegetation coverage playing a key role
in maintaining the steady-state conditions. We hypothesize that, in areas considered as being pris-
tine or protected zones (i.e., high elevated forested areas),
with AI ≥0.65, changes in water yield would majorly be
attributed to climate changes and negligibly due to land
use/cover changes. The high elevated forested areas would
then be expected to fall on the theoretical Budyko curve over
the study period. The water yield simulations were evaluated
against observation-based runoff. https://doi.org/10.5194/hess-25-5641-2021 C. N. Wamucii et al.: Land use and climate change effects on water yield However, the challenges of mod-
eling approaches, e.g., SWAT, is that the underlying pro-
cesses must be explicit, and they require complex and mul-
tiple data inputs and time-consuming calibration and valida-
tion (Zhang et al., 2012). The application of modeling ap-
proaches is, therefore, limited to small watersheds where de-
tailed streamflow observations are available or in watersheds
that are well monitored with extensive, long-term available
data on vegetation, soil, topography, land use, hydrology,
and climate (Wei and Zhang, 2011). Conceptual approaches, https://doi.org/10.5194/hess-25-5641-2021 Hydrol. Earth Syst. Sci., 25, 5641–5665, 2021 5643 2
Data and methodology The
foot slope was selected such that there is a continuous forest block from the foot slope contour to the peak. The foot
slope contour was adjusted upwards for two water towers (i.e., Aberdare Range and Bale Mountains) to 2100 and
2600 m a.s.l., respectively, to ensure that we capture the majority of the elevated forested areas presumably under
pristine conditions. of 0.5◦. The CRU-PET is calculated using the Penman–
Monteith formula (Harris et al., 2020; Ekström et al., 2007). Normalized Difference Vegetation Index (NDVI) data to es-
timate land surface characteristics were sourced from the
Global Inventory Monitoring and Modeling System third
generation (GIMMS 3 g) Advanced Very High-Resolution
Radiometer (AVHRR) sensor on board the National Oceanic
and Atmospheric Administration (NOAA) satellites at a spa-
tial resolution of 0.07◦(Pinzon and Tucker, 2014; Tucker et
al., 2005; Kalisa et al., 2019). The NDVI is derived using the
Bayesian methods with high-quality, well-calibrated SeaW-
iFS NDVI data. The resulting NDVI values give an error of
±0.005 NDVI (Pinzon and Tucker, 2014). Figure 1. The East African forest ecosystems and the location of
the selected water towers (adapted from The Nature Conservancy,
2012). The research borrows from the concept of quantifying
the long-term impact of climate and land use changes on
mean annual evapotranspiration and water yield at catchment
scales based on data and parameters that are easily measur-
able at a regional scale (Zhang et al., 2001). Forested catch-
ments generally have higher evapotranspiration than other
land covers such as grassed catchments. Therefore, changes
in land use and forest management have an impact on catch-
ment water balance and, hence, water yield (Zhang et al.,
2001; Teuling and Hoek van Dijke, 2020). Figure 1. The East African forest ecosystems and the location of
the selected water towers (adapted from The Nature Conservancy,
2012). One way of estimating water yield (Q) and actual evapo-
transpiration (ET) is to assume that evapotranspiration from
land surfaces is controlled by water availability and atmo-
spheric demand (Zhang et al., 2001). The water availability
can be approximated by precipitation; the atmospheric de-
mand represents the maximum possible evapotranspiration
and is often considered as being the potential evapotranspi-
ration (PET). Under very dry conditions, PET exceeds pre-
cipitation (P ) and actual evapotranspiration (ET) equals pre-
cipitation. 2
Data and methodology About 60 % of the world’s land surface is con-
sidered an arid area, i.e., P / PET ratio, also known as the
aridity index (AI), of below 0.65 (Convention on Biological
Diversity, 2021). Under very wet conditions, water availabil- 2
Data and methodology We used the Budyko conceptual framework to evaluate the
impacts of land use changes and climate changes on water
yield from the selected forested water towers. The study area
is the East African region. The montane forest ecosystems
are the major forest types in eastern Africa. They range from
the Ethiopian Highlands to Albertine Rift mountains stretch-
ing along the Democratic Republic of Congo (DRC) and bor-
dering Uganda, Rwanda, Burundi, and Tanzania. This study
focused on montane forest ecosystems and their moorlands. The selected water towers are shown in Fig. 1 and summa-
rized in Tables 1 and A2. Generally, the Budyko framework, either in the original
format (i.e., steady state) or in the modified format (i.e., non-
steady-state conditions) is a quick first-order tool for esti-
mating precipitation partitioning into evaporation and water https://doi.org/10.5194/hess-25-5641-2021 Hydrol. Earth Syst. Sci., 25, 5641–5665, 2021 5644
C. N. Wamucii et al.: Land use and climate change effects on water yield
Table 1. The selected water towers of East Africa. Note: a.s.l. – above sea level. Mountain ecosystems
Location
Peak elevation
Foot slope contour
a.s.l. (m)
a.s.l. (m)
Mt. Kilimanjaro
Tanzania
5895
2000
Mt. Kenya
Kenya
5199
2000
Mt. Elgon
Kenya/Uganda
4321
2000
Aberdare Range
Kenya
3999
2100
Rwenzori Mountains
Uganda/DRC
5109
2000
Mt. Meru
Tanzania
4565
2000
Virunga Mountains
DRC/Rwanda/Uganda
4507
2000
Bale Mountains
Ethiopia
4337
2600
Imatong Mountains
South Sudan/Uganda
3187
2000
Note: the selected water towers have an aridity index above 0.65 (i.e., humid) located in high elevated areas. The
foot slope was selected such that there is a continuous forest block from the foot slope contour to the peak. The foot
slope contour was adjusted upwards for two water towers (i.e., Aberdare Range and Bale Mountains) to 2100 and
2600 m a.s.l., respectively, to ensure that we capture the majority of the elevated forested areas presumably under
pristine conditions. C. N. Wamucii et al.: Land use and climate change effects on water yield 5644 C. N. Wamucii et al.: Land use and climate change effects on water yield Table 1. The selected water towers of East Africa. Note: a.s.l. – above sea level. Table 1. The selected water towers of East Africa. Note: a.s.l. – above sea level. Note: the selected water towers have an aridity index above 0.65 (i.e., humid) located in high elevated areas. C. N. Wamucii et al.: Land use and climate change effects on water yield where P , PET, and ET are the precipitation, potential evap-
oration, and actual evapotranspiration. PET / P and ET / P
are termed the dryness index and evapotranspiration ratio,
respectively. The ω parameter is an empirical parameter that
controls how much of the available water will be evaporated
given the available energy. ity exceeds PET, and ET will asymptotically approach the
potential evapotranspiration (Zhang et al., 2001; see Fig. A1
for key assumptions on energy and water limits). The Budyko
Curve provides a “business as usual” reference condition for
the water balance. Assuming that it can depict the expected
partitioning of P into ET and Q, then it is possible to ac-
count for the reasons why some points depart from the base-
line (Creed and Spargo, 2012b). The vertical deviations re-
flect a change in the partitioning of P into ET and Q; hence,
the higher the evaporative index (EI), the lesser the stream-
flow (Q). The horizontal deviations reflect the change in cli-
matic conditions (i.e., temperature and precipitation); thus,
the higher the dryness index (DI), the warmer/drier the con-
ditions. One important feature of the Budyko curve is the
assumption that, under stationary conditions (i.e., naturally
occurring fluctuations due to P and PET), study areas will
fall on the Budyko Curve. However, under non-stationary
conditions (i.e., anthropogenic influence manifested in ET
changes), each catchment will deviate from the Budyko
curve, depending on land cover and physical catchment char-
acteristics (Creed and Spargo, 2012b; Mwangi et al., 2016). This feature can be used to separate land cover change effects
from climate change. The ω parameter is the most difficult parameter to esti-
mate in Budyko framework applications (Bai et al., 2019). It reflects the impact of other factors such as land surface
characteristics and climate seasonality on water and energy
balances (Li et al., 2013). Previous studies have adopted var-
ious ways of estimating the ω parameter. Some studies used
fitted values based on the land use/cover of the areas under
investigation. For instance, Zhang et al. (2012) used values
of 2, 0.5, and 1 to represent ω for the forest, grassland, and
shrubland, respectively. Creed et al. (2014) used ω = 2 in
forested catchments, ω = 0.5 in grassland or cropland catch-
ments, and ω = 1 in mixed cover catchments. C. N. Wamucii et al.: Land use and climate change effects on water yield ω = 2.36M + 1.16,
(3) (3) where M represents the vegetation coverage, which is calcu-
lated based on NDVI indices as follows (Yang et al., 2009): where M represents the vegetation coverage, which is calcu-
lated based on NDVI indices as follows (Yang et al., 2009): M =
NDVI −NDVImin
NDVImax −NDVImin
. (4) C. N. Wamucii et al.: Land use and climate change effects on water yield Other stud-
ies calibrated it based on historical data (Gunkel and Lange,
2017; Redhead et al., 2016; Yang et al., 2014). However, for
data-limited regions, calibration-based estimations are im-
possible, and simpler methods to estimate the ω parameter
based on readily available data are desirable. Land surface
hydrology varies due to variations in different factors such
as vegetation, soil types, topography, and climate seasonality
(Li et al., 2013; Yan et al., 2020). Soil texture and topogra-
phy influence the amount of water available for vegetation;
hence, the vegetation signatures can reflect the underlying
conditions of soil water conditions, topography, seasonality,
etc. Donohue et al. (2007) argued, based on the theory of
ecohydrological equilibrium, that, in water-limited environ-
ments, vegetation is the integrated response to all processes
affecting the availability of water. Therefore, vegetation in-
formation can serve as a good integrated indicator of these
ecohydrological impacts on water and energy balances as it
reflects the integrated landscape and climatic features. Using
data from 26 major global river basins under a wide range
of climate regimes, Li et al. (2013) developed a simple pa-
rameterization for the Budyko ω parameter based solely on
vegetation information as shown in Eq. (3), as follows: In this study, we used the Budyko framework and two
recently introduced Budyko metrics (deviation and elastic-
ity; Creed et al., 2014) to study the changes in the water
yields. Similar methodologies were adopted by Helman et
al. (2017) to determine the resilience of forested catchments
and Sinha et al. (2018) to understand the involvement of an-
thropogenic stress and climatic variance on the partitioning
of precipitation. Based on these studies, catchments can be
assumed to shift predictably along the Budyko curve. This
forms the basis for interpreting the vertical and horizontal de-
viations as a result of changes in climate and anthropogenic
effects. The elasticity is defined as a measure of a catch-
ment’s ability to maintain hydroclimatic conditions as the
climate varies. In contrast to other studies using the Budyko
framework to look at different drivers of change, we used
Budyko-derived data rather than observations. Therefore, the
deviations are thereby constructed and presented as a way
to visualize and interpret the results. Beyond the maps and
graphs presented following the Budyko equation, we further
illustrate the movement of water towers within the Budyko
space. 2.1
Data and analysis Precipitation
(P )
data
were
gathered
from
the
Cli-
mate Hazards Group Infrared Precipitation with Stations
(CHIRPS v2), with a temporal coverage beginning in 1981
and a spatial resolution of 0.05◦. CHIRPS uses the Tropi-
cal Rainfall Measuring Mission Multi-satellite Precipitation
Analysis version 7 (TMPA 3B42-V7) to calibrate global cold
cloud duration (CCD) rainfall estimates (Funk et al., 2015). Potential evapotranspiration (PET) data were sourced from
the Climate Research Unit (CRU) database, with the tem-
poral coverage beginning in 1981 and a spatial resolution https://doi.org/10.5194/hess-25-5641-2021 Hydrol. Earth Syst. Sci., 25, 5641–5665, 2021 5645 2.1.3
Evaluating the effects of land use and climate
changes where 1DI represents a range in DI values, and 1EI repre-
sents a range in EI values observed in the periods of 1991–
2000, 2001–2010, and 2011–2019, using the period of 1981–
1990 as the reference period. In our study, the evaporative in-
dices (EI) for the four periods (i.e., 1981–1990, 1991–2000,
2001–2010, and 2011–2019) were calculated (based on aver-
ages of ET and P for each period). The range in EI (i.e., 1EI)
is the difference between the reference/baseline EI (of 1981–
1990) and succeeding periods of 1991–2000, 2001–2010,
and 2011–2019. The water towers with lower elasticity val-
ues indicate greater ranges in their EI values. The water tow-
ers with higher elasticity values demonstrate fewer ranges in
their EI values (i.e., larger denominator reduces elasticity). where 1DI represents a range in DI values, and 1EI repre-
sents a range in EI values observed in the periods of 1991–
2000, 2001–2010, and 2011–2019, using the period of 1981–
1990 as the reference period. In our study, the evaporative in-
dices (EI) for the four periods (i.e., 1981–1990, 1991–2000,
2001–2010, and 2011–2019) were calculated (based on aver-
ages of ET and P for each period). The range in EI (i.e., 1EI)
is the difference between the reference/baseline EI (of 1981–
1990) and succeeding periods of 1991–2000, 2001–2010,
and 2011–2019. The water towers with lower elasticity val-
ues indicate greater ranges in their EI values. The water tow-
ers with higher elasticity values demonstrate fewer ranges in
their EI values (i.e., larger denominator reduces elasticity). To evaluate the impacts of climate and land use changes,
the sensitivity of climate and land use changes was con-
ducted. The climate and land use values for the years 1981–
1990 were used as the reference conditions in the Budyko
framework. The climatic conditions (i.e., P and PET) for the
years 1981–1990 were held constant in the Budyko frame-
work to evaluate the impacts under changing land use con-
ditions in the succeeding periods of 1991–2000, 2001–2010,
and 2011–2019. Similarly, the land use conditions (i.e., ω pa-
rameters) for the years 1981–1990 were held constant in the
Budyko framework to evaluate the impacts under changing
climatic conditions in the succeeding periods of 1991–2000,
2001–2010, and 2011–2019. 2.1.2
Developing the Budyko curves To develop Budyko curves that are representative of the se-
lected forested water towers, 100 random points were gen-
erated in each of the water towers in ArcGIS. The random
points were used to extract values from raster P, PET, and
ET grids for developing the Budyko curves. For maximum
representation, the minimum allowed distance between the
random points was set to 100 m. The random points gen-
erated were assigned the respective values of PET, ET, and
P using the Extract Multi Values to Points tool in ArcGIS. The evaporative index (EI) values, calculated as a ratio of ET
and P , and dryness index (DI) values, a ratio of PET and
P , were used to draw the Budyko curves. In this study, the
Budyko curve for the 1981–1990 period was used as the ref-
erence condition for the water balance to effectively assess
the trends in the succeeding periods of 1991–1990, 1991–
2000, 2001–2010, and 2011–2019. e = 1DI
1EI ,
(7) e = 1DI
1EI , (7) C. N. Wamucii et al.: Land use and climate change effects on water yield A negative d represents
a downward shift from the Budyko curve and, hence, an in-
crease in Q (Creed and Spargo, 2012a). A positive d repre-
sents an upward shift from the Budyko curve and, hence, a
decrease in Q. Elasticity (e) was defined as being the ratio of
interdecadal variation in dryness index (DI) to interdecadal
variation in the evaporative index (EI), as shown in the fol-
lowing formula: 2.1.1
The Budyko equations (4) Several analytical equations have been proposed for the
Budyko curve. In this study, Fu’s equation was used (Eq. 2)
in a 10-year time step for 1981–2010 and 9 years for 2011–
2019. The equation has been applied in different studies
(Teuling et al., 2019; Li et al., 2013; Zhang et al., 2004). In this study, the M values were calculated for 1985, 1995,
2005, and 2015 and assumed to represent the ω parameters
for the respective periods of 1981–1990, 1991–2000, 2001–
2010, and 2010–2019. The water yield was estimated using the following equa-
tion: ET
P = 1 + PET
P
−
1 +
PET
P
ω1/ω
,
(2) (2) Q = P −ET + 1S,
(5) (5) Q = P −ET + 1S, https://doi.org/10.5194/hess-25-5641-2021 https://doi.org/10.5194/hess-25-5641-2021 Hydrol. Earth Syst. Sci., 25, 5641–5665, 2021 Hydrol. Earth Syst. Sci., 25, 5641–5665, 2021 5646 C. N. Wamucii et al.: Land use and climate change effects on water yield where Q represents the water yield (parameter to be es-
timated), P is precipitation (input from data sets), ET is
the simulated actual evapotranspiration (input from Budyko
equation), and 1S is the water storage term. Over a long pe-
riod (i.e., 5–10 years), it is reasonable to assume that changes
in soil water storage are zero under stationary climate condi-
tions (Teuling et al., 2019; Creed et al., 2014; Zhang et al.,
2001). Therefore, 1S was assumed to be zero in our study. 2012b, a). The deviation (d) and elasticity (e) are two indices
used to describe the potential departure from the theoretical
Budyko curve of a catchment’s DI and EI points with time
(Creed et al., 2014). The deviation (d) was defined as ver-
tical deviation from the Budyko curve calculated using the
following formula: d = EISim −EIBud,
(6) (6) where EISim represents EI simulated for periods in 1991–
2000, 2001–2010, and 2011–2019, and EIBud represents the
predicted theoretical Budyko value for the reference period
of 1981–1990. The EIBud therefore represents the theoretical
value (i.e., point on the Budyko curve where the water tower
was expected to fall at a particular period). The EISim repre-
sents the point where the water tower plotted in that period. The difference between the expected/reference point (EIBud)
and the actual point (EISim) was then calculated to give the
deviation (d) from the Budyko curve. A negative d represents
a downward shift from the Budyko curve and, hence, an in-
crease in Q (Creed and Spargo, 2012a). A positive d repre-
sents an upward shift from the Budyko curve and, hence, a
decrease in Q. Elasticity (e) was defined as being the ratio of
interdecadal variation in dryness index (DI) to interdecadal
variation in the evaporative index (EI), as shown in the fol-
lowing formula: where EISim represents EI simulated for periods in 1991–
2000, 2001–2010, and 2011–2019, and EIBud represents the
predicted theoretical Budyko value for the reference period
of 1981–1990. The EIBud therefore represents the theoretical
value (i.e., point on the Budyko curve where the water tower
was expected to fall at a particular period). The EISim repre-
sents the point where the water tower plotted in that period. The difference between the expected/reference point (EIBud)
and the actual point (EISim) was then calculated to give the
deviation (d) from the Budyko curve. 3.3
Simulation of actual evapotranspiration The long-term actual evapotranspiration (ET) assessment re-
vealed longitudinal differences in the spatial distribution. The
water towers towards the west were observed to be located in
regions with higher ET (examples are Mt. Elgon and the Ima-
tong, Rwenzori, and Virunga mountains). The water towers
towards the east are located in regions with relatively lower
ET (examples are Mt. Meru, Mt. Kilimanjaro, the Aberdare
Range, and Mt. Kenya; Figs. 4a and 5a, b, c, d). The changes
in ET in the region were analyzed using the 1981–1990 pe-
riod as the baseline (Fig. 4a). Decreases in ET were observed
in the southeastern parts of Ethiopia, along a north–south
gradient in Kenya, central Tanzania, and the western side of
the DRC and Burundi region, as shown in Fig. 4b, c, and
d. Increases in ET were observed in northern parts of eastern
Africa (i.e., Sudan, South Sudan, Djibouti, northern Somalia,
the Kenyan–Somali border, and parts of northwestern Kenya
bordering Uganda and South Sudan (Fig. 4b, c, and d). Changes in precipitation, relative to the 1981–1990 pe-
riod, showed a longitudinal gradient. Negative changes in
rainfall were observed in the water towers located towards
the eastern side, except for the Virunga Mountains. Positive
trends were observed in the water towers located towards the
western side with exception of the Aberdare Range. Mt. Kili-
manjaro experienced a strong mean annual rainfall reduction,
with an average annual reduction of 13.5 % and 12 % ob-
served in 2001–2010 and 2011–2019, respectively (Fig. 2e). Conversely, a steady increase in mean annual rainfall was ob-
served in Mt. Elgon, with an average increase of over 20 %
recorded in the years 2011–2019 (Fig. 2e). y
g
Long-term assessment of atmospheric demand (PET)
showed areas with relatively higher mean annual PET to co-
incide with areas of low rainfall and vice versa (Fig. 2b). Generally, atmospheric demand continued to increase over
time in all the water towers, with a peak observed in 2001–
2010 (Figs. 2d and A3). The Imatong Mountains water tower
had the highest atmospheric demand, with an average long-
term mean of approximately 1500 mm yr−1, followed closely
by Mt. Elgon, with an average long-term mean of approx-
imately 1400 mm yr−1. 3.2
Land cover characteristics over the period
1981–2019 Higher values for the Budyko parameter (ω) were derived in
the western part of the DRC and Uganda, the Ethiopian High-
lands and along the coastline of Tanzania, Kenya, and So-
malia (Fig. 3a). The land surface characteristics (ω) ranged
between 2.4 and 3.1 in the different water towers, with the
exception of the Bale Mountains where a drop to 2.3 was ob-
served in 2015 (Fig. 3b). Using 1985 as the reference period
for the land cover characteristics, different patterns of neg-
ative changes and positive changes were observed. A major
drop was observed in Mt. Meru and Mt. Kilimanjaro, while
Mt. Elgon and the Imatong Mountains maintained a positive
change (Fig. 3c). C. N. Wamucii et al.: Land use and climate change effects on water yield 1.0 %, and 0.8 % in 1991–2000, 2001–2010, and 2011–2019,
respectively, as shown in Fig. 2f. 1.0 %, and 0.8 % in 1991–2000, 2001–2010, and 2011–2019,
respectively, as shown in Fig. 2f. in ArcGIS. For maximum representation, the minimum al-
lowed distance between the random points was set to 100 m. The selected random points and their respective values of
simulated streamflow and composite runoff were compared. 3.1
Climate characteristics over the period 1981–2019
(precipitation and potential evapotranspiration) Higher
long-term
mean
annual
rainfall
of
above
1000 mm yr−1 was generally observed in the mountain-
ous forest ecosystems located in the western region and the
Ethiopian Highlands in the north of East Africa (Fig. 2a). The mountainous forest ecosystems are important rain-
fall regions in drier environments, as represented by Mt. Kilimanjaro (average 1800 mm yr−1), Mt. Meru (average
1200 mm yr−1), Mt. Kenya (average 1400 mm yr−1), and
the Aberdare Range (average 1200 mm yr−1) as shown
in Fig. 2a and c. The 10-year moving averages analysis
revealed patterns of increasing and decreasing trends in
precipitation in the different water towers (Fig. A2). 2.1.4
Comparison of simulated streamflow with
observation-based runoff Deviations from the Budyko curves were also investigated
to give a further illustration of the movement of water tow-
ers in the Budyko space. The x axis (horizontal deviations)
in the Budyko space illustrates the aridity indices and the
y axis (vertical deviations) characterizes the evaporative in-
dices (Heidari et al., 2021; Creed et al., 2014). Vertical devi-
ations from the Budyko curve indicate anthropogenic effects
which result in increases or decreases in water yield (Creed
and Spargo, 2012b, a). Horizontal deviations reflect a shift
to warmer or humid conditions, mainly due to resultant vari-
ations in temperature and precipitation (Creed and Spargo, The simulated streamflow of the water towers was compared
with composite runoff data downloaded from the Global
Runoff Data Centre (GRDC). The composite runoff fields,
developed through combining observed river discharge in-
formation with a climate-driven water balance model, pro-
vide the best estimate of terrestrial runoff over large do-
mains (Fekete et al., 2002). A total of 312 points above
2000 m a.s.l. (above sea level), which is the focus of this
study (i.e., elevated water towers), were randomly generated https://doi.org/10.5194/hess-25-5641-2021 Hydrol. Earth Syst. Sci., 25, 5641–5665, 2021 5647 https://doi.org/10.5194/hess-25-5641-2021 3.3
Simulation of actual evapotranspiration The water towers located towards
the western side exhibited lower atmospheric demand (ex-
amples are the Virunga Mountains – long-term mean of
990 mm yr−1 – and Rwenzori Mountains – long-term mean
of 1100 mm yr−1). g
g
g
Despite the longitudinal differences in the region, the in-
dividual water towers recorded varied ET values. Higher ET
values were simulated around the Imatong Mountains, with
a long-term mean of 1107 mm yr−1, Mt. Elgon, with a long-
term mean of 1097 mm yr−1,, and Mt. Kilimanjaro, with a
long-term mean of 1012 mm yr−1. The lowest ET values
were observed in the Bale Mountains, with a long-term mean
of 747 mm yr−1 (Fig. 5j). Using the 1981–1990 period as the
reference period, Mt. Elgon recorded a steady increase in an-
nual mean ET, with an average increase of the order of 10 %
between 2011 and 2019. Pronounced decreases in ET were
observed in the Mt. Kilimanjaro and Mt. Meru water towers
(Fig. 5k), consistent with the decreasing trend in precipita-
tion. All water towers experienced increases in the annual atmo-
spheric demand relative to the 1981–1990 period (Fig. 2f.)
The Bale Mountains saw a sharp increase in atmospheric de-
mand of approximately 6 % in 2001–2010 and 2011–2019. A minimal increase in atmospheric demand was observed at
Mt. Kilimanjaro, with an average annual increase of 0.1 %, Hydrol. Earth Syst. Sci., 25, 5641–5665, 2021 Hydrol. Earth Syst. Sci., 25, 5641–5665, 2021 3.4
Simulation of water yield Using the period 1981–1990 as the reference point, pos-
itive and negative changes in water yield were observed at
the different water towers. There was a consistent increase in
annual mean water yield at the Mt. Elgon water tower, with
increases of 11.4 % and 42.9 % recorded in 2001–2010 and
2011–2019, respectively (Fig. 7k). There was a decrease in
water yield in the Aberdare Range and Mt. Meru water tow-
ers during the 1991–2000 period, after which an increase in
annual mean water yield was recorded in the later years. A
consistent decline was observed at Mt. Kilimanjaro and the
Virunga Mountains during the study period. Similar to what was observed for ET, the long-term water
yield (Q) assessment also showed that the water towers lo-
cated towards the east are surrounded by regions with low
water yield potential below 200 mm yr−1 (examples are Mt. Meru, Mt. Kilimanjaro, the Aberdare Range, and Mt. Kenya;
Figs. 6a and 7a, b, c, d). Using the period 1981–1991 as the
baseline, major increases in Q were observed in areas of Su-
dan and the Kenyan–Somali border as shown in Fig. 6b, c,
and d. The elevation plays a key important role in influencing hy-
droclimatic conditions in the East African region. The aver-
age atmospheric demand increases with a reducing elevation
gradient. A steady increase in P , ET, and Q was observed as
elevation increases. For regions above 2000 m a.s.l, the pre-
cipitation exceeds potential evapotranspiration as shown in
Fig. A4. This demonstrates the importance of the elevated
humid zones in generating and sustaining water yield to the
adjacent lowland areas in the region. Despite longitudinal differences, the higher mean annual
water yield was observed at the Mt. Kilimanjaro water tower
located on the eastern side, with the long-term annual mean
of 794 mm yr−1, followed by two water towers located on the
western side (i.e., Virunga Mountains, with a long-term an-
nual mean of 676 mm yr−1, and Rwenzori Mountains, with a
long-term annual mean of 650 mm yr−1). The lowest annual
mean water yield was observed in the Bale Mountains, with a
long-term annual mean of 315 mm yr−1 (Fig. 7j). Conversion
of water yield units from millimeters per year to cubic me-
ters per second per unit area revealed that Mt. Kilimanjaro
and Mt. https://doi.org/10.5194/hess-25-5641-2021 https://doi.org/10.5194/hess-25-5641-2021 C. N. Wamucii et al.: Land use and climate change effects on water yield 5648 2. Long-term mean annual rainfall and potential evapotranspiration. The left panels show the precipitation and the observed chan
period 1981–2019 (a, c, e). The right panels show potential evapotranspiration and the observed changes for the period 1981–2019 Figure 2. Long-term mean annual rainfall and potential evapotranspiration. The left panels show the precipitation and the observed changes
for the period 1981–2019 (a, c, e). The right panels show potential evapotranspiration and the observed changes for the period 1981–2019 (b,
d, f). rth Syst. Sci., 25, 5641–5665, 2021
https://doi.org/10.5194/hess Hydrol. Earth Syst. Sci., 25, 5641–5665, 2021 https://doi.org/10.5194/hess-25-5641-2021 C. N. Wamucii et al.: Land use and climate change effects on water yield C. N. Wamucii et al.: Land use and climate change effects on water yield 5649 Figure 3. The land surface characteristics. Spatial distribution of the Budyko parameter (ω) for 1985 to 2015 (a) and the changes observed
between 1985 and 2015 (b) and (c) are shown. Figure 3. The land surface characteristics. Spatial distribution of the Budyko parameter (ω) for 1985 to 2015 (a) and the changes observed
between 1985 and 2015 (b) and (c) are shown. Figure 3. The land surface characteristics. Spatial distribution of the Budyko parameter (ω) for 1985 to 2015 (a) and the changes observed
between 1985 and 2015 (b) and (c) are shown. 3.4
Simulation of water yield Kenya are important sources of water in the drier
parts of the East African region (Table A1). Hydrol. Earth Syst. Sci., 25, 5641–5665, 2021 https://doi.org/10.5194/hess-25-5641-2021 C. N. Wamucii et al.: Land use and climate change effects on water yield 5650 Figure 4. Distribution of simulated actual evapotranspiration in East Africa. (a) ET for 1981–1990, (b) ET changes in 1991–2000, (c) ET
changes in 2001–2010, and (d) ET changes in 2011–2019. Figure 4. Distribution of simulated actual evapotranspiration in East Africa. (a) ET for 1981–1990, (b) ET changes in 1991–2000, (c) ET
changes in 2001–2010, and (d) ET changes in 2011–2019. 3.5
Comparison of simulated streamflow with existing
runoff data ers revealed that the effects of land use changes have greater
impacts on water yield outside the water towers boundaries
(Fig. 9). An example is on the eastern side of Mt. Elgon,
where there was a major reduction in water yield, especially
in the periods of 2001–2010 and 2011–2019 (Fig. 9; see the
third row in column B). Climate changes showed a reduction
in water yield in seven water towers in the periods of 1991–
2000 and 2001–2010. However, in the years 2011–2019, cli-
mate changes triggered increased water yield in seven water
towers (Fig. 10). The climate changes in Mt. Elgon resulted
in a consistent increase in water yield, while a consistent de-
crease was inferred for Mt. Kilimanjaro. The simulated streamflow was compared with composite
runoff data downloaded from the GRDC (Fekete et al., 2002). The spatial pattern of the simulated streamflow closely re-
sembles the pattern produced by GRDC composite runoff, as
shown in Fig. 8a and b. A total of 312 points above 2000 m
above sea level, the focus of this study (i.e., elevated wa-
ter towers), were randomly selected, and their respective val-
ues of simulated streamflow and composite runoff were com-
pared. The Kling–Gupta efficiency test revealed positive val-
ues (KGE = 0.33), as shown in Fig. 8c. 3.6
The effects of land use and climate changes on
water yield The analysis of vertical deviations revealed downward and
upward shifts from the Budyko curve in the different wa-
ter towers. The vertical deviations (d) ranged from negative
(−0.05) to positive (+0.02; Fig. 11a). There were no vertical
deviations observed in Mt. Elgon and the Imatong Moun-
tains, indicating that the values observed (between 1991 and The water yield was observed to be relatively more sensitive
to climate changes (i.e., P and PET) than land use changes
within the selected East African water towers. However, a
closer look at the regions surrounding the selected water tow- https://doi.org/10.5194/hess-25-5641-2021 Hydrol. Earth Syst. Sci., 25, 5641–5665, 2021 C. N. Wamucii et al.: Land use and climate change effects on water yield C. N. Wamucii et al.: Land use and climate change effects on water yield 5651 Figure 5. Distribution of long-term actual evapotranspiration. The spatial distribution of long-term ET in and around the water towers (a–i)
and the changes in the different water towers (j, k) are shown. Figure 5. Distribution of long-term actual evapotranspiration. The spatial distribution of long-term ET in and around the water towers (a–i)
and the changes in the different water towers (j, k) are shown. 4
Discussion 2019) were approximately close to those predicted by the ref-
erence Budyko curve. The elasticity (e) values ranged from
0.49 to 17.6, with most of the water towers recording lower
elasticity values as shown in Fig. 11b. The higher elastic-
ity (e) values were observed at the Mt. Kenya water tower in
1991–2000, the Bale Mountains in 2011–2019, and the Ab-
erdare Range in 1991–2000 and 2001–2010. We found that, within the water towers, water yield was more
sensitive to climate changes than to land use changes. In
contrast, outside the water towers, the water yield was ob-
served to be more sensitive to land use changes than to cli-
mate changes. This suggests that anthropogenic influences
are relatively higher outside the water towers. Contrary to our
expectation, our analysis showed that most of the water tow-
ers (i.e., seven of nine) did not plot on the reference Budyko
curve over the study period. This is a relevant finding, since
all water towers were considered pristine and protected. Only
two water towers, Mt. Elgon and the Imatong Mountains,
showed no deviations from the reference Budyko curve. Gen-
erally, our investigation highlights the importance of elevated
water towers in a semi-arid region in the generation and sup-
ply of water to adjacent lowland areas. The forested water The horizontal shifts, either to the left or to the right rel-
ative to the dryness index (DI), were observed in the water
towers. Figure A5 demonstrates that seven of the nine water
towers plotted in the left (i.e., DI values less than 1; towards
humid conditions). However, two of the water towers (i.e.,
Mt. Meru and the Bale Mountains) plotted more towards the
right (i.e., DI values greater than 1). Mt. Meru seems to have
shifted from warmer to humid conditions in the period of
2011–2019, as shown in Fig. A5. https://doi.org/10.5194/hess-25-5641-2021 https://doi.org/10.5194/hess-25-5641-2021 Hydrol. Earth Syst. Sci., 25, 5641–5665, 2021 C. N. Wamucii et al.: Land use and climate change effects on water yiel C. N. Wamucii et al.: Land use and climate change effects on water yield 5652 Figure 6. Spatial distribution of simulated water yield. (a) Q is for 1981–1990, (b) Q is for changes in 1991–2000, (c) Q is for changes in
2001–2010, and (d) Q is for changes in 2011–2019. Figure 6. Spatial distribution of simulated water yield. (a) Q is for 1981–1990, (b) Q is for changes in 1991–2000, (c) Q is for changes in
2001–2010, and (d) Q is for changes in 2011–2019. Figure 6. Spatial distribution of simulated water yield. (a) Q is for 1981–1990, (b) Q is for changes in 1991–2000, (c) Q is for changes in
2001–2010, and (d) Q is for changes in 2011–2019. towers located in drier environments (such as Mt. Kiliman-
jaro, Mt. Meru, Mt. Kenya, and the Aberdare Range) are im-
portant rainfall regions as they receive relatively higher rain-
fall than the adjacent areas. This ensures water availability
in the adjacent lowlands in the arid and semi-arid (ASAL)
regions. out. Our analysis revealed vertical deviations (d) from the
Budyko curve for seven of nine forested water towers. Ac-
cording to Creed et al. (2014), these vertical deviations may
indicate the presence of anthropogenic effects within the wa-
ter towers. The two water towers where no deviations were
observed (i.e., Mt. Elgon and the Imatong Mountains) indi-
cate that the hydroclimatic conditions in the study period did
not vary much from the reference conditions of 1981–1990,
and any changes in water yield in the two water towers can
largely be associated with climatic changes in P and PET. Our results indicate that changes in precipitation and po-
tential evapotranspiration are currently the major determi-
nants of water availability from high elevated forested wa-
ter towers in the East African region. Related observations
have been made in the region, where climate changes in
Africa have been reported to have a higher impact on wa-
ter yield compared to other drivers such as land use changes
(Alcamo et al., 2007; Niang et al., 2014). However, the lack
of evidence of sensitivity to land use changes within the wa-
ter towers themselves may be linked to existing institutional
arrangements. https://doi.org/10.5194/hess-25-5641-2021 We presume that the results would be differ-
ent if such rules would be relaxed. That said, the movement
of water towers in the Budyko space revealed that anthro-
pogenic influence within the water towers cannot be ruled Moreover, the lack of deviations in the two water towers
may indicate the resilience of forested regions (i.e., adapt-
able nature of forests), as described in Van der Velde et
al. (2014), Creed et al. (2014), and Helman et al. (2017). Such resilience (measured as elasticity) could be a key fac-
tor in forested water towers, indicating their ability to re-
sist change or bounce back to their initial natural conditions,
hence plotting along the reference Budyko curve. Long-term
adaptations of forests have been achieved by trees even in
the most water-limited forests (Helman et al., 2017). How- https://doi.org/10.5194/hess-25-5641-2021 Hydrol. Earth Syst. Sci., 25, 5641–5665, 2021 C. N. Wamucii et al.: Land use and climate change effects on water yield
5 C. N. Wamucii et al.: Land use and climate change effects on water yield
5653
Figure 7. Distribution of long-term water yield (Q). The spatial distribution in and around the water towers (a–i) and the changes in the
different water towers (j, k) are shown. C. N. Wamucii et al.: Land use and climate change effects on water yield 5653 Figure 7. Distribution of long-term water yield (Q). The spatial distribution in and around the water towers (a–i) and the changes in the
different water towers (j, k) are shown. Further illustrations can be shown in the Budyko space
based on the horizontal shifts relative to the dryness index
(DI). The horizontal shifts are important indicators of the be-
havior of the water towers towards warmer or humid condi-
tions. These horizontal deviations reflect a change in the cli-
matic conditions, specifically temperature and precipitation
(Creed and Spargo, 2012a). This study observed that many of
the water towers (seven of nine) were plotted within humid
conditions (i.e., DI < 1). On the other hand, two of the water
towers (i.e., Mt. Meru and the Bale Mountains) demonstrated
warmer conditions (i.e., DI > 1). https://doi.org/10.5194/hess-25-5641-2021 One major observation is
that water towers in eastern Africa seem to shift towards the
left in the Budyko space, an indication of the increased hu- ever, our investigations on elasticity (which refers to the de-
gree of initial change using 1981–1990 as the reference pe-
riod) did not support the above science as lower elasticity
values were observed in most of the water towers. Given that
low elasticity indicates broad ranges in the evaporative in-
dex (EI) compared to the dryness index (DI), this may fur-
ther indicate the presence of anthropogenic influence within
the water towers. According to Creed et al. (2014), elastic
catchments are expected to plot along the Budyko curve (i.e.,
high elasticity = resilient to climate changes), while inelas-
tic catchments (i.e., low elasticity = non-resilience to climate
changes) would deviate from the Budyko curve. ever, our investigations on elasticity (which refers to the de-
gree of initial change using 1981–1990 as the reference pe-
riod) did not support the above science as lower elasticity
values were observed in most of the water towers. Given that
low elasticity indicates broad ranges in the evaporative in-
dex (EI) compared to the dryness index (DI), this may fur-
ther indicate the presence of anthropogenic influence within
the water towers. According to Creed et al. (2014), elastic
catchments are expected to plot along the Budyko curve (i.e.,
high elasticity = resilient to climate changes), while inelas-
tic catchments (i.e., low elasticity = non-resilience to climate
changes) would deviate from the Budyko curve. https://doi.org/10.5194/hess-25-5641-2021 Hydrol. Earth Syst. Sci., 25, 5641–5665, 2021 C. N. Wamucii et al.: Land use and climate change effects on water yield 5654 Figure 8. Comparison of simulated streamflow and observation-based runoff. (a) Simulated streamflow, (b) observation-based composite
runoff, and (c) Kling–Gupta efficiency calculation are shown. Figure 8. Comparison of simulated streamflow and observation-based runoff. (a) Simulated streamflow, (b) observation-based composite
runoff, and (c) Kling–Gupta efficiency calculation are shown. mid conditions, especially in the period of 2011–2019. At
the same time, a gradual increase in PET was observed in all
the water towers. A climate shift to wetter conditions and si-
multaneous increases in regional temperatures have also been
reported in the East African region and projected to increase
by the end of the 21st century (Niang et al., 2014; Giannini
et al., 2018; Omambia et al., 2012). https://doi.org/10.5194/hess-25-5641-2021 incide with the recent reports on the rising lake levels phe-
nomenon in the eastern Africa region (Patel, 2020, 2021;
Wambua, 2020; Chebet, 2020; Chepkoech, 2020). We, how-
ever, do not believe we have the results to link the climatic
shifts and swelling of lakes to ENSO variations in our study
which requires further scientific investigations. The simulated actual evapotranspiration (ET) and water
yield (Q) revealed longitudinal differences with low to high
values ranging from east to west. A related pattern on cli-
mate varying across East Africa from arid conditions in the
east to more humid conditions in the west was also observed
by Daron (2014). However, the individual water towers re-
vealed independent variations that do not follow the longitu-
dinal pattern. For instance, a higher mean annual water yield
was observed at Mt. Kilimanjaro despite being in a drier en-
vironment on the eastern side. This emphasizes the impor-
tance of elevated forested areas in ensuring water availability
in semi-arid areas. This study revealed that in high elevated
forested zones, the precipitation exceeds potential evapotran-
spiration, which ensures a surplus of water that eventually
flows downstream. The effects of increasing temperatures have already been
identified to have decreased the surface area of glaciers by
80 % in East African water towers (EAC et al., 2016), af-
fecting runoff and water resources downstream. According to
Niang et al. (2014), the temperatures in Africa are projected
to rise faster than other parts of the world, which could ex-
ceed 2 ◦C by the mid-21st century and 4 ◦C by the end of the
21st century. Therefore, the water towers are under pressure
from climate changes and PET is proving to be an impor-
tant climate driver influencing water availability in the re-
gion. There are chances that the shifts to wetter conditions
in the water towers may also be as a result of the extended
impact of increasing PET on the El Niño–Southern Oscil-
lation (ENSO), a phenomenon that influences precipitation
in the East African region. Li et al. (2016) investigated an-
nual flood frequencies, from 1990 to 2014, and observed up-
ward trends that were linked to the ENSO phenomenon in
Africa. Furthermore, the shifts to wetter conditions also co- The extreme opposite temporal trends observed in water
yields from the different water towers confirm a strong vari-
ation in the regional climatic patterns. https://doi.org/10.5194/hess-25-5641-2021 Figure 9. Effects of land use and climate change on water yield (Q). C. N. Wamucii et al.: Land use and climate change effects on water yield 5656 Figure 10. Relative change in water yield for East African water towers. (a) 1991–2000, (b) 2001–2010, (c) 2011–2019. Figure 11. Illustration of the water towers in the Budyko space. (a) The deviation from the Budyko curve and (b) elasticity are shown. Figure 10. Relative change in water yield for East African water towers. (a) 1991–2000, (b) 2001–2010, (c) 2011–2019. Figure 10. Relative change in water yield for East African water towers. (a) 1991–2000, (b) 2001–2010, (c) 2011–2019. Figure 11. Illustration of the water towers in the Budyko space. (a) The deviation from the Budyko curve and (b) elasticity are shown. f the water towers in the Budyko space. (a) The deviation from the Budyko curve and (b) elasticity are shown. Figure 11. Illustration of the water towers in the Budyko space. (a) The deviation from the Budyko curve an Mt. Elgon, the opposite was true at Mt. Kilimanjaro, where a
steady decline in water yield was observed. Our results fur-
ther revealed that precipitation (P ) is the dominant driver in
the East African region. For instance, a consistent increase
in Q at Mt. Elgon coincided with a steady increase in land
surface characteristics (ω), as shown in Fig. 3c. Ideally, a
decrease in Q would have occurred due to the increases in
ET (associated with increases in land surface characteristics),
but this was diffused by the increases in rainfall, as shown in
Fig. 2c. At the Mt. Kilimanjaro water tower, a continuous de-
crease in Q coincided with a steady reduction in ω. Again,
an increase in Q would have been expected due to decreases
in ET. Therefore, precipitation is the dominant driver in the
generation and supply of water from the forested water tow-
ers in the East African region. timate water yield in drier regions. Moreover, other factors,
such as interception, soil type, topography, seasonality, wa-
ter storage, etc., were not accounted for in the quantitative
framework, which can affect the simulations in the selected
forested water towers. Canopy interception, for instance, plays an important role
in the water balance of forested ecosystems as noted in sev-
eral studies (Teuling et al., 2019; Gash et al., 1980; Zimmer-
mann et al., 1999; Astuti and Suryatmojo, 2019). In their
study, Teuling et al. (2019) found many forested points to
have average yearly observed evapotranspiration (ET) that
exceeds the average potential evapotranspiration (PET). Van
Dijk et al. https://doi.org/10.5194/hess-25-5641-2021 For instance, while
there was a consistent increase in annual mean water yield at https://doi.org/10.5194/hess-25-5641-2021 Hydrol. Earth Syst. Sci., 25, 5641–5665, 2021 C. N. Wamucii et al.: Land use and climate change effects on water yield 5655 Figure 9. Effects of land use and climate change on water yield (Q). Hydrol. Earth Syst. Sci., 25, 5641–5665, 2021 https://doi.org/10.5194/hess-25-5641-2021 C. N. Wamucii et al.: Land use and climate change effects on water yield C. N. Wamucii et al.: Land use and climate change effects on water yield For instance,
the CRU data set is fairly coarse and contains rather few ob-
servations in Africa. One substantial weakness of the cur-
rent CHIRPS algorithm is the lack of uncertainty informa-
tion provided by the inverse distance weighting algorithm
used to blend the CHIRP data and station data (Funk et al.,
2015). The overall NDVI 3g uncertainty comes from spa-
tial and temporal coherence variability, which gives approxi-
mately an error of ±0.002 NDVI units. However, this NDVI
error is considered low uncertainty and, hence, applicable for
studying seasonal and interannual non-stationary phenomena
(Pinzon and Tucker, 2014). Uncertainties may also arise in
the general assumption that estimation of land surface char-
acteristics (ω) based on NDVI formulation provides values
that represent integrated conditions for soil, topography, and
climate seasonality. Some studies using various hydrologi-
cal approaches have reported the significance of these fac-
tors in influencing catchment hydrology (Kirkby et al., 2002;
Woods, 2002; Western et al., 2004; Troch et al., 2013). There
is a need for more research to come up with methodologi-
cal consistency in estimating ω parameters when using the
Budyko framework. Although the focus of the study was in
the elevated forested areas, an empirical adjustment of the
Budyko framework may be needed to capture special fea-
tures, such as desert wadis, in the application of the Budyko
equation in the lowland areas. The Budyko framework provides primary results that can
inform detailed hydrological assessments. For instance, our
findings show that elevated forested water towers are impor-
tant areas for maintaining high ET in the region. This finding
can be explored further by studying the role of water tow-
ers in the supply of rainfall water in the region (i.e., the role
of water towers in regional rainfall/moisture recycling; Elli-
son et al., 2017; Keys et al., 2014), including the effect of
mountain rain shadows on water yield (Van den Hende et
al., 2021). The major reference period for this study was the
1981–1990 period, based on the CHIRPS rainfall data set,
with data beginning from 1981 onwards. We believe the re-
sults would be different if an older reference period was used,
e.g., 100 years ago (presumably actual pristine conditions). This would help to strengthen the findings of this study, espe-
cially after the evidence of climatic shifts towards wetter con-
ditions in all the water towers. C. N. Wamucii et al.: Land use and climate change effects on water yield Yuan et al., 2017). However, detailed investigations are rec-
ommended within the East African region. Other factors that
may affect our results include the human alteration to wa-
ter usage. Kiteme et al. (2008) reported unregulated abstrac-
tion of water upstream of Mt. Kenya water tower, leading
to hydrological droughts downstream. An intensification of
irrigated agriculture and a growing human population was
reported at the foot slopes of the water towers (Ulrich et
al., 2012; Liniger et al., 2005). The effects of anthropogenic
presence at the foot slope of the water towers were not ac-
counted for, and further studies are needed to understand how
humans living at the foot slope of protected water towers af-
fect the pristine conditions of the water towers at high ele-
vations. Notwithstanding these limitations, our study offers
important findings on the sensitivity of water yield to climate
and land use changes and the importance of these water tow-
ers in the generation and supply of water to adjacent lowland
areas. These results can be used by decision-makers, policy-
makers, stakeholders, and scientists to emphasize the need
to protect and conserve the high elevated forested areas in
the region, particularly forest ecosystems above 2000 m a.s.l. where there is a surplus of water. (Røhr and Killingtveit, 2003). Locally based runoff measure-
ments would have helped to interpret if there is indeed an
overestimation in our study. We, however, observed a positive KGE, which indicates a
good model performance (Knoben et al., 2019). Therefore,
we considered the Budyko simulations as being acceptable. However, it should be noted that this comparison is added for
reference only and should not be seen as validation. This is
because the global composite runoff (Fekete et al., 2002) is
not a strictly observational data set, and it is used here as the
best estimate available for long-term estimates of streamflow
in the East African region. The fact the Budyko framework
uses fewer data and parameters that are easily measurable at
a regional scale makes it a suitable approach for data-limited
regions such as East Africa. g
Besides the strengths of using the Budyko approach, un-
certainties may exist which could have affected our results. The study used data from different data sets (CHIRPS, CRU,
and GIMMS/AVHRR) at various scales, which could po-
tentially affect results due to various assumptions and ap-
proaches in the processing of each data set. C. N. Wamucii et al.: Land use and climate change effects on water yield The anthropogenic presence
both inside and outside the forested water towers indicates
the relevance of local context, and ground research for un-
derstanding the forest–water–people nexus (Van Noordwijk
et al., 2020) is recommended. This will help in understanding
in detail the dynamics and co-evolution of coupled human–
forest–water systems. We also recognize other factors that may influence the
results of this study. For instance, increasing atmospheric
CO2 concentrations may affect terrestrial water cycling
through changes in climate and changes in transpiration (i.e.,
stomatal conductance; Frank et al., 2015; Mamuye, 2018;
Huntington, 2008). We also note that if CO2 leads to higher
NDVI, then this effect is accounted for in our modeling ap-
proach. Some studies have reported that NDVI linear trends
can be linked to increasing CO2 levels (Krakauer et al., 2017; C. N. Wamucii et al.: Land use and climate change effects on water yield (2015) opined that this is possible due to un-
derestimation of evapotranspiration which was attributed to
evaporation of interception water by energy not captured in
the formulation of PET. The forest evapotranspiration para-
dox is further discussed in Teuling (2018). The correction of
underestimation in Teuling et al. (2019) indicates the need
for long-term lysimeter observations for studies focusing on
forested ecosystems. The availability of meteorological data
in the upper slopes of the East African mountains is a big gap
as the majority of meteorological observations are conducted
below 1500 m a.s.l., and most of the upper slopes data rely
on the extrapolation of hydrological analysis in the lowlands As a first-order tool, the Budyko framework provides
an important reference point for relating variations in wa-
ter yield to variations in climatic conditions and catchment
properties. In this study, the spatial pattern of the simulated
streamflow in the Budyko framework closely resembles the
pattern observed in the GRDC composite runoff. We, how-
ever, noted an overestimation of water yield in the compar-
ison. This type of observation was also reported by Teng et
al. (2012), where the Budyko equation was found to overes- https://doi.org/10.5194/hess-25-5641-2021 Hydrol. Earth Syst. Sci., 25, 5641–5665, 2021 5657 C. N. Wamucii et al.: Land use and climate change effects on water yield 5658 water yield are larger in the adjacent regions surrounding
the water towers. The majority of East African water tow-
ers are under pressure from human influences both within
and outside the water towers. Generally, the patterns in water
yield showed a strong longitudinal difference (east to west),
though the elevation is a key factor that ensures the gener-
ation of water in the water towers located in drier environ-
ments. A hydroclimatic phenomenon is occurring in the East
African region as the water towers show a strong shift to-
wards wetter conditions (especially in the period of 2011–
2019), while at the same time the atmospheric demand is
gradually increasing. Given that most of the water towers
were identified as non-resilient to changes, it means there are
greater possibilities of extreme variations in water yield un-
der changing climatic conditions. The Budyko framework is
an important first-order tool that uses fewer data inputs and
provides important illustrations of hydroclimatic conditions
of areas under study. The primary results contribute to the
general understanding that can further inform detail hydro-
logical studies. Therefore, Budyko is a suitable approach, es-
pecially for data-deficit regions such as East Africa. Figure A2. The 10-year moving averages of mean annual mean pre-
cipitation at the different water towers. Figure A2. The 10-year moving averages of mean annual mean pre-
cipitation at the different water towers. Figure A3. The 10-year moving averages of mean annual PET at
the different water towers. Figure A3. The 10-year moving averages of mean annual PET at
the different water towers. Figure A3. The 10-year moving averages of mean annual PET at
the different water towers. Appendix A: Extended figures and tables
Figure A1. The Budyko curve. The water limit (ET = P ) is shown;
a site cannot plot above the blue line unless there is input of wa-
ter beyond precipitation. The energy limit (ET = PET) is shown; a
site cannot plot above the red line unless precipitation is being lost
from system by means other than discharge (adapted from Creed
and Spargo, 2012a). Higher levels of annual precipitation were observed in
1990–1992, 1998–1999, and 2013–2015. Lower levels of an-
nual precipitation were observed in 1987, 1995–1996, 2004–
2005, and 2017. Mt. Kilimanjaro was observed to have a
consistent decline in annual mean rainfall between 1981 and
2017. The Mt. 5
Conclusions The forested water towers provide important water sources
for the surrounding semi-arid area through both runoff gen-
eration and regional rainfall recycling. Climate changes (i.e.,
changes in precipitation and potential evapotranspiration)
have a relatively large effect on water yield within the East
African water towers. The effects of land use changes on https://doi.org/10.5194/hess-25-5641-2021 Hydrol. Earth Syst. Sci., 25, 5641–5665, 2021 i et al.: Land use and climate change effects on water yield
Figure A2. The 10-year moving averages of mean annual mean pre-
cipitation at the different water towers. C. N. Wamucii et al.: Land use and climate change effects on water yield C. N. Wamucii et al.: Land use and climate change effects on water yield C. N. Wamucii et al.: Land use and climate change effects on water yield 5659 C. N. Wamucii et al.: Land use and climate change effects on water yield
5659
mary of simulated evapotranspiration (ET) and water yield (Q), given the precipitation (P ) between 1981 and 2019. s
PE
FSC
A
1981–1990
1991–2000
2001–2010
2011–2019
P
ET
Q
Q
RR
P
ET
Q
Q
RR
P
ET
Q
Q
RR
P
ET
Q
Q
RR
m
m
km2
mm/yr
FR
%
mm/yr
FR
%
mm/yr
FR
%
mm/yr
FR
%
5895
2000
1513.0
1989.1
1056.3
919.1
0.029
46
1811.2
1019.2
779.3
0.025
43
1720.7
980.7
727.9
0.023
42
1751.4
991.4
748.0
0.024
43
5199
2000
3298.3
1429.0
867.5
551.5
0.017
39
1350.5
828.5
512.7
0.016
38
1365.0
861.7
494.6
0.016
36
1466.4
884.0
573.7
0.018
39
4321
2000
2548.0
1538.4
1051.3
480.8
0.015
31
1560.0
1068.7
483.6
0.015
31
1653.8
1110.2
535.5
0.017
32
1856.9
1159.5
686.9
0.022
37
3999
2100
6671.8
1161.0
804.3
353.0
0.011
30
1110.2
772.1
335.2
0.011
30
1169.3
804.7
360.9
0.011
31
1352.8
869.7
479.1
0.015
35
5109
2000
1465.0
1609.5
934.1
653.6
0.021
41
1610.1
931.1
656.1
0.021
41
1546.0
944.7
580.4
0.018
38
1705.7
970.2
710.0
0.023
42
4565
2000
225.5
1244.4
898.2
330.6
0.010
27
1171.1
866.2
290.2
0.009
25
1156.1
803.7
338.0
0.011
29
1340.6
894.8
427.2
0.014
32
4507
2000
5288.2
1591.8
847.6
737.1
0.023
46
1512.4
833.5
672.0
0.021
44
1498.2
861.4
629.0
0.020
42
1523.9
850.0
666.8
0.021
44
4337
2600
5030.7
1099.4
755.3
343.3
0.011
31
1040.5
741.9
299.7
0.010
29
1034.4
755.2
279.6
0.009
27
1071.3
733.5
338.4
0.011
32
3187
2000
396.7
1464.9
1084.4
369.2
0.012
25
1482.5
1102.1
368.8
0.012
25
1416.7
1097.1
305.7
0.010
22
1576.6
1145.6
416.8
0.013
26
Figure A5. Graphical representation of the baseline Budyko curve
(estimated for 1981–1990) and the trends of water towers in the
different years. Table A1. Summary of simulated evapotranspiration (ET) and water yield (Q), given the precipitation (P ) between 1981 and 2019. Mountain ecosystems
PE
FSC
A
1981–1990
1991–2000
2001–2010
2011–2019
P
ET
Q
Q
RR
P
ET
Q
Q
RR
P
ET
Q
Q
RR
P
ET
Q
Q
RR
Units
m
m
km2
mm/yr
FR
%
mm/yr
FR
%
mm/yr
FR
%
mm/yr
FR
%
Mt. Kilimanjaro
5895
2000
1513.0
1989.1
1056.3
919.1
0.029
46
1811.2
1019.2
779.3
0.025
43
1720.7
980.7
727.9
0.023
42
1751.4
991.4
748.0
0.024
43
Mt. C. N. Wamucii et al.: Land use and climate change effects on water yield Elgon water tower recorded a consistent in-
crease in annual mean rainfall during the study period. Figure A3. The 10-year moving averages of mean annual PET at
the different water towers. Figure A4. The impact of elevation on hydroclimatic conditions in
the East African region. Appendix A: Extended figures and tables
Figure A1. The Budyko curve. The water limit (ET = P ) is shown;
a site cannot plot above the blue line unless there is input of wa-
ter beyond precipitation. The energy limit (ET = PET) is shown; a
site cannot plot above the red line unless precipitation is being lost
from system by means other than discharge (adapted from Creed
and Spargo, 2012a). Appendix A: Extended figures and tables Figure A3. The 10-year moving averages of mean annual PET at
the different water towers. Figure A4. The impact of elevation on hydroclimatic conditions in
the East African region. Figure A1. The Budyko curve. The water limit (ET = P ) is shown;
a site cannot plot above the blue line unless there is input of wa-
ter beyond precipitation. The energy limit (ET = PET) is shown; a
site cannot plot above the red line unless precipitation is being lost
from system by means other than discharge (adapted from Creed
and Spargo, 2012a). Higher levels of annual precipitation were observed in
1990–1992, 1998–1999, and 2013–2015. Lower levels of an-
nual precipitation were observed in 1987, 1995–1996, 2004–
2005, and 2017. Mt. Kilimanjaro was observed to have a
consistent decline in annual mean rainfall between 1981 and
2017. The Mt. Elgon water tower recorded a consistent in-
crease in annual mean rainfall during the study period. Figure A4. The impact of elevation on hydroclimatic conditions in
the East African region. https://doi.org/10.5194/hess-25-5641-2021 Hydrol. Earth Syst. Sci., 25, 5641–5665, 2021 C. N. Wamucii et al.: Land use and climate change effects on water yield C. N. Wamucii et al.: Land use and climate change effects on water yield Table A2. Description of the selected water towers. Mt. Kilimanjaro
The water tower is of volcanic origin, located in northeastern Tanzania, near the border with Kenya. High
rainfall and extensive forests make the water tower a critical water catchment for both Kenya and Tanzania. Forest fires linked to both human and climate warming are reported in the higher slopes, while forest clearing
is reported along the forest boundary. The surrounding area is home to over 1 million people (Lambrechts et
al., 2002; EAC et al., 2016). The water tower attracts more than 35 000 climbers a year, and 5000 visitors from
around the world (EAC et al., 2016). Hence, the location is a major source of foreign exchange earnings for
Tanzania. Mt. Kenya
A volcanic water tower located just south of the Equator in the central region of Kenya. The vegetation include
savanna grasslands at the base, forests of tall and broadleaf trees on the lower mountain slopes, Podocarpus
and cedar trees in the middle slopes, a belt of bamboo from about 2200 to 3100 m surrounded by thin forests
of Hagenia and Hypericum trees above and below the belt, and the alpine zone above 4000 m. Above 5000 m,
the landscape is bare rock and glaciers (UNEP, 2014). The water tower provides fresh water to about 7 million
people (EAC et al., 2016). The total population of the communities that live within the districts that border the
water tower was estimated at 24.4 million in 2009 (Nyongesa and Vacik, 2019). Mt. Elgon
A volcanic water tower located on the Kenya–Uganda boundary. The moorland zone, containing tree heaths,
giant groundsels, and lobelias, extends down to 3050 m, where it is succeeded by bamboo forest. Below 2550 m
is a temperate deciduous forest. The Ugandan side of Mt. Elgon has an average population density of 900 people
per square kilometer. Aberdare Range
A volcanic mountain, stretching for approximately 100 km from north to south, is mainly made up of alkaline
rock types (UNEP, 2014). Diverse forests grow above 2500 m, including cedar and riverine forests. From about
2700 to 2900 m, there is a belt of bamboo and, above 2900 m, cloud forests. Above 3100 m is the upper limit of
the montane forests. The millions of residents of Nairobi (capital city), 50 km away, depend on the water tower
for water supplies. C. N. Wamucii et al.: Land use and climate change effects on water yield Rwenzori Mountains
A block mountain, also known as mountains of the moons, the water tower is located just north of the Equator
bordering Uganda and the DRC. The range is about 120 km long and 65 km wide. Up to about 2300 m in the
park, the vegetation is mixed broadleaf forest. Between 2300 and 3200 m, the vegetation is characterized by
Afromontane forest trees. Between 3200 and 3800 m, there is a bamboo forest zone. Above this is a grassland
zone and cloud woodland. Afroalpine moorland extends to the snow line at 4400 m. The population density of
the area surrounding the mountain is between 150 and 430 persons per square kilometer (Yichuan, 2011). Mt. Meru
A volcanic mountain located 70 km west of Mt. Kilimanjaro. The forest covers about 9000 ha of land and is
bordered by 32 villages (Ndomba et al., 2015). The population of the area surrounding Mt. Meru is concentrated
in the southern flanks, with an estimated population of over 1.6 million (Vye-Brown et al., 2014). Virunga Mountains
Located within the Albertine Rift, Virunga is a crescent-shaped collection of mountains covering parts of
Uganda, the DRC, Rwanda, Burundi, and Tanzania. The vegetation belts in the Virunga Mountains are cat-
egorized into alpine, subalpine (ericaceous), and montane forest. The region surrounding Virunga has one of
the highest human population densities in Africa, with as many as 1000 people per square kilometer living in
the southern sections (USAID, 2013). Bale Mountains
The Bale Mountains are located in the southern highlands of Ethiopia (06◦41′ N, 39◦03′ E and 07◦18′ N,
40◦00′ E), and 400 km southeast of Addis Ababa. The water tower represents the largest area in Africa of
afroalpine vegetation over 3000 m a.s.l. (Tallents and Macdonald, 2011). The grassland area is situated between
an altitude of 2600–3000 m a.s.l. There are 25 villages surrounding the water tower. Bale watershed flows to
more than 12 million people in southern Ethiopia and western Somalia (Integrating Population, Health, and
Environment in Ethiopia’s Bale Mountains, 2021). Imatong Mountains
The Imatong Mountains are located in southeastern South Sudan and extend into the northern region of the
Ugandan border between 3◦57′ N 32◦54′ E and 3◦57′ N 32◦54′ E. Vegetation in the lower areas includes wood-
lands and mixed lowland semi-evergreen forest up to 1000 m. Above 1000 m there is montane forest up to
2900 m. C. N. Wamucii et al.: Land use and climate change effects on water yield Kenya
5199
2000
3298.3
1429.0
867.5
551.5
0.017
39
1350.5
828.5
512.7
0.016
38
1365.0
861.7
494.6
0.016
36
1466.4
884.0
573.7
0.018
39
Mt. Elgon
4321
2000
2548.0
1538.4
1051.3
480.8
0.015
31
1560.0
1068.7
483.6
0.015
31
1653.8
1110.2
535.5
0.017
32
1856.9
1159.5
686.9
0.022
37
Aberdare Range
3999
2100
6671.8
1161.0
804.3
353.0
0.011
30
1110.2
772.1
335.2
0.011
30
1169.3
804.7
360.9
0.011
31
1352.8
869.7
479.1
0.015
35
Rwenzori Mountains
5109
2000
1465.0
1609.5
934.1
653.6
0.021
41
1610.1
931.1
656.1
0.021
41
1546.0
944.7
580.4
0.018
38
1705.7
970.2
710.0
0.023
42
Mt. Meru
4565
2000
225.5
1244.4
898.2
330.6
0.010
27
1171.1
866.2
290.2
0.009
25
1156.1
803.7
338.0
0.011
29
1340.6
894.8
427.2
0.014
32
Virunga Mountains
4507
2000
5288.2
1591.8
847.6
737.1
0.023
46
1512.4
833.5
672.0
0.021
44
1498.2
861.4
629.0
0.020
42
1523.9
850.0
666.8
0.021
44
Bale Mountains
4337
2600
5030.7
1099.4
755.3
343.3
0.011
31
1040.5
741.9
299.7
0.010
29
1034.4
755.2
279.6
0.009
27
1071.3
733.5
338.4
0.011
32
Imatong Mountains
3187
2000
396.7
1464.9
1084.4
369.2
0.012
25
1482.5
1102.1
368.8
0.012
25
1416.7
1097.1
305.7
0.010
22
1576.6
1145.6
416.8
0.013
26 Figure A5. Graphical representation of the baseline Budyko curve
(estimated for 1981–1990) and the trends of water towers in the
different years. Figure A5. Graphical representation of the baseline Budyko curve
(estimated for 1981–1990) and the trends of water towers in the
different years. Hydrol. Earth Syst. Sci., 25, 5641–5665, 2021 https://doi.org/10.5194/hess-25-5641-2021 5660 C. N. Wamucii et al.: Land use and climate change effects on water yield 5661 Data availability. All climatic data used in this study (i.e., P,
PET, and NDVI) are publicly available. Precipitation data (P ) were
downloaded from Climate Hazards Group Infrared Precipitation
with Stations (CHIRPS v2) https://www.chc.ucsb.edu/data/chirps
(University
of
California,
2020). Potential
evapotranspira-
tion (PET) data were downloaded from the CRU database
https://crudata.uea.ac.uk/cru/data/hrg/
(Climatic
Research
Unit
(University of East Anglia) and NCAS, 2020). The Normalized Dif-
ference Vegetation Index (NDVI) was sourced from Global Inven-
tory Monitoring and Modeling System third generation (GIMMS
3 g) Advanced Very High-Resolution Radiometer (AVHRR) sensor
on board the National Oceanic and Atmospheric Administration
(NOAA)
satellites
https://climatedataguide.ucar.edu/climate-
data/ndvi-normalized-difference-vegetation-index-3rd-generation-
nasagfsc-gimms (National Center for Atmospheric Research Staff,
2018). The water towers analyzed data sets are summarized in
Table A1 for each water tower, and the full analysis is available
in the SESAM project SharePoint. The data can be provided upon
request from the first author (charles.wamucii@wur.nl). Aleman, J. C., Jarzyna, M. A., and Staver, A. C.: Forest extent and
deforestation in tropical Africa since, Nat. Ecol. Evol., 2, 26–33,
https://doi.org/10.1038/s41559-017-0406-1, 2018. Astuti, H. P. and Suryatmojo, H.: Water in the forest: Rain-
vegetation interaction to estimate canopy interception in a trop-
ical borneo rainforest, IOP Conf. Ser. Earth Environ. Sci., 361,
012035, https://doi.org/10.1088/1755-1315/361/1/012035, 2019. Bai, P., Zhang, D., and Liu, C.: Estimation of the Budyko model
parameter for small basins in China, Hydrol. Process., 34, 125–
138, https://doi.org/10.1002/hyp.13577, 2019. Booij, M. J., Schipper, T. C., and Marhaento, H.: Attributing
changes in streamflow to land use and climate change for 472
catchments in australia and the United States, Water, 11, 1059,
https://doi.org/10.3390/w11051059, 2019. Bosch, J. M. and Hewlett, J. D.: A review of catchment ex-
periments to determine the effect of vegetation changes on
water yield and evapotranspiration, J. Hydrol., 55, 3–23,
https://doi.org/10.1016/0022-1694(82)90117-2, 1982. Budyko, M. I.: Climate and Life, Acad. Press, New York, 18, 1st
Edition, 1974. Chebet, C.: Environmental degradation to blame for swelling of Rift
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English
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A Facile, Dry-Processed Lithium Borate-Based Cathode Coating for Improved All-Solid-State Battery Performance
|
Journal of the Electrochemical Society
| 2,020
|
cc-by
| 3,938
|
ISSN
0013-4651
Authors
Wu, Erik A
Jo, Chiho
Tan, Darren HS
et al.
Publication Date
2020-01-10
DOI
10.1149/1945-7111/abb8b3
Peer reviewed ISSN
0013-4651
Authors
Wu, Erik A
Jo, Chiho
Tan, Darren HS
et al. Publication Date
2020-01-10
DOI
10.1149/1945-7111/abb8b3
Peer reviewed Powered by the California Digital Library
University of California UC San Diego
UC San Diego Previously Published Works
Title
A Facile, Dry-Processed Lithium Borate-Based Cathode Coating for Improved
State Battery Performance
Permalink
https://escholarship.org/uc/item/2xf0b5gk
Journal
Journal of The Electrochemical Society, 167(13)
ISSN
0013-4651
Authors
Wu, Erik A
Jo, Chiho
Tan, Darren HS
et al.
Publication Date
2020-01-10
DOI
10.1149/1945-7111/abb8b3
Peer reviewed UC San Diego
UC San Diego Previously Published Works
Title
A Facile, Dry-Processed Lithium Borate-Based Cathode Coating for Improved
State Battery Performance
Permalink
https://escholarship.org/uc/item/2xf0b5gk
Journal
Journal of The Electrochemical Society, 167(13)
ISSN
0013-4651
Authors
Wu, Erik A
Jo, Chiho
Tan, Darren HS
et al. Publication Date
2020-01-10
DOI
10.1149/1945-7111/abb8b3
Peer reviewed Journal of The Electrochemical
Society
OPEN ACCESS
A Facile, Dry-Processed Lithium Borate-Based Cathode Coating for
Improved All-Solid-State Battery Performance
To cite this article: Erik A. Wu et al 2020 J. Electrochem. Soc. 167 130516
View the article online for updates and enhancements. Journal of The Electrochemical
Society This content was downloaded from IP address 137.110.61.44 on 11/11/2020 at 00:54 Powered by the California Digital Library
University of California eScholarship.org Methods y
However, many of the recently well-studied SSEs are sulfide-based;
while sulfide SSEs have an advantage over oxides in terms of ionic
conductivity and ease of processability,4–6 one main drawback is
higher chemical reactivity and lower electrochemical stability,
especially when used in conjunction with oxide cathode materials,
as sulfides will inherently electrochemically oxidize once subjected
to higher voltages during charging.7,8 Materials preparation.—As sulfide-based materials are sensitive
to air and moisture (decomposing to form toxic gases such as H2S),
all synthesis and characterization steps were done within an argon-
filled glovebox (MBraun MB 200B, H2O < 0.5 ppm, O2 < 5.0 ppm)
unless otherwise stated. Commercial LPSCl was obtained from NEI Corporation and used
as received. Commercial, bare LiNi0.8Co0.1Mn0.1O2 (NCM811) was
obtained from LG Chem. The dry coating process was conducted as
follows: Boric acid (>99.5%, Sigma Aldrich) was mixed with the
uncoated NCM811 cathode in an agate mortar and pestle for 5 min
according to the predetermined final boron parts per million (ppm),
hereby designated as: LBO B1 (875 ppm), LBO B2 (1800 ppm), and
LBO B3 (3500 ppm). The mixture was subsequently heated at 300 °
C for 5 h under ambient conditions to produce the LBO-coated
NCM811. One common strategy to mitigate unwanted reactions between
the SSE and the oxide cathode is the implementation of a chemically
inert, Li-ion conducting, and electronically-insulating coating on the
cathode particles. Such a coating needs to have a much lower
reactivity with the SSE (compared to the cathode) and must also be
thin enough (∼5 nm) to not drastically raise the overall impedance of
the ASSB. For this purpose, cathode coatings such as LiNbO3
(LNO), LiAlO2, LiTaO3, Li4Ti5O12 (LTO), lithium borate (LBO),
and others have been explored.7,9–14 However, it is important to note
that many of these cathode coatings are prepared via a solvent-based
solution process. In this work, a new dry coating process for LBO
was investigated on the cathode LiNi0.8Co0.1Mn0.1O2 (NCM811)
The
electrochemical
performance
of
ASSBs,
prepared
using
Li6PS5Cl (LPSCl) as the electrolyte and LiIn alloy as the anode,
showed that the LBO coating improved the first cycle discharge
capacity from 40 mAh/g to 126 mAh/g when compared to a similar
cell with bare NCM811. zE-mail: shmeng@ucsd.edu
=These authors contributed equally to this work.
*Electrochemical Society Member.
**Electrochemical Society Fellow. Erik A. Wu,1,= Chiho Jo,1,2,= Darren H. S. Tan,1 Minghao Zhang,1,* Jean-Marie Doux,1 Yu-
Ting Chen,3 Grayson Deysher,3 and Ying Shirley Meng1,2,4,**,z All-solid-state batteries (ASSBs) have gained much research
interest due to their potential for higher energy density, the
possibility of using metallic anodes, and improved safety since the
solid-state electrolyte (SSE) is non-flammable and non-corrosive.1–3 All-solid-state batteries (ASSBs) have gained much research
interest due to their potential for higher energy density, the
possibility of using metallic anodes, and improved safety since the
solid-state electrolyte (SSE) is non-flammable and non-corrosive.1–3
However, many of the recently well-studied SSEs are sulfide-based;
while sulfide SSEs have an advantage over oxides in terms of ionic
conductivity and ease of processability,4–6 one main drawback is
higher chemical reactivity and lower electrochemical stability,
especially when used in conjunction with oxide cathode materials,
as sulfides will inherently electrochemically oxidize once subjected
to higher voltages during charging.7,8 To cite this article: Erik A. Wu et al 2020 J. Electrochem. Soc. 167 130516 To cite this article: Erik A. Wu et al 2020 J. Electrochem. Soc. 167 130516 View the article online for updates and enhancements. Journal of The Electrochemical Society, 2020 167 130516 A Facile, Dry-Processed Lithium Borate-Based Cathode Coating
for Improved All-Solid-State Battery Performance
Erik A. Wu,1,= Chiho Jo,1,2,= Darren H. S. Tan,1 Minghao Zhang,1,* Jean-Marie Doux,1 Yu-
Ting Chen,3 Grayson Deysher,3 and Ying Shirley Meng1,2,4,**,z
1Department of Nano-Engineering, University of California San Diego, La Jolla, California 92093, United States of America
2LG Chem Research Park, Battery R&D, Daejeon, Republic of Korea
3Department of Materials Science and Engineering, University of California San Diego, La Jolla, California 92093, United
States of America
4Sustainable Power & Energy Center (SPEC), University of California San Diego, La Jolla, California 92093, United States
of America Erik A. Wu,1,= Chiho Jo,1,2,= Darren H. S. Tan,1 Minghao Zhang,1,* Jean-Marie Doux,1 Yu-
Ting Chen,3 Grayson Deysher,3 and Ying Shirley Meng1,2,4,**,z 1Department of Nano-Engineering, University of California San Diego, La Jolla, California 92093, United States of America
2LG Chem Research Park, Battery R&D, Daejeon, Republic of Korea
3 y
j
p
f
Department of Materials Science and Engineering, University of California San Diego, La Jolla, California 92093, Unite
States of America 4Sustainable Power & Energy Center (SPEC), University of California San Diego, La Jolla, California 92093, United States
of America Sulfide-based solid electrolytes are known to have narrow electrochemical windows which limit their practical use in all-solid-state
batteries (ASSBs). Specifically, when paired with a high-voltage transition metal oxide (TMO) cathode, the electrolyte will
typically undergo unwanted degradation via chemical reactions or electrochemical oxidation, especially upon charging to voltages
beyond the electrochemical stability window of the electrolyte. To mitigate these undesired reactions, thin (<10 nm), conformal,
ionically-conducting, and electronically-insulating oxide-based protective coating layers have been applied on the cathode,
typically via a solution process. In this work, a lithium borate-based (LBO) coating, prepared instead with a dry coating process,
was shown to have the same beneficial properties. As evidenced by electrochemical characterization, the developed LBO coating
shows good cycling performance and even performs better than the LiNbO3 coating commonly used in the literature. This new
solvent-free coating method can thus be used to fabricate longer-lasting ASSBs. g
g
g
© 2020 The Author(s). Published on behalf of The Electrochemical Society by IOP Publishing Limited. This is an open access
article distributed under the terms of the Creative Commons Attribution 4.0 License (CC BY, http://creativecommons.org/licenses/
by/4.0/), which permits unrestricted reuse of the work in any medium, provided the original work is properly cited. [DOI: 10.1149/
1945-7111/abb8b3] [ Manuscript submitted July 16, 2020; revised manuscript received September 8, 2020. Published September 24, 2020. Manuscript submitted July 16, 2020; revised manuscript received September 8, 2020. Published September 24, 2020. S
l
t
t i l f
thi
ti l i
il bl
li Manuscript submitted July 16, 2020; revised manuscript received September 8, 2020. Published September 24, 2020. Supplementary material for this article is available online Manuscript submitted July 16, 2020; revised manuscript received September 8, 2020. Published September 24, 2020. Supplementary material for this article is available online by the coating. This dry process opens a new avenue to fabricate
protective cathode coatings to enable longer-lasting ASSBs. Methods Other characterization methods, such as
scanning transmission electron microscopy (STEM) with electron
energy loss spectroscopy (EELS), revealed a ∼1–2 nm-thick
conformal LBO coating and X-ray diffraction (XRD) demonstrated
that chemical reactions between LPSCl and NCM811 were mitigated To prepare the solution-processed LiNbO3 (LNO) coating,
stoichiometric
amounts
of
lithium
ethoxide
(>95%,
Sigma
Aldrich) and niobium ethoxide (>99.95%, Sigma Aldrich) were
added to NCM811 before dispersing in anhydrous ethanol. The
solution was then stirred for 1 h and dried under vacuum, before
heating at 450 °C for 1 h under ambient conditions to produce the
2 wt% LNO-coated NCM811 powder. To prepare the composite cathodes, LPSCl was mixed with the
LBO/LNO-coated NCM811 in an agate mortar and pestle (using
either a 60:40 weight ratio of LPSCl:cathode without carbon, or a
66:33:1 weight ratio of LPSCl:cathode:carbon) for 5 min. To prepare
the Li0.5In anode, stoichiometric amounts of stabilized lithium metal
powder (FMC Lithium) and indium powder (>99.99%, Sigma
Aldrich) were mixed in a glass vial using a vortex mixer for about
5 min. Cell fabrication and electrochemical characterization.—To
prepare the cells, LPSCl powder was pressed at 370 MPa in a
10 mm polyether ether ketone (PEEK) die using two titanium Journal of The Electrochemical Society, 2020 167 130516 diffusing beyond the LBO coating. This cation mixing layer is
4–5 nm in thickness for LBO B2 (Fig. 2c) but 10–12 nm for LBO
B3; such an increased thickness could be potentially detrimental to
cell performance as the resistance would increase. For this reason,
further increases in the boron content were not considered. plungers. Subsequently, 10 mg of the as-prepared cathode composite
was added on one side of the LPSCl pellet and pressed at 370 MPa,
followed by the application of 40 mg of LiIn on the other side of the
electrolyte pellet and pressing at 120 MPa. The cell configuration
was secured into a cell holder and connected to a Landhe CT2001
Battery cycler and analyzed with the Land v7.3 software. All cells
were cycled at room temperature, inside the Ar glovebox, at
2.5–4.3 V vs Li/Li+. For the rate studies, the first charge was done
at 0.1 C, while the subsequent charging steps were done at 0.5 C. All
the specific capacities mentioned in this work were calculated based
on the electrode active material amount. Electrochemical impedance
spectroscopy (EIS) was performed with a Solartron 1260 impedance
analyzer for assembled half cells. Methods An applied AC potential of 30 mV
over a frequency range from 1 MHz to 0.1 Hz was used for the EIS
measurement. As there is a wide variety of lithium borate compounds, EELS
spectra were obtained to deduce which specific borate species was
synthesized during the coating process (Fig. 3). LBO B2 most
closely matches with the spectra from Li2B4O7 and LiBO2, while for
LBO B3, a peak shift is observed (due to the increasing B content
during the coating process). Nevertheless, since boron is a light
element and many LBO compounds yield similar spectra, it is
challenging to narrow down the exact species present. g g
p
p
To test its electrochemical performance, LBO-coated NCM811
was used in an ASSB configuration that contains Li0.5In as the anode
and LPSCl as the SSE. Figure 4 shows the first cycle charge-
discharge voltage profile, rate capability, capacity retention, and EIS
of the full cells. From the data, the LBO-coated NCM811 out-
performed bare NCM811 in all instances, and LBO B2 was shown to
have the highest capacity among the three concentrations tested,
regardless of the C rate (Figs. 4b and S3). The lower capacity of
LBO B1 can be attributed to a coating that is too thin, increasing the
chances of incomplete coverage, and thus not all unwanted reactions
are mitigated. As for LBO B3, the lower capacity compared to LBO
B2 can be attributed to the significantly thicker coating layer, which
would increase charge transfer resistance in the cell. EIS of the bare
and LBO B2-coated cells are shown in Fig. 4d; significant
impedance growth after 20 cycles is observed for the bare
NCM811 cell, indicative of unwanted chemical reactions that result
in resistive CEI products unfavorable for cell performance. There is
still impedance growth in the LBO B2 cell (due to unavoidable
LPSCl oxidation)15 but it is comparatively mitigated. To verify that
the performance degradation of the bare NCM811 and LPSCl is
from a chemical reaction, air-sensitive capillary XRD was conducted
and the data are shown in Fig. S4. It is clear that LPSCl has degraded
when mixed with the bare cathode but remained intact when mixed
with LBO B2. Thus, unwanted chemical reactions are prevented by
the coating. Characterization.—STEM-EELS was performed on a JEOL
JEM-ARM300CF at 300 kV, equipped with double correctors. Methods EELS spectra were acquired from a square area of ∼2 × 2 nm
near the surface layer with an acquisition time of 0.01 s. To
minimize possible electron beam irradiation effects, EELS spectra
presented in this work were acquired from areas without pre-beam
irradiation. XRD was carried out by loading the powder sample into a
0.5 mm Boron-rich glass capillary tube (Charles Supper). The
sample was flame-sealed to ensure no ambient air contamination. The samples were measured on a Bruker Kappa goniometer
equipped with a Bruker Vantec 500 detector. The sample was placed
in the Bragg−Brentano θ−θ configuration and the measurement was
carried out using Mo Kα radiation. Results and Discussion The schematic of the dry coating process is shown in Fig. 1. As
detailed in the experimental part, the boron concentrations used in
this study were 875 ppm (LBO B1), 1800 ppm (LBO B2) , and 3500
ppm (LBO B3). To show that the surface impurities were removed
by the dry coating process via reaction with boric acid, STEM
imaging and EELS were performed on both the bare and LBO-
coated NCM811 and the data are shown in Fig. 2. It is important to note that by using more active material and
adding conductive additive (66:33:1 NCM:LPSCl:C weight ratio),
the initial discharge capacity further increased from 124 to 160 mAh
g−1 (Fig. S5). These initial results are promising and suggest that
continued optimization of the cell setup would be even more
beneficial for cycling performance. g
From the STEM images and STEM-EELS mapping of the bare
NCM811 (Figs. 2d and 2f), there is a layer of carbon on the surface
of the NCM811 particles that comes from surface species such as
Li2CO3. The LBO-coated NCM EELS spectrum (Fig. 2g) does not
exhibit a signal from C, but only signal from B. This is an indication
that Li2CO3 is consumed during the coating process to ultimately
form the LBO coating on the cathode material. Similar results were
obtained with the LBO B3 coating and the results are shown in Fig. S1 (available online at stacks.iop.org/JES/167/130516/mmedia) of
the Supplementary Information. STEM images and intensity plots
along the surface of the coated NCM particle (Fig. S2, for both LBO
B2 and LBO B3) show the presence of cation mixing, i.e. TM Furthermore, a comparison between the commonly used, solu-
tion-processed LNO coating and the dry-coated LBO B2 NCM811
was conducted and shown in Fig. 5, with longer-term cycling in Fig. S6, which shows the superior cycling performance of LBO B2
compared with LNO. The dry-processed LBO thus shows great
promise as a coating material for long-lasting Li ASSBs. Figure 1. Schematic of the LBO coating process on NCM811 cathode particles through the simple dry coating method. Li-containing surface impurities are
consumed during the coating process. Figure 1. Schematic of the LBO coating process on NCM811 cathode particles through the simple dry coating method. Li-containing surface impurities are
consumed during the coating process. Journal of The Electrochemical Society, 2020 167 130516 Figure 2. Conclusions Figure 3. EELS spectra for LBO B2-coated NCM, LBO B3-coated NCM, Figure 3. EELS spectra for LBO B2-coated NCM, LBO B3-coated NCM,
LiBO2, and Li2B4O7 (latter two displayed for reference). In sulfide-based all-solid-state batteries (ASSBs) that use an
oxide cathode, it has been demonstrated that thin, conformal, and
chemically inert oxide-based cathode coatings are beneficial for
cycling performance as they mitigate unwanted chemical reactions
between the SSE and the oxide cathode. Many coatings have been
previously explored and applied to the cathode, usually via a
solvent-based solution process. In this work, a dry-processed,
lithium borate-based coating on NCM811 was shown to have the
same desirable properties and even showed a higher first cycle
capacity compared to the LiNbO3-based solution-processed coat-
ings. The LBO coating was synthesized by the solid-state reaction of
boric acid with Li-containing impurities (such as Li2CO3) on the
surface of NCM811, without the need for any washing or subsequent
solvent removal. These results demonstrate the promise of both the
facile dry-coating process and the LBO coating itself toward longer-
lasting and better-performing ASSBs. Results and Discussion HAADF-STEM images of (a) Bare NCM811, (b) LBO B2-coated NCM, and (c) Boron-doped interlayer at the LBO B2-NCM surface. STEM-EELS
elemental mapping of: (d) bare NCM at C K-edges and Li K-edges, (e) Intersection of three cathode particles of LBO B2-NCM for B K-edges and Ni L-edges. EELS spectra at the surface regions of: (f) bare NCM and (g) LBO B2-NCM. Figure 2. HAADF-STEM images of (a) Bare NCM811, (b) LBO B2-coated NCM, and (c) Boron-doped interlayer at the LBO B2-NCM surface. STEM-EELS
elemental mapping of: (d) bare NCM at C K-edges and Li K-edges, (e) Intersection of three cathode particles of LBO B2-NCM for B K-edges and Ni L-edges. EELS spectra at the surface regions of: (f) bare NCM and (g) LBO B2-NCM. 8. J. Lau, R. H. DeBlock, D. M. Butts, D. S. Ashby, C. S. Choi, and B. S. Dunn,
“Sulfide solid electrolytes for lithium battery applications.” Adv. Energy Mater., 8,
1800933 (2018). Acknowledgments Figure 3. EELS spectra for LBO B2-coated NCM, LBO B3-coated NCM,
LiBO2, and Li2B4O7 (latter two displayed for reference). This study is financially supported by LG Chem through Battery
Innovation Contest (BIC) program. TEM work was performed at the
UC Irvine Materials Research Institute (IMRI). The authors would
like to acknowledge the UCSD Crystallography Department for their Journal of The Electrochemical Society, 2020 167 130516 Figure 4. (a) Charge-discharge voltage profiles, (b) Rate capability, and (c) Cycle performance at 0.1 C of bare and LBO-coated NCM811 (with different boron
concentrations) at 0.1 C. (d) Nyquist plots of bare and LBO B2-coated NCM811 before and after the 1st and 20th cycles. The cathode composites were prepared
with a 60:40 weight ratio of NCM:SSE. Figure 4. (a) Charge-discharge voltage profiles, (b) Rate capability, and (c) Cycle performance at 0.1 C of bare and LBO-coated NCM811 (with different boron
concentrations) at 0.1 C. (d) Nyquist plots of bare and LBO B2-coated NCM811 before and after the 1st and 20th cycles. The cathode composites were prepared
with a 60:40 weight ratio of NCM:SSE. Figure 5. Comparison between bare, LBO B2 and LNO coating at 1 wt% and 2 wt%: (a) Charge-discharge voltage profiles. (b) Discharge voltage profile at
various C rates. Figure 5. Comparison between bare, LBO B2 and LNO coating at 1 wt% and 2 wt%: (a) Charge-discharge voltage profiles. (b) Discharge voltage profile at
various C rates , LBO B2 and LNO coating at 1 wt% and 2 wt%: (a) Charge-discharge voltage profiles. (b) Discharge voltage profile a assistance on the capillary XRD. The data is available from the
corresponding author upon reasonable request. assistance on the capillary XRD. The data is available from the
corresponding author upon reasonable request. assistance on the capillary XRD. The data is available from the
corresponding author upon reasonable request. 6. H. Nguyen, A. Banerjee, X. Wang, D. Tan, E. A. Wu, J.-M. Doux, R. Stephens,
G. Verbist, and Y. S. Meng, “Single-step synthesis of highly conductive Na3PS4
solid electrolyte for sodium all solid-state batteries.” J. Power Sources, 435, 126623
(2019). ,
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(
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5. D. H. S. Tan et al., “Enabling thin and flexible solid-state composite electrolytes by
the scalable solution process.” ACS Appl. Energy Mater., 2, 6542 (2019). (
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7. W. D. Richards, L. J. Miara, Y. Wang, J. C. Kim, and G. Ceder, “Interface stability
in solid-state batteries.” Chem. Mater., 28, 266 (2016). 4. Y. J. Nam, S.-J. Cho, D. Y. Oh, J.-M. Lim, S. Y. Kim, J. H. Song, Y.-G. Lee, S.-
Y. Lee, and Y. S. Jung, “Bendable and thin sulfide solid electrolyte film: a new
electrolyte opportunity for free-standing and stackable high-energy all-solid-state
lithium-ion batteries.” Nano Lett., 15, 3317 (2015). 4. Y. J. Nam, S.-J. Cho, D. Y. Oh, J.-M. Lim, S. Y. Kim, J. H. Song, Y.-G. Lee, S.-
Y. Lee, and Y. S. Jung, “Bendable and thin sulfide solid electrolyte film: a new
electrolyte opportunity for free-standing and stackable high-energy all-solid-state
lithium-ion batteries.” Nano Lett., 15, 3317 (2015).
5. D. H. S. Tan et al., “Enabling thin and flexible solid-state composite electrolytes by
the scalable solution process.” ACS Appl. Energy Mater., 2, 6542 (2019).
6. H. Nguyen, A. Banerjee, X. Wang, D. Tan, E. A. Wu, J.-M. Doux, R. Stephens,
G. Verbist, and Y. S. Meng, “Single-step synthesis of highly conductive Na3PS4
solid electrolyte for sodium all solid-state batteries.” J. Power Sources, 435, 126623
(2019).
7. W. D. Richards, L. J. Miara, Y. Wang, J. C. Kim, and G. Ceder, “Interface stability
in solid-state batteries.” Chem. Mater., 28, 266 (2016).
8. J. Lau, R. H. DeBlock, D. M. Butts, D. S. Ashby, C. S. Choi, and B. S. Dunn,
“Sulfide solid electrolytes for lithium battery applications.” Adv. Energy Mater., 8,
1800933 (2018). ORCID Ying Shirley Meng
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high-energy batteries.” Adv. Mater., 29, 1700007 (2017). 1. Y. Guo, H. Li, and T. Zhai, “Reviving lithium-metal anodes for next-generation
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2. J.-M. Doux, H. Nguyen, D. H. S. Tan, A. Banerjee, X. Wang, E. A. Wu, C. Jo,
H. Yang, and Y. S. Meng, “Stack pressure considerations for room-temperature all-
solid-state lithium metal batteries.” Adv. Energy Mater., 10, 1903253 (2020). 3. L. Fan, S. Wei, S. Li, Q. Li, and Y. Lu, “Recent progress of the solid-state
electrolytes for high-energy metal-based batteries.” Adv. Energy Mater., 8, 1702657
(2018). 14. H.-W. Chan, J.-G. Duh, and S.-R. Sheen, “Electrochemical performance of LBO-
coated spinel lithium manganese oxide as cathode material for li-ion battery.” Surf.
Coat. Technol., 188–189, 116 (2004). Journal of The Electrochemical Society, 2020 167 130516 9. K. Takada, N. Ohta, L. Zhang, K. Fukuda, I. Sakaguchi, R. Ma, M. Osada, and
T. Sasaki, “Interfacial modification for high-power solid-state lithium batteries..”
Solid State Ion., 179, 1333 (2008). 13. X. Li, J. Liu, M. N. Banis, A. Lushington, R. Li, M. Cai, and X. Sun, “Atomic layer
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768 (2014). 10. N. Ohta, K. Takada, I. Sakaguchi, L. Zhang, R. Ma, K. Fukuda, M. Osada, and
T. Sasaki, “LiNbO3-coated LiCoO2 as cathode material for all solid-state lithium
secondary batteries.” Electrochem. Commun., 9, 1486 (2007). 14. H.-W. Chan, J.-G. Duh, and S.-R. Sheen, “Electrochemical performance of LBO-
coated spinel lithium manganese oxide as cathode material for li-ion battery.” Surf. Coat. Technol., 188–189, 116 (2004). 11. B. Huang, Z. Zhao, Y. Sun, M. Wang, L. Chen, and Y. Gu, “Lithium-ion conductor
LiAlO2 coated LiNi0.8Mn0.1Co0.1O2 as cathode material for lithium-ion batteries..”
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X. Wang, H. Yang, A. Banerjee, and Y. S. Meng, “Elucidating reversible
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Ceram. Int., 39, 3087 (2013).
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Table S5 from Transcriptome-wide Landscape of Pre-mRNA Alternative Splicing Associated with Metastatic Colonization
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Table S5: Enriched GO in differentially expressed genes
Downregulated gene list 1505 vs. all 13984 genes
Category
Term
Count
%
P‐Value
Benjamini‐
Hochberg FDR
GOTERM_BP_FAT
epidermis development
29
2.2
0.00000056
0.00094
GOTERM_BP_FAT
ectoderm development
31
2.3
0.00000048
0.0016
GOTERM_BP_FAT
response to wounding
54
4.1
0.0000049
0.0054
GOTERM_BP_FAT
defense response
47
3.5
0.000011
0.0094
GOTERM_BP_FAT
immune response
55
4.1
0.00002
0.013
GOTERM_BP_FAT
inflammatory response
32
2.4
0.000024
0.013
GOTERM_BP_FAT
epithelium development
32
2.4
0.000028
0.013
GOTERM_BP_FAT
embryonic organ development
25
1.9
0.000051
0.019
GOTERM_BP_FAT
leukocyte chemotaxis
10
0.8
0.000046
0.019
GOTERM_BP_FAT
cell chemotaxis
10
0.8
0.000079
0.026
GOTERM_BP_FAT
response to steroid hormone
stimulus
28
2.1
0.000089
0.027
GOTERM_BP_FAT
positive regulation of response
to external stimulus
13
1
0.00017
0.046
GOTERM_CC_FAT
extracellular region
140
10.6
1.9E‐10
0.000000091
GOTERM_CC_FAT
plasma membrane
271
20.4
0.00000007
0.000016
GOTERM_CC_FAT
extracellular region part
78
5.9
0.00000068
0.000081
GOTERM_CC_FAT
plasma membrane part
188
14.2
0.00000057
0.00009
GOTERM_CC_FAT
extracellular space
56
4.2
0.0000038
0.00036
GOTERM_CC_FAT
apical junction complex
22
1.7
0.000054
0.0042
GOTERM CC FAT
intrinsic to plasma membrane
92
6 9
0 000086
0.0051 Table S5: Enriched GO in differentially expressed genes
Downregulated gene list 1505 vs. all 13984 genes
Category
Term
Count
%
P‐Value
Benjamini‐
Hochberg FDR
GOTERM_BP_FAT
epidermis development
29
2.2
0.00000056
0.00094
GOTERM_BP_FAT
ectoderm development
31
2.3
0.00000048
0.0016
GOTERM_BP_FAT
response to wounding
54
4.1
0.0000049
0.0054
GOTERM_BP_FAT
defense response
47
3.5
0.000011
0.0094
GOTERM_BP_FAT
immune response
55
4.1
0.00002
0.013
GOTERM_BP_FAT
inflammatory response
32
2.4
0.000024
0.013
GOTERM_BP_FAT
epithelium development
32
2.4
0.000028
0.013
GOTERM_BP_FAT
embryonic organ development
25
1.9
0.000051
0.019
GOTERM_BP_FAT
leukocyte chemotaxis
10
0.8
0.000046
0.019
GOTERM_BP_FAT
cell chemotaxis
10
0.8
0.000079
0.026
GOTERM_BP_FAT
response to steroid hormone
stimulus
28
2.1
0.000089
0.027
GOTERM_BP_FAT
positive regulation of response
to external stimulus
13
1
0.00017
0.046
GOTERM_CC_FAT
extracellular region
140
10.6
1.9E‐10
0.000000091
GOTERM_CC_FAT
plasma membrane
271
20.4
0.00000007
0.000016
GOTERM_CC_FAT
extracellular region part
78
5.9
0.00000068
0.000081
GOTERM_CC_FAT
plasma membrane part
188
14.2
0.00000057
0.00009
GOTERM_CC_FAT
extracellular space
56
4.2
0.0000038
0.00036
GOTERM_CC_FAT
apical junction complex
22
1.7
0.000054
0.0042
GOTERM CC FAT
intrinsic to plasma membrane
92
6 9
0 000086
0.0051 Table S5: Enriched GO in differentially expressed genes Table S5: Enriched GO in differentially expressed genes Downregulated gene list 1505 vs. Table S5: Enriched GO in differentially expressed genes
Downregulated gene list 1505 vs. all 13984 genes
Category
Term
Count
%
P‐Value
Benjamini‐
Hochberg FDR
GOTERM_BP_FAT
epidermis development
29
2.2
0.00000056
0.00094
GOTERM_BP_FAT
ectoderm development
31
2.3
0.00000048
0.0016
GOTERM_BP_FAT
response to wounding
54
4.1
0.0000049
0.0054
GOTERM_BP_FAT
defense response
47
3.5
0.000011
0.0094
GOTERM_BP_FAT
immune response
55
4.1
0.00002
0.013
GOTERM_BP_FAT
inflammatory response
32
2.4
0.000024
0.013
GOTERM_BP_FAT
epithelium development
32
2.4
0.000028
0.013
GOTERM_BP_FAT
embryonic organ development
25
1.9
0.000051
0.019
GOTERM_BP_FAT
leukocyte chemotaxis
10
0.8
0.000046
0.019
GOTERM_BP_FAT
cell chemotaxis
10
0.8
0.000079
0.026
GOTERM_BP_FAT
response to steroid hormone
stimulus
28
2.1
0.000089
0.027
GOTERM_BP_FAT
positive regulation of response
to external stimulus
13
1
0.00017
0.046
GOTERM_CC_FAT
extracellular region
140
10.6
1.9E‐10
0.000000091
GOTERM_CC_FAT
plasma membrane
271
20.4
0.00000007
0.000016
GOTERM_CC_FAT
extracellular region part
78
5.9
0.00000068
0.000081
GOTERM_CC_FAT
plasma membrane part
188
14.2
0.00000057
0.00009
GOTERM_CC_FAT
extracellular space
56
4.2
0.0000038
0.00036
GOTERM_CC_FAT
apical junction complex
22
1.7
0.000054
0.0042
GOTERM CC FAT
intrinsic to plasma membrane
92
6 9
0 000086
0.0051 all 13984 genes
Category
Term
Count
%
P‐Value
Benjamini‐
Hochberg FDR
GOTERM_BP_FAT
epidermis development
29
2.2
0.00000056
0.00094
GOTERM_BP_FAT
ectoderm development
31
2.3
0.00000048
0.0016
GOTERM_BP_FAT
response to wounding
54
4.1
0.0000049
0.0054
GOTERM_BP_FAT
defense response
47
3.5
0.000011
0.0094
GOTERM_BP_FAT
immune response
55
4.1
0.00002
0.013
GOTERM_BP_FAT
inflammatory response
32
2.4
0.000024
0.013
GOTERM_BP_FAT
epithelium development
32
2.4
0.000028
0.013
GOTERM_BP_FAT
embryonic organ development
25
1.9
0.000051
0.019
GOTERM_BP_FAT
leukocyte chemotaxis
10
0.8
0.000046
0.019
GOTERM_BP_FAT
cell chemotaxis
10
0.8
0.000079
0.026
GOTERM_BP_FAT
response to steroid hormone
stimulus
28
2.1
0.000089
0.027
GOTERM_BP_FAT
positive regulation of response
to external stimulus
13
1
0.00017
0.046
GOTERM_CC_FAT
extracellular region
140
10.6
1.9E‐10
0.000000091
GOTERM_CC_FAT
plasma membrane
271
20.4
0.00000007
0.000016
GOTERM_CC_FAT
extracellular region part
78
5.9
0.00000068
0.000081
GOTERM_CC_FAT
plasma membrane part
188
14.2
0.00000057
0.00009
GOTERM_CC_FAT
extracellular space
56
4.2
0.0000038
0.00036
GOTERM_CC_FAT
apical junction complex
22
1.7
0.000054
0.0042
GOTERM_CC_FAT
intrinsic to plasma membrane
92
6.9
0.000086
0.0051
GOTERM_CC_FAT
apicolateral plasma membrane
22
1.7
0.000083
0.0056
GOTERM_CC_FAT
cell‐cell junction
32
2.4
0.00013
0.0061
GOTERM_CC_FAT
integral to plasma membrane
89
6.7
0.00013
0.0067
GOTERM_CC_FAT
integral to membrane
351
26.5
0.00044
0.019
GOTERM_CC_FAT
intrinsic to membrane
361
27.2
0.00079
0.031 Upregulated gene list 1954 vs. Table S5: Enriched GO in differentially expressed genes
Downregulated gene list 1505 vs. all 13984 genes
Category
Term
Count
%
P‐Value
Benjamini‐
Hochberg FDR
GOTERM_BP_FAT
epidermis development
29
2.2
0.00000056
0.00094
GOTERM_BP_FAT
ectoderm development
31
2.3
0.00000048
0.0016
GOTERM_BP_FAT
response to wounding
54
4.1
0.0000049
0.0054
GOTERM_BP_FAT
defense response
47
3.5
0.000011
0.0094
GOTERM_BP_FAT
immune response
55
4.1
0.00002
0.013
GOTERM_BP_FAT
inflammatory response
32
2.4
0.000024
0.013
GOTERM_BP_FAT
epithelium development
32
2.4
0.000028
0.013
GOTERM_BP_FAT
embryonic organ development
25
1.9
0.000051
0.019
GOTERM_BP_FAT
leukocyte chemotaxis
10
0.8
0.000046
0.019
GOTERM_BP_FAT
cell chemotaxis
10
0.8
0.000079
0.026
GOTERM_BP_FAT
response to steroid hormone
stimulus
28
2.1
0.000089
0.027
GOTERM_BP_FAT
positive regulation of response
to external stimulus
13
1
0.00017
0.046
GOTERM_CC_FAT
extracellular region
140
10.6
1.9E‐10
0.000000091
GOTERM_CC_FAT
plasma membrane
271
20.4
0.00000007
0.000016
GOTERM_CC_FAT
extracellular region part
78
5.9
0.00000068
0.000081
GOTERM_CC_FAT
plasma membrane part
188
14.2
0.00000057
0.00009
GOTERM_CC_FAT
extracellular space
56
4.2
0.0000038
0.00036
GOTERM_CC_FAT
apical junction complex
22
1.7
0.000054
0.0042
GOTERM CC FAT
intrinsic to plasma membrane
92
6 9
0 000086
0.0051 all 13984 genes
Category
Term
Count
%
P‐Value
Benjamini‐
Hochberg FDR
GOTERM_BP_FAT
biological adhesion
99
6.4
6.3E‐16
1.1E‐12
GOTERM_BP_FAT
cell adhesion
99
6.4
5.6E‐16
1.9E‐12
GOTERM_BP_FAT
cell surface receptor linked
signal transduction
151
9.8
1.1E‐12
1.3E‐09
GOTERM_BP_FAT
G‐protein coupled receptor
protein signaling pathway
64
4.2
1.3E‐10
0.000000089
GOTERM_BP_FAT
response to wounding
70
4.5
1.3E‐10
0.00000011
GOTERM_BP_FAT
cell‐cell signaling
66
4.3
2.3E‐09
0.0000013
GOTERM_BP_FAT
regulation of adenylate cyclase
activity
17
1.1
0.000000066
0.000032
GOTERM_BP_FAT
regulation of cyclase activity
17
1.1
0.00000012
0.000051
GOTERM_BP_FAT
regulation of cyclic nucleotide
biosynthetic process
18
1.2
0.00000015
0.000058
GOTERM_BP_FAT
regulation of nucleotide
biosynthetic process
18
1.2
0.00000015
0.000058
GOTERM_BP_FAT
regulation of cAMP biosynthetic
process
17
1.1
0.00000022
0.000073
GOTERM_BP_FAT
regulation of lyase activity
17
1.1
0.00000022
0.000073
GOTERM_BP_FAT
G‐protein signaling, coupled to
cAMP nucleotide second
messenger
16
1
0.00000029
0.00009
GOTERM_BP_FAT
neurological system process
79
5.1
0.00000038
0.000092
GOTERM_BP_FAT
regulation of cell proliferation
102
6.6
0.00000046
0.000092
GOTERM_BP_FAT
regulation of cell migration
36
2.3
0.00000044
0.000092
GOTERM_BP_FAT
regulation of cyclic nucleotide
metabolic process
18
1.2
0.00000042
0.000095
GOTERM_BP_FAT
regulation of cell motion
40
2.6
0.00000038
0.000099
GOTERM_BP_FAT
wound healing
35
2.3
0.00000035
0.000099
GOTERM_BP_FAT
cell‐cell adhesion
36
2.3
0.00000068
0.00011
GOTERM_BP_FAT
regulation of cAMP metabolic
process
17
1.1
0.00000062
0.00012
GOTERM_BP_FAT
regulation of locomotion
37
2.4
0.00000066
0.00012
GOTERM_BP_FAT
regulation of nucleotide
metabolic process
18
1.2
0.0000016
0.00025
GOTERM_BP_FAT
cAMP‐mediated signaling
17
1.1
0.0000016
0.00026
GOTERM_BP_FAT
homophilic cell adhesion
19
1.2
0.0000034
0.0005
GOTERM_BP_FAT
extracellular structure
organization
28
1.8
0.0000047
0.00066 Upregulated gene list 1954 vs. Table S5: Enriched GO in differentially expressed genes
Downregulated gene list 1505 vs. all 13984 genes
Category
Term
Count
%
P‐Value
Benjamini‐
Hochberg FDR
GOTERM_BP_FAT
epidermis development
29
2.2
0.00000056
0.00094
GOTERM_BP_FAT
ectoderm development
31
2.3
0.00000048
0.0016
GOTERM_BP_FAT
response to wounding
54
4.1
0.0000049
0.0054
GOTERM_BP_FAT
defense response
47
3.5
0.000011
0.0094
GOTERM_BP_FAT
immune response
55
4.1
0.00002
0.013
GOTERM_BP_FAT
inflammatory response
32
2.4
0.000024
0.013
GOTERM_BP_FAT
epithelium development
32
2.4
0.000028
0.013
GOTERM_BP_FAT
embryonic organ development
25
1.9
0.000051
0.019
GOTERM_BP_FAT
leukocyte chemotaxis
10
0.8
0.000046
0.019
GOTERM_BP_FAT
cell chemotaxis
10
0.8
0.000079
0.026
GOTERM_BP_FAT
response to steroid hormone
stimulus
28
2.1
0.000089
0.027
GOTERM_BP_FAT
positive regulation of response
to external stimulus
13
1
0.00017
0.046
GOTERM_CC_FAT
extracellular region
140
10.6
1.9E‐10
0.000000091
GOTERM_CC_FAT
plasma membrane
271
20.4
0.00000007
0.000016
GOTERM_CC_FAT
extracellular region part
78
5.9
0.00000068
0.000081
GOTERM_CC_FAT
plasma membrane part
188
14.2
0.00000057
0.00009
GOTERM_CC_FAT
extracellular space
56
4.2
0.0000038
0.00036
GOTERM_CC_FAT
apical junction complex
22
1.7
0.000054
0.0042
GOTERM CC FAT
intrinsic to plasma membrane
92
6 9
0 000086
0.0051 all 13984 genes GOTERM_BP_FAT
positive regulation of cell
proliferation
58
3.8
0.0000055
0.00074
GOTERM_BP_FAT
defense response
51
3.3
0.000008
0.001
GOTERM_BP_FAT
G‐protein signaling, coupled to
cyclic nucleotide second
messenger
16
1
0.0000091
0.0011
GOTERM_BP_FAT
cyclic‐nucleotide‐mediated
signaling
18
1.2
0.000011
0.0013
GOTERM_BP_FAT
metal ion homeostasis
26
1.7
0.000012
0.0014
GOTERM_BP_FAT
response to hormone stimulus
53
3.4
0.000015
0.0017
GOTERM_BP_FAT
vasculature development
43
2.8
0.000018
0.0019
GOTERM_BP_FAT
transmission of nerve impulse
39
2.5
0.000018
0.002
GOTERM_BP_FAT
behavior
48
3.1
0.000023
0.0023
GOTERM_BP_FAT
blood vessel development
42
2.7
0.000027
0.0027
GOTERM_BP_FAT
cellular metal ion homeostasis
24
1.6
0.000031
0.0029
GOTERM_BP_FAT
regulation of body fluid levels
22
1.4
0.000031
0.003
GOTERM_BP_FAT
lung development
22
1.4
0.00004
0.0036
GOTERM_BP_FAT
morphogenesis of a branching
structure
18
1.2
0.000041
0.0036
GOTERM_BP_FAT
hemostasis
19
1.2
0.000045
0.0037
GOTERM_BP_FAT
negative regulation of lyase
activity
11
0.7
0.000044
0.0038
GOTERM_BP_FAT
negative regulation of adenylate
cyclase activity
11
0.7
0.000044
0.0038
GOTERM_BP_FAT
negative regulation of cyclase
activity
11
0.7
0.000044
0.0038
GOTERM_BP_FAT
neuron differentiation
53
3.4
0.000045
0.0038
GOTERM_BP_FAT
immune response
58
3.8
0.00005
0.004
GOTERM_BP_FAT
blood coagulation
18
1.2
0.000055
0.0041
GOTERM_BP_FAT
coagulation
18
1.2
0.000055
0.0041
GOTERM_BP_FAT
cell morphogenesis involved in
differentiation
36
2.3
0.000054
0.0041
GOTERM_BP_FAT
response to endogenous
stimulus
55
3.6
0.000054
0.0042
GOTERM_BP_FAT
regulation of cell adhesion
25
1.6
0.000058
0.0043
GOTERM_BP_FAT
respiratory tube development
22
1.4
0.000064
0.0045
GOTERM_BP_FAT
blood vessel morphogenesis
37
2.4
0.000065
0.0046
GOTERM_BP_FAT
second‐messenger‐mediated
signaling
28
1.8
0.000071
0.0049
GOTERM_BP_FAT
tube development
36
2.3
0.000073
0.0049
GOTERM_BP_FAT
inflammatory response
33
2.1
0.000075
0.0049 GOTERM_BP_FAT
positive regulation of cell
migration
21
1.4
0.000081
0.0052
GOTERM_BP_FAT
response to steroid hormone
stimulus
30
1.9
0.000086
0.0054
GOTERM_BP_FAT
positive regulation of cell
motion
22
1.4
0.0001
0.0062
GOTERM_BP_FAT
cell motion
59
3.8
0.00011
0.0065
GOTERM_BP_FAT
synaptic transmission
31
2
0.00011
0.0066
GOTERM_BP_FAT
response to oxygen levels
27
1.8
0.00013
0.0078
GOTERM_BP_FAT
skeletal system development
43
2.8
0.00015
0.0086
GOTERM_BP_FAT
locomotory behavior
31
2
0.00015
0.0086
GOTERM_BP_FAT
positive regulation of
locomotion
21
1.4
0.00016
0.0089
GOTERM_BP_FAT
extracellular matrix organization
19
1.2
0.00017
0.0091
GOTERM_BP_FAT
response to organic substance
84
5.4
0.00018
0.01
GOTERM_BP_FAT
respiratory system development
22
1.4
0.00019
0.01
GOTERM_BP_FAT
calcium ion homeostasis
22
1.4
0.00019
0.01
GOTERM_BP_FAT
response to estrogen stimulus
21
1.4
0.0002
0.01
GOTERM_BP_FAT
cellular calcium ion homeostasis
21
1.4
0.00025
0.013
GOTERM_BP_FAT
intracellular signaling cascade
142
9.2
0.00026
0.013
GOTERM_BP_FAT
response to virus
19
1.2
0.00026
0.013
GOTERM_BP_FAT
axon guidance
19
1.2
0.00026
0.013
GOTERM_BP_FAT
response to cytokine stimulus
18
1.2
0.00034
0.017
GOTERM_BP_FAT
enzyme linked receptor protein
signaling pathway
48
3.1
0.00036
0.017
GOTERM_BP_FAT
cell morphogenesis
47
3
0.00037
0.018
GOTERM_BP_FAT
metal ion transport
46
3
0.00039
0.018
GOTERM_BP_FAT
cellular component
morphogenesis
51
3.3
0.00039
0.018
GOTERM_BP_FAT
muscle organ development
30
1.9
0.00041
0.019
GOTERM_BP_FAT
neuron projection
morphogenesis
30
1.9
0.00047
0.021
GOTERM_BP_FAT
calcium ion transport
20
1.3
0.00048
0.021
GOTERM_BP_FAT
epithelial cell differentiation
20
1.3
0.00048
0.021
GOTERM_BP_FAT
negative regulation of cell
proliferation
49
3.2
0.00047
0.021
GOTERM_BP_FAT
branching morphogenesis of a
tube
15
1
0.00054
0.023 GOTERM_BP_FAT
heart morphogenesis
16
1
0.00055
0.023
GOTERM_BP_FAT
positive regulation of DNA
replication
10
0.6
0.00069
0.029
GOTERM_BP_FAT
di‐, tri‐valent inorganic cation
transport
24
1.6
0.00069
0.029
GOTERM_BP_FAT
axonogenesis
27
1.8
0.00078
0.032
GOTERM_BP_FAT
neuron projection development
34
2.2
0.0008
0.032
GOTERM_BP_FAT
cellular cation homeostasis
28
1.8
0.00079
0.032
GOTERM_BP_FAT
neuron development
41
2.7
0.00085
0.033
GOTERM_BP_FAT
response to estradiol stimulus
13
0.8
0.0011
0.043
GOTERM_BP_FAT
cell morphogenesis involved in
neuron differentiation
28
1.8
0.0012
0.046
GOTERM_BP_FAT
bone development
21
1.4
0.0013
0.049
GOTERM_BP_FAT
cell projection morphogenesis
33
2.1
0.0013
0.049
GOTERM_CC_FAT
extracellular region
205
13.3
2.3E‐31
1.1E‐28
GOTERM_CC_FAT
extracellular region part
124
8
9E‐24
2.1E‐21
GOTERM_CC_FAT
plasma membrane
342
22.2
1.7E‐18
2.6E‐16
GOTERM_CC_FAT
intrinsic to membrane
467
30.3
2.7E‐15
3.1E‐13
GOTERM_CC_FAT
extracellular matrix
61
4
3.5E‐15
3.2E‐13
GOTERM_CC_FAT
extracellular space
79
5.1
8.2E‐14
6.4E‐12
GOTERM_CC_FAT
intrinsic to plasma membrane
131
8.5
1E‐13
7E‐12
GOTERM_CC_FAT
integral to membrane
445
28.9
1.9E‐13
1.1E‐11
GOTERM_CC_FAT
integral to plasma membrane
127
8.2
2.8E‐13
1.3E‐11
GOTERM_CC_FAT
proteinaceous extracellular
matrix
54
3.5
2.8E‐13
1.5E‐11
GOTERM_CC_FAT
plasma membrane part
224
14.5
6.4E‐11
2.8E‐09
GOTERM_CC_FAT
extracellular matrix part
27
1.8
0.000000028
0.0000011
GOTERM_CC_FAT
basement membrane
19
1.2
0.000015
0.00053
GOTERM_CC_FAT
cell projection
88
5.7
0.000019
0.00065
GOTERM_CC_FAT
neuron projection
50
3.2
0.000037
0.0012
GOTERM_CC_FAT
external side of plasma
membrane
22
1.4
0.00058
0.017
GOTERM_CC_FAT
synapse part
29
1.9
0.00069
0.019
GOTERM_CC_FAT
cell surface
39
2.5
0.0014
0.036
GOTERM_MF_FAT
calcium ion binding
121
7.8
8.6E‐18
9.2E‐15
GOTERM_MF_FAT
growth factor binding
26
1.7
0.00000022
0.00012
GOTERM_MF_FAT
ion channel activity
36
2.3
0.000035
0.0041
GOTERM_MF_FAT
endopeptidase inhibitor activity
20
1.3
0.000024
0.0042
GOTERM_MF_FAT
channel activity
38
2.5
0.000033
0.0044 GOTERM_MF_FAT
passive transmembrane
transporter activity
38
2.5
0.000033
0.0044
GOTERM_MF_FAT
peptidase inhibitor activity
20
1.3
0.000032
0.0048
GOTERM_MF_FAT
gated channel activity
30
1.9
0.000024
0.005
GOTERM_MF_FAT
neurotransmitter binding
11
0.7
0.000047
0.005
GOTERM_MF_FAT
peptide binding
25
1.6
0.000068
0.006
GOTERM_MF_FAT
substrate specific channel
activity
37
2.4
0.000023
0.0061
GOTERM_MF_FAT
neurotransmitter receptor
activity
10
0.6
0.000065
0.0063
GOTERM_MF_FAT
peptide receptor activity
12
0.8
0.00002
0.0069
GOTERM_MF_FAT
peptide receptor activity, G‐
protein coupled
12
0.8
0.00002
0.0069
GOTERM_MF_FAT
calcium channel activity
13
0.8
0.000086
0.007
GOTERM_MF_FAT
cation channel activity
27
1.8
0.00022
0.016
GOTERM_MF_FAT
glycosaminoglycan binding
19
1.2
0.00023
0.017
GOTERM_MF_FAT
serine‐type endopeptidase
inhibitor activity
13
0.8
0.00037
0.024
GOTERM_MF_FAT
integrin binding
13
0.8
0.00037
0.024
GOTERM_MF_FAT
lipid binding
56
3.6
0.00062
0.038
GOTERM_MF_FAT
growth factor activity
21
1.4
0.00073
0.042
GOTERM_MF_FAT
polysaccharide binding
19
1.2
0.00084
0.046
GOTERM_MF_FAT
pattern binding
19
1.2
0.00084
0.046
|
https://openalex.org/W4388833745
|
https://microbiologyjournal.org/download/86202/
|
English
| null |
Prevalence and Risk Assessment of Human Papillomavirus Infection in a Bengali Cohort
|
Journal of pure and applied microbiology
| 2,023
|
cc-by
| 7,311
|
Prevalence and Risk Assessment of Human
Papillomavirus Infection in a Bengali Cohort Nabamita Chaudhury1, Tanusri Biswas1, Koushik Bose2,
Prabir Sengupta3, Arghya Nath4, Nivedita Mukherjee4,
Anupam Basu5* and Subhra Kanti Mukhopadhyay6* 1Department of Microbiology, Burdwan Medical College, Burdwan, West Bengal, India. 2Department of Pathology, Burdwan Medical College, Burdwan, West Bengal, India. 3Burdwan Medical College, Burdwan, West Bengal, India. 4ICMR-DHR VRDL (Viral Research and Diagnostic Laboratory) Department of Microbiology,
Burdwan Medical College, Burdwan, West Bengal, India. 5Department of Zoology The University of Burdwan West Bengal India 1Department of Microbiology, Burdwan Medical College, Burdwan, West Bengal, India. 2Department of Pathology, Burdwan Medical College, Burdwan, West Bengal, India. 3Burdwan Medical College, Burdwan, West Bengal, India.i 3Burdwan Medical College, Burdwan, West Bengal, India. 4ICMR-DHR VRDL (Viral Research and Diagnostic Laboratory) Department of Microbiology,
Burdwan Medical College, Burdwan, West Bengal, India. 4ICMR-DHR VRDL (Viral Research and Diagnostic Laboratory) Department of Microbiology,
Burdwan Medical College, Burdwan, West Bengal, India. g ,
,
g ,
5Department of Zoology, The University of Burdwan, West Bengal, India. 6Department of Microbiology, The University of Burdwan, Burdwan, West Bengal, India. Chaudhury et al | Article 8642
J Pure Appl Microbiol. 2023;17(4):2281-2294. doi: 10.22207/JPAM.17.4.25
Received: 21 April 2023 | Accepted: 31 October 2023
Published Online: 20 November 2023 Chaudhury et al | Article 8642
J Pure Appl Microbiol. 2023;17(4):2281-2294. doi: 10.22207/JPAM.17.4.25
Received: 21 April 2023 | Accepted: 31 October 2023
Published Online: 20 November 2023 Abstract 2281 www.microbiologyjournal.org Chaudhury et al | J Pure Appl Microbiol. 2023;17(4):2281-2294. https://doi.org/10.22207/JPAM.17.4.25 INTRODUCTION essential nowadays. Hence, in this study, we
carried out both the cytological study along with
molecular detection of HPV side by side.4,5 Recently, cervical cancer has become
a burgeoning burden on the health of women. Human papillomaviruses (HPV) infect mucosal
and cutaneous epithelium and induce cellular
proliferation.1 Over 170 types of HPV circulate
worldwide.2 Among these, many types have
been found to cause cervical cancer, however,
HPV-16 and -18 are the two most high-risk HPV
types. The overexpression of two oncoproteins,
E6 and E7, are responsible for causing neoplasia. There are many risk factors associated with the
development of cervical neoplasia including
smoking, being married at early age, multiple
pregnancies, early age at first pregnancy, and
multiple sexual partners. Vaccination against
HPV-16 and -18 infections, that primarily targets
adolescent females, are very effective in reducing
the incidence of cervical cancer.2i Place of study Department of Microbiology, Burdwan
Medical College and Department of Microbiology,
Burdwan University, Burdwan, West Bengal, India,
from January 2019 to December 2021. Ethics committee approvalii The Institutional Ethical Committee (IEC)
of BMC and IEC of Burdwan University approved
the study. Written consent was obtained from each
of the patients. Types of HPV belonging to phylogenetically
“high-risk” species of the mucosotropic alpha
genus, namely alpha-5,-6,-7,-9, and -11, are known
to cause cervical carcinoma. The types most
frequently found in cervical cancer are alpha-16,
-18,-31,-33,-35, -45, -52, and -58, while rare types
include alpha-39, -51, -56, and -59. The remaining
types of HPV in the “high-risk” group are classified
as “possibly carcinogenic”(group 2: 2A (alpha-68)
and 2B (alpha-26, -30,-34, -53, -66, -67, -70, -73,
-82, -85, and -97).3 Abstract Cervical cancer is a notable cause of mortality and morbidity among women of reproductive age. Human papillomavirus (HPV) is the leading cause of cervical cancer among women. Among 170 types
of HPV; HPV-16 and -18 are responsible for cervical cancer. The overexpression of oncoproteins E6
and E7 are predominantly responsible for causing neoplasia. The presence of koilocytosis/koilocytotic
atypia is the diagnostic point of HPV infection in pap smears. To identify the circulating types of HPV
and determine the various risk factors associated with HPV infection, 100 vaginal biopsies or swabs
were taken from patients suspected with cervical cancer, and qualitative and semi-quantitative real-
time PCR were performed. PCR primers (GP5+/GP6+) based on a conserved region of the HPV-L1open
reading frame(ORF) gene were used for the detection of HPV strains, while another set of primers was
used for detecting the E6 gene (HPV-16) and E7 gene (HPV-18). The results showed an HPV infection
rate of 23%. Furthermore, the prevalent genotype was found to be HPV-16 (73.91%), followed by HPV-
18 (26.1%), while mixed infections of both HPV-16 and -18 accounted for 21.74%. In addition, an age
of above 45 years, multiple pregnancies, low socioeconomic status, postmenopausal state, anemia,
and early coitarche were significantly associated with HPV infection. These results provide the basis
for the formulation of an appropriate strategy for disease monitoring to determine the frequency and
distribution pattern of HPV infection. Keywords: Human Papillomaviruses, Molecular Techniques, HPV16, HPV18, Risk Factors Citation: Chaudhury N, Biswas T, Bose K, et al. Prevalence and Risk Assessment of Human Papillomavirus Infection in a Bengali
Cohort. J Pure Appl Microbiol. 2023;17(4):2281-2294. doi: 10.22207/JPAM.17.4.25
© The Author(s) 2023. Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License which
permits unrestricted use, sharing, 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. www.microbiologyjournal.org
2281
Journal of Pure and Applied Microbiology
*Correspondence: abasu@zoo.buruniv.ac.in; skmukhopadhyay@microbio.buruniv.ac.in *Correspondence: abasu@zoo.buruniv.ac.in; skmukhopadhyay@microbio.buruniv.ac.in e Author(s) 2023. Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License which
mits unrestricted use, sharing, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and
ource, provide a link to the Creative Commons license, and indicate if changes were made. Source of data A total of 100 vaginal biopsies or swabs
were collected in viral transport media at the
Department of Gynecology and Obstetrics,
Burdwan Medical College and Hospital and sent
to the Department of Microbiology, Burdwan
Medical College for further laboratory diagnosis
in Burdwan Medical College and Hospital. Study population The widespread use of cervical cytology
screening to detect both premalignant and
malignant lesions has reduced the incidence of
cervical cancer globally. The detection of HPV
largely depends on cytology,from which abnormal
koilocytosis or koilocytotic atypia, comprised of the
combination of nuclear atypia and the formation
of a perinuclear halo, can be identified. However,
the sensitivity of cytology testing depends on the
quality of the staining technique and the stage of
CIN at which the smear is collected from patients. Nevertheless, cytology methods can often be
overburdened by the presence of dyskaryotic
smears, which may have a low productive value. This highlights the importance of the molecular
detection of HPV as well as the identification of
the type of HPV for early detection and treatment. In such scenario, the importance of molecular
detection of HPV along with its typing is very Clinically suspected patients of cervical
neoplasia, visiting the colposcopy unit for a pap
smear or admitted to the Department of Obstetrics
and Gynecology in Burdwan Medical College and
Hospital. Journal of Pure and Applied Microbiology Pap smear A sample was taken from the junction of
the ectocervix by rotating a wooden Ayre spatula
at 360°. The sample was then smeared onto
a labelled glass slide and fixed with 95% ethyl
alcohol. The smears were then stained using the
Papanicolaou method.6 The Bethesda Classification Data for epidemiological surveyi Patients who were clinically suspected
to have cervical lesions or cervical cancer were
selected and provided a questionnaire to obtain
their medical history including clinical signs
and symptoms,as well as personal information
including age, residency, occupation, income,
number of pregnancies, use of contraception,
coitarche,menstrual history, and age at the time
of marriage. We have also recorded the comorbid
conditions including the history of diabetes
mellitus, anemia, hypertension, hypothyroidism,
and hyperthyroidism. 2282 www.microbiologyjournal.org www.microbiologyjournal.org Chaudhury et al | J Pure Appl Microbiol. 2023;17(4):2281-2294. https://doi.org/10.22207/JPAM.17.4.25 of Cervical Cytology was used for reporting cervical
cytology (Figure 1 and 2, Table 1 and 2).7 Molecular diagnosis
Primer set design Studies have shown that the HPV-L1
gene is highly conserved among various HPV
genotypes, and a total of 10% of the HPV-L1
sequence can vary in distinct HPV genotypes.8
Moreover, the conserved region of E6 and E7
is comprised of open reading frames (ORFs) for
HPV-16 and -18, respectively. The full genome
sequences were downloaded from GenBank for
alignment. Subsequently, species-specific primer
sets for HPV-L1, 16, and 18 were designed using
primer 3. Further, the specificity of three pairs
of primers was verified using the Primer-BLAST
program (Table 3). 2
Journal of Pure and Applied Microbiology
Table 1. Bethesda system for reporting cervical cytology
(Ref-6)
Pap smear
Bethesda
classification
(n=100)(n ,%)
1. Negative for Intraepithelial
84, (84%)
lesion or malignancy ) NILM
A. Organisms –
68, (81%)
a. Shift in flora suggestive
42, (62%)
of bacterial vaginosis
b. Trichomonalas vaginalis
8, (12%)
c. Fungal organism
18, (26%)
d. Actinomycosis
0, (0%)
e. HSV
0, (0%)
f. CMV
0, (0%)
B.Other non neoplastic finding
1
6, (16%)
a.Reactive changes associated
16, (100%)
with inflammation
b. glandular cells status post
0, (0%)S
hysterectomy
2. Epithelial cell abnormalities
16, (16%)
A. Squamous cell
16, (100%)
a. Atypical squamous cells of
2, (13%)
undermined significance (ASCUS)
b. Atypical squamous cells cannot
2, (13%)
exclude HSIL (ASC-H)
c. Low grade squamous
1, (6%)
intraepithelial lesion (LSIL)
d. High grade squamous
2, (13%)
intraepithelial lesion HSIL
e. Squamous cell carcinoma
9, (56%)
B. Glandular cell
0, (0%)
a. Atypical glandular cells
0, (0%)
(endocervical/endometrial
and not otherwise specified)
b. Atypical glandular cells, favor
0, (0%)
neoplastic (endocervical /NOS)
c. Endocervical adenocarcinoma
0, (0%)
in situ (AIS)
d. Adenocarcinoma
0, (0%)
3. Others
0, (0%)
A. Endometrial cells in women
0, (0%) Table 1. Bethesda system for reporting cervical cytology
(Ref-6) Journal of Pure and Applied Microbiology www.microbiologyjournal.org HPV DNA extraction The viral DNA was isolated using
a High Pure PCR Template Preparation Kit
(cat. no. 11796828001, according to the
manufacturer’s instructions (Roche). Detection of HPV-L1 gene using qualitative PCR i
g
g qi
The HPV-L1 gene is conserved in all HPV
genotypes. Thus, to cover all of the required
positions,the PCR primers GP5+ and GP6+ were
used for the amplification of the HPV-L1 gene
(Table 3). Qualitative PCR (qPCR) was performed
using Emerald Amp® GT PCR Master Mix (RR310B;
TAKARA Bio.).Briefly, in a reaction volume of 50 µl,
100 ng of template DNA was mixed with 25 µl of
2׳Emerald Amp GT PCR Master Mix, 0.8 µl of 2 mM
MgCl2, and 0.2 µM (final concentration) of forward Table 2. Distribution of cases according to
colposcopic finding (n=100) 2283 Chaudhury et al | J Pure Appl Microbiol. 2023;17(4):2281-2294. https://doi.org/10.22207/JPAM.17.4.25 Real-Time PCR Detection System (Bio-Rad) was
used for real-time PCR with the following cycling
conditions: initial denaturation incubation at 95°C
for 2 min, followed by 38 cycles of alternating
incubation periods at 95°C for 5 s, at 56°C for 30
s, and at 72°C for 10 s. Fluorescence was detected
after each period of extension incubation at 72°C. All the clinical samples were screened using this
method to verify the presence of HPV viral DNA. Only one HPV-L1 gene-positive sample (which was
identified positive according to the NCBI BLASTN
result for human papillomavirus (ID: 10566)),
was sent for sequencing. This sample was used
as positive control. Subsequently, genotyping of
HPV-18 and -16 were carried out by using qPCR. β-actin was used as the SYBR Green test control. and reverse primers; the remaining volume was
comprised of ultra pure molecular grade water. The PCR cycling conditions were as follows: initial
denaturation at 95°C (3 min), 35 cycles including
denaturation at 94°C (30 s), annealing at 56°C
(30 s), and elongation at 72°C (45 s), followed by
72°C for 5 min. Aliquots (10 µl) of the amplified
PCR products were then run in 1.0 % agarose gel
stained by ethidium bromide. The suitability of
each sample for PCR amplification was verified by
the presence of housekeeping gene β-actin, used
as a control (Figure 3). Real-time PCR of HPV-L1 using SYBR Green Real-time PCR of HPV-L1 using SYBR Green
Qualitative real-time PCR reactions
targeting the L1 region of HPV were performed
using TB Green Premix Ex Taq II (TiRNase H Plus). Each reaction consisted of 1xTB Green Premix
Master Mix and the Gp5+ and Gp6+ primers in a
reaction volume of 25 µl.12,13 Regions showing high
rates of homology between different types of HPV
were identified and used to design degenerative
“broad spectrum” HPV primers. A CFX96 Touch HPV-16 and -18 genotyping by qPCRi The L1-positive samples obtained using
SYBR Green were further processed for HPV-16
and -18 screening. The E6 and E7 genes were used
to classify the HPV-16 and -18 variants present
in the clinical samples. Two E6 PCR targets were www microbiologyjournal org
2284
Journal of Pure and Applied Microbiology
Table 3. Detailed sequences of the designed primer sets
Gene
Primer
Sequence
Product
Ref. Length
L1 gene
GP5+
5’-TTTGTTACTGTGGTAGATACTAC-3’
142 bp. (9)
GP6+
5’-GAAAAATAAACTGTAAATCATATTC-3’
E6 gene
Forward
5’-AGGACCCACAGGAGCGAC-3’
126 bp. (10)
Reverse
5’-AGTCATATACCTCACGTCGCAGT-3’
E7 gene
Forward
5’-TGCCAGAAACCGTTGAATCC-3’
268 bp. (11)
Reserve
5’-TCTGAGTCGCTTAATTGCTC-3’
Figure 1. Pap smear: Showing HSIL (High grade
squamous intraepithelial lesion)
Figure 2. Pap smear showing SCC ( Squamous cell
carcinoma) Table 3. Detailed sequences of the designed primer sets Table 3. Detailed sequences of the designed primer sets
Gene
Primer
Sequence
Product
Ref. Length
L1 gene
GP5+
5’-TTTGTTACTGTGGTAGATACTAC-3’
142 bp. (9)
GP6+
5’-GAAAAATAAACTGTAAATCATATTC-3’
E6 gene
Forward
5’-AGGACCCACAGGAGCGAC-3’
126 bp. (10)
Reverse
5’-AGTCATATACCTCACGTCGCAGT-3’
E7 gene
Forward
5’-TGCCAGAAACCGTTGAATCC-3’
268 bp. (11)
Reserve
5’-TCTGAGTCGCTTAATTGCTC-3’ Figure 2. Pap smear showing SCC ( Squamous cell
carcinoma) Figure 1. Pap smear: Showing HSIL (High grade
squamous intraepithelial lesion) Figure 1. Pap smear: Showing HSIL (High grade
squamous intraepithelial lesion) Figure 2. Pap smear showing SCC ( Squamous cell
carcinoma) Figure 1. Pap smear: Showing HSIL (High grade
squamous intraepithelial lesion) Figure 2. Pap smear showing SCC ( Squamous cell
carcinoma) Journal of Pure and Applied Microbiology Journal of Pure and Applied Microbiology www.microbiologyjournal.org 2284 Chaudhury et al | J Pure Appl Microbiol. 2023;17(4):2281-2294. https://doi.org/10.22207/JPAM.17.4.25 www.microbiologyjournal.org
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Journal of Pure and Applied Microbiology
Table 4. Journal of Pure and Applied Microbiology HPV-16 and -18 genotyping by qPCRi Distribution of cases according to frequency of co-morbidities
Co-morbidities
According to RT-PCR report
Statistical Test Cytological findings
Total samples
(n=100)
HPV positive
Rate of
(Negative
Fisher’s Exact
P value NILM (n=84)
Epithelial cell
n, (%)
cases (n=23)
positivity
samples
Test value,
abnormalities
(n, %)
(n, %)
n=77) :
df
Organism
Other non
(n=16) (n, %)
(n, %)
(n=68):
neoplastic
(n, %)
findings
(n=16) (n, %)
Hypertension
5, (21.74%)
27.78%
13, (16.88%)
3.257,4
0.583
12, (17.64%)
4, (25%)
4,(25%)
18, (18%)
Diabetes mellitus
6, (26.09%)
25%
18, (23.37%)
1.354,4
0.001*
16, (23.53%)
5, (31.25%)
5,(31.25%)
24, (24%)
Hypothyroidism
2, (8.7%)
16.66%
10, (12.99%)
5.692,4
0.241
10, (14.71%)
1, (6.25%)
1, (6.25%)
12, (12%)
Hyperthyroidism
2, (8.7%)
40%
3, (3.9%)
7.982,4
0.369
3,(4.41%)
1, (6.25%)
1, (6.25%)
5, (5%)
Anaemia
8, (34.78%)
19.51%
33, (42.86%)
1.289,4
0.026*
27, (39.71%)
5,(31.25%)
5, (31.25%)
41, (41%)
*Significant at p<0.05, Fisher’s Exact Test; No significant difference was observed between the groups HPV-positive cases and negative samples in terms of hypertension, hypothyroidism, and
hyperthyroidism. However, a significant difference was observed between the groups HPV-positive cases and negative samples in terms of diabetes mellitus and anemia (p <0.001 and p = 0.026,
respectively). Table 5. Distribution of cases according to coitarche
Coitarche
According to RT-PCR report Statistical Test Cytological findings
Total samples
(years)
(n=100)
HPV positive
Rate of
(Negative
Fisher’s Exact
P value NILM (n=84)
Epithelial cell
n, (%)
cases (n=23)
positivity
samples
Test value,
abnormalities
(n, %)
(n, %)
n=77) :
df
Organism
Other non
(n=16) (n, %)
(n, %)
(n=68):
neoplastic
(n, %)
findings
(n=16) (n, %)
<18
3, ( 13.04 %)
27.27%
8, (10.39%)
7.529,2
0.963
5 ,(7.35%)
4, ( 25%)
2,( 12.5%)
11, (11%)
18-25
14, ( 60.87%)
30.43%
32, (41.56%)
1.057,2
0.001*
27, (39.71%)
8,( 50%)
11, (68.75%)
46, (46%)
>25
6, (26.09%)
13.95%
37, (48.1%)
8.524,2
0.582
36, (52.94%)
4,( 25%)
3, (18.75%)
43, (43%)
*Significant at p<0.05, Fisher’s Exact TestA significant difference was observed between the HPV-positive cases and negative samplesin terms of coitarche (years) for 18–25 years(p <0.001) www.microbiologyjournal.org 2285 Chaudhury et al | J Pure Appl Microbiol. 2023;17(4):2281-2294. https://doi.org/10.22207/JPAM.17.4.25 www.microbiologyjournal.org
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urnal of Pure and Applied Microbiology
Table 6. Journal of Pure and Applied Microbiology HPV-16 and -18 genotyping by qPCRi Distribution of cases according to the parity
Parity
According to RT-PCR report Statistical Test Cytological findings
Total samples
(n=100)
HPV positive
Rate of
(Negative
Fisher’s Exact
P value NILM (n=84)
Epithelial cell
n, (%)
cases (n=23)
positivity
samples
Test value,
abnormalities
(n, %)
(n, %)
n=77) :
df
Organism
Other non
(n=16) (n, %)
(n, %)
(n=68):
neoplastic
(n, %)
findings
(n=16) (n, %)
P1+0
2, ( 8.7 %)
14.29%
12,( 15.58%)
11.574,3
0.452
11 ,(16.18%)
2, ( 12.5%)
1, ( 6.25%)
14, (14%)
P2+0
5 , (21.74 %)
12.82%
34,(44.16%)
7.853,3
0.836
32 ,(47.06%)
3, (18.75%)
4, ( 25%)
39, (39%)
P3+0
13, ( 56.52 %)
37.14%
22,(28.57%)
8.414,3
0.023*
17, (25%)
8, (50%)
10, (62.5%)
35, (35%)
P4+0
3 , (13.04 %)
25%
9,(11.69%)
4.617,3
2.547
8, (11.76%)
3, (18.75%)
1, (6.25%)
12, (12%)
between the groups in terms of parity for P3+0(p = 0.023). Table 7. Distribution of cases according to occupation
Occupation
According to RT-PCR report Statistical Test Cytological findings
Total samples
(n=100)
HPV positive
Rate of
(Negative
Fisher’s Exact
P value NILM (n=84)
Epithelial cell
n, (%)
cases (n=23)
positivity
samples
Test value,
abnormalities
(n, %)
(n, %)
n=77) :
df
Organism
Other non
(n=16) (n, %)
(n, %)
(n=68):
neoplastic
(n, %)
findings
(n=16) (n, %)
Agriculture
6, (26.1%)
24%
19, (24.68%)
5.367,4
3.245
18, ( 26.47%)
4, ( 25%)
4, ( 25%)
25, (25%)
Daily worker
10, (43.48%)
27.78%
26, (33.77%)
11.257,4
1.254
25, (36.76%)
6, (37.5%)
6, (37.5%)
36, (36%)
Teacher
2, (8.7%)
14.29%
12, (15.58%)
6.392,4
2.581
10, (14.71%)
1, ( 6.25%)
1, ( 6.25%)
14, (14%)
House wives
4, (17.39%)
40%
6, (7.79%)
1.257,4
0.235
2, (2.94%)
4, ( 25%)
4, ( 25%)
10, (10%)
Trade
1,(4.35%)
6.67%
14, (18.18%)
2.365,4
0.743
13, ( 19.12%)
1, (6.25%)
1, ( 6.25%)
15, (15%)
*Significant at p<0.05, Fisher’s Exact Test; No significant difference was observed between the groups in terms of occupation Journal of Pure and Applied Microbiology www.microbiologyjournal.org 2286 Chaudhury et al | J Pure Appl Microbiol. 2023;17(4):2281-2294. https://doi.org/10.22207/JPAM.17.4.25 www.microbiologyjournal.org
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Journal of Pure and Applied Microbiology
Table 8. Journal of Pure and Applied Microbiology HPV-16 and -18 genotyping by qPCRi Distribution of cases according to monthly income
Monthly
According to RT-PCR report Statistical Test Cytological findings
Total samples
income
(n=100)
HPV positive
Rate of
(Negative
Fisher’s Exact
P value NILM (n=84)
Epithelial cell
n, (%)
cases (n=23)
positivity
samples
Test value,
abnormalities
(n, %)
(n, %)
n=77) :
df
Organism
Other non
(n=16) (n, %)
(n, %)
(n=68):
neoplastic
(n, %)
findings
(n=16) (n, %)
<=10000/-
11,(48%)
23.91%
35,(45.45%)
7.325,3
2.014
32 ,(47.06%)
6 ,(37.5%)
8, (50%)
46,(46%)
>10000-20000/-
8 ,(35%)
36.36%
14, (18.18%)
2.853,3
0.125
11 ,(16.18%)
6,(37.5%)
5,(31.25%)
22, (22%)
>20000-30000/-
3 ,(13%)
12.5 %
21,(27.27%)
4.258,3
0.532
19,( 27.94%)
3 ,(18.75%)
2,( 12.5%)
24, (24%)
>30000 /-
1 ,(4.35%)
12.5%
7,(9.09%)
1.246,3
0.412
6, (8.82%)
1,( 6.25%)
1,( 6.25%)
8, (8%)
*Significant at p<0.05, Fisher’s Exact Test; In addition, nosignificant difference was observed between the groups in terms of monthly income
Table 9. Distribution of cases according to the use of contraceptive methods
Contraceptive
According to RT-PCR report Statistical Test Cytological findings
Total samples
methods
(n=100)
HPV positive
Rate of
(Negative
Fisher’s Exact
P value NILM (n=84)
Epithelial cell
n, (%)
cases (n=23)
positivity
samples
Test value,
abnormalities
(n, %)
(n, %)
n=77) :
df
Organism
Other non
(n=16) (n, %)
(n, %)
(n=68):
neoplastic
(n, %)
findings
(n=16) (n, %)
Barrier methods 8 (34.78%)
25.81%
23 (29.87%)
6.244,2
1.249
23 (33.82%)
3 (18.75%)
5 ( 31.25%)
31, (31%)
Non-barrier
12 (52.17%)
23.53%
39(50.64%)
3.248,2
0.034*
30,(44.12%)
11 (68.75%)
10, (62.5%)
51, (51%)
methods
Not used any
3 (13.04%)
16.66%
15(19.48%)
1.946,2
0.001*
15,(22.06%)
2( 12.5%)
1( 6.25%)
18, (18%)
contraceptive
*Significant at p<0.05, Fisher’s Exact Test; However, a significant difference was observed between the HPV-positive cases and the negative samplesin terms of the use of non-barrier
methods of contraception compared to not–used any method of contraceptive (p = 0.034 and p <0.001, respectively) 2287 Chaudhury et al | J Pure Appl Microbiol. 2023;17(4):2281-2294. https://doi.org/10.22207/JPAM.17.4.25 www.microbiologyjournal.org
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Journal of Pure and Applied Microbiology
Table 10. Journal of Pure and Applied Microbiology HPV-16 and -18 genotyping by qPCRi Frequency of distribution of cases according to the age group (n=100)
Age groups
According to RT-PCR report Statistical Test Cytological findings
Total samples
in years
(n=100)
HPV positive
Rate of
(Negative
Fisher’s Exact
P value NILM (n=84)
Epithelial cell
n, (%)
cases (n=23)
positivity
samples
Test value,
abnormalities
(n, %)
(n, %)
n=77) :
df
Organism
Other non
(n=16) (n, %)
(n, %)
(n=68):
neoplastic
(n, %)
findings
(n=16) (n, %)
<20-30 years
4 (17.39%)
14.81%
23 (29.87%)
2.148,3
2.142
22(32.35%)
2( 12.5%)
3 (18.75%)
27, (27%)
31-40 years
5 (21.74%)
19.23%
21,(27.27%)
2.127,3
0.274
21 (30.88%)
3 (18.75%)
2( 12.5%)
26, (26%)
41-50 years
11 (47.83%)
28.95%
27 (35.06%)
3.582,3
0.965
19 (27.94%)
9 (56.25%)
10 (62.5%)
38, (38%)
>50 years
3 (13.04%)
33.33%
6,(7.79%)
4.125,4
0.716
6(8.82%)
2( 12.5%)
1(6.25%)
9, (9%)
*Significant at p<0.05, Fisher’s Exact Test; No significant difference was observed between the groups in terms of age. Table 11. Distribution of cases according to frequency of presenting symptoms
Symptoms
According to RT-PCR report Statistical Test Cytological findings
Total samples
(n=100)
HPV positive
Rate of
(Negative
Fisher’s Exact
P value NILM (n=84)
Epithelial cell
n, (%)
cases (n=23)
positivity
samples
Test value,
abnormalities
(n, %)
(n, %)
n=77) :
df
Organism
Other non
(n=16) (n, %)
(n, %)
(n=68):
neoplastic
(n, %)
findings
(n=16) (n, %)
White discharge
12, (52.17%)
40%
30, (38.96%)
2.654,4
0.039*
26, (38.24%)
8 ,(50%)
8, (50%)
42, (42%)
Post coital bleeding
4 ,(17.39%)
23.53%
17,(22.08%)
3.651,4
1.022
17, (25%)
2 ,( 12.5%)
2, ( 12.5%)
21, (21%)
Menstrual irregularity
2, (8.7%)
28.57%
7,(9.09%)
1.247,4
0.026*
7, (10.29%)
1, ( 6.25%)
1, ( 6.25%)
9, (9%)
Post menopausal
1, (4.35%)
9.09%
11, (14.29%)
1.582,4
0.964
9, (13.24%)
2, ( 12.5%)
1, ( 6.25%)
12, (12%)
bleeding
Abdominal pain
4, (17.39%)
33.33%
12, (15.58%)
1.381,4
0.001*
9, (13.24%)
3, (18.75%)
4 , (25%)
16, (16%)
*Significant at p<0.05, Fisher’s Exact Test; Finally, asignificant difference was observed between the HPV-positive cases and the negative samplesin terms of white discharge, menstrual irregularity,
and abdominal pain (p = 0.039, p = 0.026, and p <0.001, respectively) 2288 Chaudhury et al | J Pure Appl Microbiol. 2023;17(4):2281-2294. https://doi.org/10.22207/JPAM.17.4.25 designed to cover the required positions using the
corresponding forward and reverse primers (Table
3). HPV-16 and -18 genotyping by qPCRi Subsequently, other two primers were used for
the detection of HPV E7 gene for the genotyping
of HPV-18 types (Table 3).9 qPCR was performed
using Emerald Amp® GT PCR Master Mix (RR310B;
TAKARA Bio.). Briefly, in a reaction volume of 50
µl, 100 ng of template DNA was mixed with 25 µl
of 2׳Emerald Amp GT PCR Master Mix, 0.8 µl of
2 mM MgCl2, and 0.2 M (final concentration) of
both forward and reverse primers;the remaining
volume was comprised of ultra pure molecular
grade water. The cycling conditions for qPCR
were as follows: initial denaturation at 95°C (3
min), 35 cycles including denaturation at 94°C
(30 s), annealing at 56°C (30 s), and elongation at
72°C (45 s), followed by 72°C in 5 min. Aliquots
of the amplified PCR products were run in 1.0%
agarose gel stained with ethidium bromide. Since all the amplified products had different lengths,
genotypes of the virus were analyzed by using
electrophoresis and visualized via an ultraviolet
light trap-illuminator.11 Bands of appropriate size
were identified by comparing with DNA molecular
weight markers. The adequacy of the DNA in each
specimen for PCR amplification was determined by
the detection of the housekeeping gene β-actin. Journal of Pure and Applied Microbiology RESULTS The prevalence of HPV infection in 100
samples was 23% (n= 23), among which the
most prevalent genotype was HPV-16 (76.91%,
n= 17), followed by HPV-18 (26.1%, n=6). Mixed
infections of both HPV-16 and -18 were identified
in 21.74% (n=5) of the samples. Among the total
HPV positive cases, 34.78% patients had reported
an underlying comorbidity of anemia, followed
by 26.09% with diabetes mellitus (Table 4). Among the patients,60.87% had their first sexual
encounter at an age of 18–25 years (Table 5). The rate of HPV positivity was higher in women
who had experienced multiple pregnancies
(Table 6). Among those patients positive for HPV
infection,43.48% were daily workers,followed by
26.1% who were involved in agricultural labor
(Table 7). In terms of the monthly income of
patients positive for HPV infection, the monthly
income of 48% of these patients was ≤10,000
rupees, whereas 35% had a monthly income
>10,000-20,000 rupees (Table 8). In terms of
the type of contraception method, used by HPV
positive women, 52.17% used non barrier methods
of contraception, while 34.78% used barrier
methods of contraception (Table 9). The highest
prevalence (47.83%)of HPV infection was observed
among patients aged 41–50 years, followed by
21.74%in patients aged 31-40 years (Table 10). Among the patients positive for HPV, 52.17%
reported experiencing white discharge over a long
time, followed by 17.39% who reported a history
of post-coital bleeding and abdominal pain (Table
11). A comparison between the RT-PCR results and
the pap smear samples is provided in Table 12. The
sensitivity, specificity,and positive predictive value
of RT-PCR method were significantly higher the pap
smear (72.53% vs.65.29%, 98.14% vs. 91.76%, and
82.91% vs.70.86%, respectively). Many types of oncogenic cancer are
prevalent worldwide, among which the seventh
most common type of cancer is cervical cancer,
which is the fourth most prevalent type in
women.14 Approximately 85% of cervical cancer
cases are found in developing and underdeveloped
countries.15 The worldwide prevalence of cervical
cancer is 11.7%, with countries including South
Africa (17.4.0%), Eastern Africa (33.6%), Eastern
Europe (21.4%), Western Europe (9.0%), Eastern
Europe (21.4%), and the Caribbean (35.4%)
reporting the highest rates of HPV prevalence.16,17
In this study, the prevalence of HPV infection
among a cohort of 100 patients clinically suspected
of having cervical cancer in Burdwan, India,
was 23%. Similarly,another study conducted
in Southern India by Franceschi reported a
prevalence of HPV infection of 16.9%.18 However,
in a study by Senapati et al. Real-time PCR of HPV-16 and -18 using SYBR
Greeni Real-time PCR of HPV-16 and -18 using SYBR
Greeni Positive samples for L1 obtained through
a previous round of qPCR were screened for their
HPV type since the L1 gene is a conserved region in
different HPV types, including HPV-16 and -18.10,11
The quantitative Sybr Green TB Green Premix
Ex Taq II (TiRNase H Plus) was used to perform
real-time PCR targeting the E6 and E7 regions of
HPV. Briefly, in a reaction volume of 25 µl, 1xTB
Green Premix Master Mix was supplemented
with generic primers of a short sequence of the
HPV E6 and E7 genes. HPV16 and HPV18 have
highly preserved regions. 10,11 The following cycling
conditions were used: initial denaturation at 95°C
for 2 min, followed by 38 cycles of alternating
incubation periods at 95°C for 5 s, at 56°C for 30
s, and at 72°C for 10 s. After each 72°C extension
incubation, fluorescence was recorded. Positive
clinical samples derived from HPV-L1 genes
were chosen for this approach, confirming the
presence of HPV-16 and HPV-18 type viral DNA. As previously mentioned, the adequacy of the
DNA in each specimen for PCR amplification was
determined by the detection of the housekeeping
gen β-actin as control. Figure 3. Gel Electrophoresis image of HPV L1, E6 and E7 Figure 3. Gel Electrophoresis image of HPV L1, E6 and E7 www.microbiologyjournal.org
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Journal of Pure and Applied Microbiology
Table 12. Comparison between RTPCR with pap smear (N=100)
Screening
Sensitivity
Specificity
Accuracy
PPV
NPV
result
(%)
(%)
(%)
(positive
(Negative
( n=100)
predictive
predicted
value ) (%)
value ) (%)
RT PCR
n=100
Positive
23
72.53%
98.14%
96.59%
82.91%
96.25%
Negative
77
Pap smear
NILM
84
65.29%
91.76%
89.47%
70.86%
90.19%
Positive
16
(epithelial cell
abnormalities) Table 12. Comparison between RTPCR with pap smear (N=100) 2289 Chaudhury et al | J Pure Appl Microbiol. 2023;17(4):2281-2294. https://doi.org/10.22207/JPAM.17.4.25 DISCUSSION All statistical analyses were performed
using Statistical Package for Social Sciences(SPSS)
(version 23). Fisher’s exact test was used to analyze
categorical data. A p-value of less than 0.05 was
used to indicate statistical significance. Cancer has become one of the largest
burdens on public health in recent decades. Among
the different types of cancer, 20% are known
to originate from viruses, known as oncogenic
viruses.1 Oncogenic viruses are responsible for
transforming normal cells into malignant cells
that, instead of growing in a monolayer, pileup in
multilayers,resulting in the formation of tumors. These cancerous cells encode proteins that
encourage the transformation of normal cells
into cancerous cells, the genetic basis of which is
mutations.11 www.microbiologyjournal.org RESULTS in the eastern region
of India, an overall prevalence of 60.33% was
reported for HPV infection.19 Persistent infection with a high-risk
HPV type is a mandatory factor, although not
the determining reason, for the development of
cervical carcinoma. Premalignant lesions are likely
to transform into malignant lesions if they remain
unidentified in the cervix for a prolonged time
period.3 A study conducted by Centers for Disease
Control and Prevention (CDC) in 2008 revealed
that, each year, despite a large number of HPV
infections (6.2 million), only a small proportion
of women infected with a high-risk HPV type
developed precancerous lesions, with even fewer
developing cervical cancer.20,21 Similar findings 2290 Journal of Pure and Applied Microbiology www.microbiologyjournal.org www.microbiologyjournal.org Chaudhury et al | J Pure Appl Microbiol. 2023;17(4):2281-2294. https://doi.org/10.22207/JPAM.17.4.25 the process of cervical carcinogenesis in individuals
infected with HPV.24,25 were observed in the present study, wherein
among a total of 23 patients positive for HPV
infection, only 39.13% (n=9) developed cervical
cancer. Nutrition also plays a pivotal role in
immunity, which is directly related to HPV
infection. In this study, the highest rate of HPV
infection was observed in patients with a monthly
income <10,000rupees, with some families having
a meager monthly income of 2,000–3,000 rupees,
most of which were daily workers and farmers,
accounting for 43.48% and 26.1% of HPV-positive
cases, respectively. Furthermore, nutrition also
plays an important role in pregnancy. A low
nutritional status is associated with low levels of
antioxidants, such as beta-carotene; lycopene;
and vitamins A, C, and E (alpha-tocopherol)
which can be obtained from fruits and vegetables
and which interact with free radicals to prevent
cancer.26,27 Thus, in individuals with diets that
lackfruit and vegetables,free radicals can damage
cell components,including DNA, protein, and
cell membranes.28Conversely,a healthy and
well-maintained nutritional status can boost the
immune system to counteract this. Various risk factors are known to
accelerate this progression into cervical cancer. In this study, several risk factors related to HPV
infection and cervical cancer were identified,
among which the most important risk factors
were age (>45years), multigravida, low socio-
economic status, post-menopausal state, anemia,
diabetes mellitus, using oral contraceptive pills
(OCP) for many years, and long years of coitarche. RESULTS Lertcharernrit reported similar demographic data,
revealing that HPV infection was more common in
women under the age of 30, with multiple sexual
partners, with a low socioeconomic status, with
sexually transmitted diseases, who did not use
contraceptives, having had multiple pregnancies,
and long years of coitarche and malnutrition.22 In the present study, among the total
HPV-positive cases, the leading comorbidities
were anemia (34.78%), diabetes mellitus (26.09%),
and hypertension (21.74%). Furthermore, as
reported in the literature, nutrition and comprised
immunity due to other co-morbid conditions may
also accelerate the development of precancerous
lesions into cervical cancer.23 Here, we also found that women who use
non-barrier methods of contraception (e.g. OCP)
were more prone to HPV infection. The daily use
of an oral contraceptive for a long period appears
to increase the risk of cervical cancer, which
suggests that the long-term use of OCP is directly
proportional to the risk of cervical cancer. This may
be due to the fact that the levels of estrogen and
progesterone interact with hormones receptors
situated in cervical tissue,thereby playing in role
in susceptibility to HPV infection. Sex steroid
hormones are considered to be responsible
for enhancing the expression of oncogenes (E6
and E7), which triggers the degradation of p53
tumor suppressor genes, there by increasing the
ability of viral DNA to transform cells and induce
carcinogenesis.29-31 Lertcharernrit et al. found that early
coitarche was associated with the onset of HPV
infection.22 Similarly, in the present study, women
who had their first exposure to sexual relations at
the ages of 18–25 were found to be more likely
to be positive for HPV infection. We have also
noted that the women that started having sexual
relations at an early age were more likely to have
many children. These multigravida women were
found to be more prone to HPV infection,which
suggests that multiparity is proportionately related
to HPV infection. As previously suggested, elevated
levels of hormones, particularly progesterone,
at the time of pregnancy causes alterations to
the squamocolumnar junction. Journal of Pure and Applied Microbiology www.microbiologyjournal.org ACKNOWLEDGMENTS The authors would like to thank the
Medical Technologist (Lab), who diligently helped
us to accomplish this study. Cuzick J, Sasieni P, Davies P, et al. A systematic review
of the role of human papilloma virus (HPV) testing
within a cervical screening programme: summary and
conclusions. Br J Cancer. 2000;83(5):561-565. doi:
10.1054/bjoc.2000.1375 FUNDING
None Finally, in this study, approximately
52.17% of HPV-positive women reported
experiencing white discharge for an extended
period of time, followed by 17.39% who had a
history of post-coital bleeding and abdominal pain. Similarly,in a study conducted by Sachan, 36.96%
of women found to be HPV-positive complained
of white discharge during history taking.6i ETHICS STATEMENT Concludingly, the patients at the highest
risk of HPV infection are multigravida-middle
aged Bengali women from a low socioeconomic
background with early coitarche, who had the co-
morbid conditions of anemia or diabetes mellitus,
and who used non-barrier contraceptive methods
(e.g.,OCP). This study was approved by the
Institutional Ethical Committee (IEC) of BMC
and IEC of Burdwan University, vide memo
no.:BMC-1200, dated 25/05/18 and approval
No.:IEC/BU/2019/01, dated 05.07.2019. REFERENCES This study demonstrates that molecular
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the sample collection. NC, SKM, AB, KB and NM
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M, Telser J. Free radicals and antioxidants in normal
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29. International Collaboration of Epidemiological Studies
of Cervical Cancer, Appleby P, Beral V, de Gonzalez AB,
et al. Cervical cancer and hormonal contraceptives:
collaborative reanalysis of individual data for 16,573
women with cervical cancer and 35,509 women
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10.1016/S0140-6736(07)61684-5
dl
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h 28. Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur
M, Telser J. Free radicals and antioxidants in normal
physiological functions and human disease. Int J
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average, 12% of women worldwide had a detectable
cervical HPV infection varying by geography and
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of Cervical Cancer, Appleby P, Beral V, de Gonzalez AB,
et al. Cervical cancer and hormonal contraceptives:
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women with cervical cancer and 35,509 women
without cervical cancer from 24 epidemiological
studies. Lancet. 2007;370(9599):1609-1621. doi:
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conflict of interest The authors declare that there is no
conflict of interest 6. Sachan PL, Singh M, Patel ML, Sachan R. A Study
on Cervical Cancer Screening Using Pap Smear
Test and Clinical Correlation. Asia Pac J Oncol Nurs. Journal of Pure and Applied Microbiology 2292 www.microbiologyjournal.org Chaudhury et al | J Pure Appl Microbiol. 2023;17(4):2281-2294. https://doi.org/10.22207/JPAM.17.4.25 20. Jeon S, Lambert PF. Integration of Human Papillomavirus
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(HPV), genital warts and cervical cancer. J Infect. 2013;66(3):207-217. doi: 10.1016/j.jinf.2012.10.024l 11. 11. Settheetham-Ishida W, Kanjanavirojkul N, Kularbkaew
C, Ishida T. Human papillomavirus genotypes and
the p53 codon 72 polymorphism in cervical cancer
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Hormonal Factors on the Risk of Developing Cervical
Cancer and Pre-Cancer: Results from the EPIC Cohort. PLoS One. 2016;11(1):e0151427. doi: 10.1371/journal. pone.0147029 12. Hesselink AT, Berkhof J, van der Salm ML,et al. Trottier H, Franco EL. Human papillomavirus
and cervical cancer: burden of illness and basis
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Dempsey AF. Human papillomavirus: the usefulness of
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Rev Obstet Gynecol. 2008;1(3):122-128. therapy use and cervical carcinogenesis: a review of
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604. doi: 10.3109/09513590.2011.558953 Castle PE, Schiffman M, Herrero R, et al. A prospective
study of age trends in cervical human papillomavirus
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study of age trends in cervical human papillomavirus
acquisition and persistence in Guanacaste, Costa
Rica. J Infect Dis.2005;191(11):1808-1816. doi:
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604. doi: 10.3109/09513590.2011.558953
32.
Dempsey AF. Human papillomavirus: the usefulness of
risk factors in determining who should get vaccinated.
Rev Obstet Gynecol. 2008;1(3):122-128. CONFLICT OF INTEREST de Sanjose S, Diaz M, Castellsague X, et al. Worldwide
prevalence and genotype distribution of cervical human
papillomavirus DNA in women with normal cytology:
a meta-analysis. Lancet Infect Dis. 2007;7(7):453-459. doi: 10.1016/S1473-3099(07)70158-5 18. Franceschi S, Rajkumar R, Snijders PJF, et al. Papillomavirus infection in rural women in southern
India. Br J Cancer. 2005;92(3):601-606. doi: 10.1038/
sj.bjc.6602348i 30. 30. Moodley M, Moodley J, Chetty R, Herrington CS. The role of steroid contraceptive hormones in the
pathogenesis of invasive cervical cancer: a review. Int J Gynecol Cancer. 2003;13(2):103-110. doi:
10.1046/j.1525-1438.2003.13030.xti 19. 19. Senapati R, Nayak B, Kar SK, Dwibedi B. HPV Genotypes
distribution in Indian women with and without cervical
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Genazzani A. Smoking habit, immune suppression,
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Navigating Violence: Fear and Everyday Life in Colombia and Mexico
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International journal for crime, justice and social democracy
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Abstract Violence and insecurity are often read as totalising narratives of communities in parts of
Latin America, flattening the complexity of everyday life and the responses of occupants
who suffer from fear. In this article we draw on ethnographic research undertaken in los
Altos de Cazucá in Colombia and in San Luis Potosí in Mexico. While both sites are distinct
locations with different historic, economic, social and political contexts they share features
of communities affected by violence and insecurity: distrust of institutions of the state;
rationalisations for managing violence in daily life; and narratives of fear that appear woven
through the fabric of conversations. However, fear and violence are not all‐encompassing
experiences and individuals in both these communities describe practices of navigation of
violence that draw on positive communal experiences. This article explores how, in these
communities where violence comes to be expected but never normalised, people navigate
their everyday lives. Helen Berents, Charlotte ten Have
Queensland University of Technology, Australia Helen Berents, Charlotte ten Have
Queensland University of Technology, Australia Helen Berents, Charlotte ten Have
Queensland University of Technology, Australia Please cite this article as:
Berents H and ten Have C (2017) Navigating violence: Fear and everyday life in Colombia and
Mexico. International Journal for Crime, Justice and Social Democracy 6(1): 103‐117. DOI:
10.5204/ijcjsd.v6i1.373. www.crimejusticejournal.com IJCJ&SD 2017 6(1): 103‐117 www.crimejusticejournal.com IJCJ&SD 2017 6(1): 103‐117 ISSN 2202–8005 Navigating Violence: Fear and Everyday Life in
Colombia and Mexico This work is licensed under a Creative Commons Attribution 4.0 Licence. As an
open access journal, articles are free to use, with proper attribution, in educational and other non‐
commercial settings. ISSN: 2202‐8005 Introduction Both Colombians and Mexicans have long experienced the effects of violence in their countries. While Colombia’s conflict has lasted more than half a century the parties to the conflict, both the
leftist guerrillas and the right‐wing paramilitary organisations (as well as corrupt state officials)
have also become involved in drugs and arms trafficking (Palacios 2006). The consequences of
five decades of conflict and violence have been large scale displacement (approximately six
million people (Edwards 2016) or approximately one in ten Colombians (Internal Displacement
Monitoring Centre 2015) have been displaced due to the conflict), the wide scale abnegation of
human rights, underdevelopment, poverty, and the undermining of the government’s authority
and capacity to serve all its citizens (see, amongst others, Bouvier 2009; Pecaut 2006; Profamilia
2005; Richani 2002; Romero 2007). In parts of the country the state has lost its authority and
illegal groups control communities. Since 2012 there has been a peace process underway
between the government and the leftist guerrillas, the Revolutionary Armed Forces of Colombia
(known by their Spanish acronym of FARC); however, however, even with a peace deal signed in
late 2016 and being implemented, insecurity and inequality will persist in many places. In Mexico the self‐declared war on organised crime and drugs launched by President Felipe
Calderón, newly elected in 2006, has seen an unprecedented wave of violence and insecurity
convulse the nation (Morris 2009). Following the highly disputed elections Calderón dispatched
tens of thousands of military troops and federal police to key narcotic‐trafficking states during
his presidency, resulting in an estimated 60,000 to 70,000 deaths with a further 26,000 people
missing (Gayón 2015; Rosen and Martínez 2014). In more recent years civilians, authorities and
journalists have increasingly become the target of the growing insecurity with drug cartels
branching out into extortion, human smuggling and kidnapping. This is to the exclusion of the
extensive list of violations attributed directly to state forces (Beittel 2011; Human Rights Watch
2015). Similar to Colombia in the 1990s, the Mexican state has lost its monopoly over the use of
force which is evidenced by the private armed groups, self‐defence groups, corrupt state forces
and drug trafficking organisation which also routinely use violence as a tool. As a result Mexican
citizens have become trapped in networks of extortion and coercion, in which both state forces
and criminal organisations prey with impunity (Magaloni et al. 2015: 28). Keywords Violence; social navigation; Colombia; Mexico; insecurity; everyday life. This work is licensed under a Creative Commons Attribution 4.0 Licence. As an
open access journal, articles are free to use, with proper attribution, in educational and other non‐
commercial settings. ISSN: 2202‐8005 © The Author(s) 2017 Helen Berents, Charlotte ten Have: Navigating Violence: Fear and Everyday Life in Colombia and Mexico Helen Berents, Charlotte ten Have: Navigating Violence: Fear and Everyday Life in Colombia and Mexico Online version via www.crimejusticejournal.com Introduction In both countries the violence has affected people unevenly but both situations are also
characterised by widespread insecurity, the targeting of individuals such as human rights
advocates and journalists (Molzahn et al. 2012; Human Rights Watch 2014; Tate 2007), the
prevalence of stories of death, torture and violence in print and visual media (Brambila 2014),
and the undermining of a sense of security and of rights. This article draws upon ethnographic research conducted by the authors, one working in an
informal community on the outskirts of Colombia’s capital Bogotá, and the other in the
metropolitan area of San Luis Potosí City, Mexico. While the sites differ in a range of significant
ways including size and demographic features, occupants of both communities demonstrate the
cost of living with fear and insecurity, the impact of violence, and the techniques people adopt to
navigate their daily lives. We challenge both a flattened narrative of violence as totalising and an understanding of violence
as ever normalised, which is sometimes implicit in engagements with the global South. Instead
we propose a way of understanding the experiences of structural and direct violence that takes
its starting point with the experiences of individuals who have learned to navigate their everyday
lives despite, and because of, the insecurity and fear that impacts their lives. We offer this as a
way of countering the ideas that these kinds of places that experience violence are inherently
violent, or that sites of conflict and insecurity in the global South are intrinsically or totally sites
of despair or danger, or that people are nothing more than passive victims in the face of violence
in their daily lives. The forms of navigation – of agency and decision‐making – that we explore 104 IJCJ&SD
© 2017 6(1) Online version via www.crimejusticejournal.com Helen Berents, Charlotte ten Have: Navigating Violence: Fear and Everyday Life in Colombia and Mexico here are generally mundane; they are practices in everyday life of moving through insecure and
unpredictable terrains. The occupants of both communities recognise the challenges of living
with violence and, as we discuss below, have developed strategies built on communication with
other community members, on‐the‐spot decision making, and weighing of risk, to become skilled
navigators in these contexts. Introduction We argue that attention to the often small, everyday agency
exercised by individuals offers alternative perceptions of communities and circumstances
provided by other research in similar locations and situations where those affected by violence
collectively and openly organise against violence. We do not wish to suggest that the individuals
in either community are passive; rather, their agency is shaped by their experiences of insecurity
and violence and, in different ways, they navigate through and over these uncertain seas. To do this, we briefly introduce the two sites, explain our research processes and situate
ourselves as researchers; we then proceeds in two parts. In the first part we explore notions of
violence and fear to theorise them as complex and ever‐changing experiences. While violence can
break individuals and communal bonds, it is not a normal or totalising experience. We also
forward here a notion of a skilled navigator, drawing on Henrik Vigh’s (2009) work. In the second
part we explore accounts of structural and direct violence experienced by participants in both
Colombia and Mexico and the consequences for their lives. These experiences form the basis for
understanding these individuals as skilled navigators of their everyday lives. In this article we explore how, in these sites, violence, fear and distrust are not extra‐ordinary
conditions but part of everyday life. In acknowledging this, we do not seek to exoticise the
communities presented in this research or reinforce a totalising narrative. Rather, we aspire to
prompt more critical reflection on how these topics are described and understood, and argue for
the capacity of individuals to navigate their everyday lives in ways that respond to and ameliorate
the effects of violence and insecurity. Research process and positionality Original research for both sites was conducted independently for two different projects and is
brought together in conversation here.1 In both cases the researchers spent several months in the
community and adopted ethnographic methods. Such research is predicated on respect for those
involved, recognition of expertise of the local community in issues that affect them, and
commitment to reflexively engage in the complexity of lived experiences of participants. All
names used here are pseudonyms. Both researchers speak Spanish fluently and research was
conducted in Spanish in Mexico and Colombia. In Colombia Helen Berents spent time in the community of los Altos de Cazucá between
September and December 2010 participating in daily life through a school in the community and
a foundation, Fundacion Pies Descalzos, which supported students through school and community
engagements. This original research was focused on young people’s responses to violence and
construction of peace, and thus youth formed the bulk of the fieldwork data quoted here. Observation and, later, interviews with young people, their parents, teachers and guardians as
well as other key community figures informed the research. In Mexico Charlotte ten Have conducted fieldwork between January and March 2013 with the use
of participant observation and qualitative focus groups, and individual interviews. The first two
months of fieldwork were conducted in San Luis Potosí and the last month in Guanajuato, with
frequent visits back to the original fieldwork site. The Universidad Autónoma de San Luis Potosí,
and the help of Dr Daniel Solís Domínguez were crucial in gaining access to respondents. The
majority of the interviews were conducted at the university and two high schools in lower socio‐
economic suburbs of the city. The original research focused on coping mechanisms in relation to
increasing violence. There are important considerations in undertaking research of this nature, which we
acknowledge and respect. As two researchers from the global North (Australia and the
Netherlands), conducting research in the global South requires recognition of perhaps uneven
power relations and privileges that cannot be ‘solved’. There has been excellent research
conducted by academics from within these countries about the situations we research, and
avoiding extractive, disrespectful research was a key consideration for us both. Fieldwork sites As a result of this unprecedented increase, San Luis 105 IJCJ&SD
© 2017 6(1) Online version via www.crimejusticejournal.com Helen Berents, Charlotte ten Have: Navigating Violence: Fear and Everyday Life in Colombia and Mexico Helen Berents, Charlotte ten Have: Navigating Violence: Fear and Everyday Life in Colombia and Mexico Helen Berents, Charlotte ten Have: Navigating Violence: Fear and Everyday Life in Colombia and Mexico Potosí was added to the list of Mexico’s most dangerous cities in the 2013 National Social Plan of
Prevention of Violence and Crime. During daytime, however, the only visible aspect of the
violence were the posters of missing persons in the city and the enormous show of force by the
many pick‐up trucks with masked municipal, state and federal police forces carrying automatic
weapons. At a first glance life seemed to continue as normal in the vibrant colonial city centre
with its many bookstores, busy public transport and many food carts but, despite this first
appearance, the insecurity had seeped through into an underlying layer that deeply impacted
everyday life in the form of fear and coping mechanisms. Violence was thus more often a visceral
than a visual experience; nonetheless, one that was constantly present and constantly changing. Within this situation San Luis Potosí occupants, the Potosínos, had become knowledgeable and
skillful in moving with and through this uncertain environment to continue their daily routines. Despite the differences between the two communities and their broader contexts, we argue that
it is fruitful to bring together the experiences of these two communities to explore occupants’
navigation of everyday life in situations of insecurity and unpredictable violence. Fieldwork sites In Colombia, the community of los Altos de Cazucá (often referred to simply as Cazucá) is an
illegally established community that forms part of Comuna 4 of the municipality of Soacha, on the
periphery of Bogotá. There are over 60,000 inhabitants of Comuna 4 (Alcaldia Municipial de
Soacha 2008: 8). The Comuna is one of the poorest in Colombia; it is also one of the largest
recipient communities for people internally displaced by Colombia’s long‐running conflict. The
poverty and neglect of the community is visible in the lack of paved roads, the ad‐hoc construction
of houses, presence of rubbish, and lack of communal spaces. Most occupants do not have running
water, and access to government services is often complex and fraught. Fear of violence by armed
gangs is a constant concern of the occupants, and the violence and altercations between gangs
and state forces paint a picture – for those outside the space of the community – of endemic
violence and delinquent or threatening occupants. Despite these challenges, such a description is
not a totalising account of a community that for the most part is comprised of families trying to
live day‐to‐day, often displaced, negotiating poverty and the absence of state care (see Berents
2015). There is pride in the community and a desire to counter negative narratives. San Luis Potosí in Mexico is the state capital of San Luis Potosí State and has a population of
approximately 772,604 inhabitants. The city is located halfway between Mexico City and the
United States border and is in the middle of the triangle formed by Mexico City and the two other
largest cities in Mexico, Guadalajara and Monterrey. While the city’s central location has made it
an attractive location for legitimate investment, its good connections to the tumultuous northern
regions, and ready access to the main railroad to the United States has also attracted criminal
organisations. The region remained reasonably stable until the drug‐related violence increased
by 49 per cent in the first four months of 2013, just before and during the fieldwork period
(Overseas Security Advisory Council 2013). Complex notions of violence and fear Violence is complex and the ways in which people respond to violence are also complex. Scheper‐
Hughes and Bourgois (2003: 2) argue that ‘violence defies easy categorization’, and while
violence performed on ‘grand’ scales such as war, genocide or state‐sanctioned oppression are
‘graphic and transparent’, it is the ‘everyday violence of infant mortality, slow starvation, disease,
despair, and humiliation that destroys socially marginalized humans with even greater frequency
[that] are usually invisible or misrecognized’. We acknowledge that both forms of violence – the
‘grand’ ‘graphic’ violence and the ‘usually invisible’ everyday violence – affect the lives of those in
both communities. Violence, then, is not only deliberate and conscious but also arises from
systems of oppression that are maintained and condoned through the maintenance of particular
social orders (Farmer 2004: 307). Such an understanding is also linked to Galtung’s (1990) three
pillars of violence. Galtung sees ‘direct violence’, such as murder or physical abuse, as one pillar. Secondly, ‘structural violence’ recognises that things such as inequality, repression and exclusion
are part of structures that enable violence. Finally, ‘cultural violence’ for Galtung legitimises direct
and structural violence through cultural norms. Galtung (1990: 294) argues that ‘direct violence
is an event; structural violence is a process with ups‐and‐downs and cultural violence is an
invariant or a permanence remaining essentially the same for long periods, given the slow
transformations of basic culture’. Violence also has an emotional component. The threat or risk of violence creates an atmosphere
of fear and insecurity. Fear spreads distrust and breaks communal bonds as people fear speaking
up and thus suffer further. This self‐censorship not only occurs in the public space but also filters
into the private sphere. Beyond the direct human suffering caused by conflict and drug‐related
violence, violence thus also undermines people’s sense of security (Morris 2009). In such
environments ‘fear, like pain, is overwhelmingly present to the person experiencing it, but it may
be barely perceptible to anyone else and almost defies objectification. Subjectively the mundane
experience of chronic fear wears down one’s sensibility to it’ (Green 1994: 230). The concept of
‘pain’, in this sense, is introduced as a bridging notion between violence and fear or, rather, as
part of a relationship between violence and fear. In such situations pain can be emotional as well
as physical. People hide their pain in response to fear and the silencing impact violence can have. Theorising violence, fear and navigation In this section we explore theorisations of violence and fear, understanding them as neither static
nor simplistic but, instead, as complex, kinetic processes which impact people and to which
people respond. Violence and fear can silence, debilitate and deconstruct people and
relationships (Koonings and Kruijt 1999). However, in this section we also forward a notion of
navigation. Building on Vigh’s (2009) work, we explore navigation as a way of understanding
people’s everyday responses to violence and fear. Such a configuration challenges the passive
construction implied by ‘coping’ and, alternatively, recognises agency and capacity. Through this
theoretical exploration we arrive at the notion of a skilled navigator, a person who moves through
experiences of violence and insecurity by drawing on their knowledge and expertise. Research process and positionality Engaging with
important local research – not to assimilate it or extract that which is useful within our
epistemological framework but to challenge our understanding, our assumptions – is part of an
academic and creative practice that can perhaps respond to the inadequacies of North‐to‐South
research that remains extractive and colonial.2 IJCJ&SD
© 2017 6(1) 106 Online version via www.crimejusticejournal.com Helen Berents, Charlotte ten Have: Navigating Violence: Fear and Everyday Life in Colombia and Mexico Helen Berents, Charlotte ten Have: Navigating Violence: Fear and Everyday Life in Colombia and Mexico Ethnographic research can, to some extent, mediate these expectations as the researcher
becomes better understood by the community, as they come to understand the community.3 As
part of the social world under study, reflexivity allows us to acknowledge and question what role
we play as researchers and beyond (see Gouldner 1971). Recognising our positionality we
acknowledge we can never avoid it, and accept that we may be unable to fully move beyond
understandings rooted in the traditions of the global North. However, we offer these explorations
both in terms of methodological reflexivity here, as well as within our theoretical engagement by
foregrounding the voices and experiences of those from Colombia and Mexico who shared parts
of their experiences with us, and concede partiality in this endeavour. Complex notions of violence and fear Elaine Scarry notes that violence is a silencing act: ‘pain does not simply resist language but 107 IJCJ&SD
© 2017 6(1) Online version via www.crimejusticejournal.com elen Berents, Charlotte ten Have: Navigating Violence: Fear and Everyday Life in Colombia and Mex actively destroys it’ (1985: 4). The threat or previous experience of violence silences people who
fear further violence. Fear of being a victim of violence pervades all the inhabitants of a city. However, the impact is not
equally distributed throughout the city’s geographic area. The poor, for example, are
disproportionately victimised (Briceño‐León and Zubillaga 2002). Green (1994: 227), in writing
about Guatemala, argues that fear ‘penetrat[es] the social memory’ and, thus, rather than being
an acute, individualised response, becomes a ‘chronic condition’. Such an understanding of fear
as linked to violence evidences the complexity of the rubric of insecurity, violence and fear within
which the occupants of San Luis Potosí and los Altos de Cazucá exist. In these communities,
everyday life is uncertain; while occupants can anticipate violence and insecurity, they cannot
predict it. Michael Taussig calls such situations ‘the nervous system’: ‘illusions of order, congealed
by fear’ (1992: 2) where social life is defined by constant unease, and ‘attention towards change
in the shape of possible acts of power and social forces’ (Vigh 2009: 422). Vigh (2009: 422)
explains that the nervous system in Taussig’s configuration refers to both an unbalanced system
(one that acts nervously) and a ‘sensory faculty, constantly focused on movement and necessarily
feeling its way through unsettled environments’. Thus, the violences and insecurity of these environments and the fear and pain provoked are not
experienced as acute or isolated episodes. Violence and fear here can be understood as a chronic
condition, never normalised but as an expected part of daily life. It is important to emphasise
violence is not a totalising experience – people find multiple ways of responding to and resisting
the totalisation of violence, as we discuss below – but part of an ongoing, daily existence, to be
managed like chronic pain or malady. Navigation of everyday violence The experience of multiple forms of violence and the accompanying fear create an insecure
topography, one which people must navigate in their everyday lives. In this, we forward the
specific idea of ‘navigation’ of violence, as distinct from ‘coping’ or ‘normalising’ violence. We take
the idea from Vigh (2009: 420) who engages the idea of ‘social navigation’, understood as a
concept that: … highlights motion within motion; it is the act of moving in an environment that
is wavering and unsettled, and when used to illuminate social life it directs our
attention to the fact that we move in social environments of actors and actants,
individuals and institutions, that engage and move us as we move along. If we conceive of those impacted by violence and insecurity not as individuals fixed within a
landscape in which they are battered by external forces of violence – structural, direct, cultural –
but, rather, as individuals and collectives who move through and between an insecure,
threatening topography, and who navigate social relationships, insecure terrain and
unpredictable violence, we recognise that those impacted by violence are not passive. Although
they lack structural power to effect large scale change, their everyday navigation is vital to
maintaining daily life and resisting violence beyond their control. Navigation, as Vigh conceives of it, is not only topographical but also temporal. Social navigation
‘encompasses both the assessment of the dangers and possibilities of one’s present position as
well as the process of plotting and attempting to actualise routes into an uncertain and
changeable future’ (Vigh 2009: 425). This relies on a notion of radical interactivity, of ‘motion
within motion’ that involves ‘implicating oneself in the ongoing life of the social and material
world’ (Guyer 2007: 409). It is important to recognise that the landscape, the topography, in
which people are navigating is similarly not fixed but constantly shifting. Navigation, in its Latin
origins refers to wayfaring on the sea (Vigh 2009) and this gives us an analogy that is useful in 108 IJCJ&SD
© 2017 6(1) Online version via www.crimejusticejournal.com Helen Berents, Charlotte ten Have: Navigating Violence: Fear and Everyday Life in Colombia and Mexico imagining the shifting, swelling, choppy, even stagnant, ocean of everyday life navigated by
individuals and communities. Lefebvre (1991: 91‐92) argues for a similar conceptualisation of
our social relations and spaces as ‘complex mobilities’ that form ‘convergence[s] of waves and
currents’. Navigation of everyday violence This is not to minimise the crucial fact that, to extend the metaphor, some individuals
have better vessels or find themselves on calmer seas. The ‘intensity and visibility of our
navigational efforts’, argues Vigh, is dependent on ‘the speed and/or opacity of social change and
our ability to control oncoming movement’ (2009: 430). Structural forces can limit this; however,
knowledge of a local topography and temporality of violence and insecurity can enable better
navigation. We forward this notion of navigation of everyday violence, adopted from Vigh (2009) combined
with others, to recognise that those affected by violence and fear are not without capacity to
respond, resist and reform their everyday ‘seascapes’. Using this notion, which builds from a
scaffold of key work in the area of everyday violences and social suffering, we now turn to explore
how this manifests in the communities of los Altos de Cazucá and San Luis de Potosí. Everyday expertise: San Luis Potosí and los Altos de Cazucá In both Colombia and Mexico participants have a fraught relationship with the institutions of the
state and illegal actors. While the police are present in both communities, individuals expressed
strong reservations about seeking their help and, in both sites, explained their distrust of agents
of the state who frequently not only fail to provide security but often act in ways which exacerbate
the insecurity of people. In los Altos de Cazucá most participants mentioned unpredictability of
the police and, in San Luis Potosí, respondents tried to avoid having to interact or be confronted
with state forces. In Cazucá, a conversation with two young women included the following
exchange: Laura (aged 17 years): ... the police are useless here on the hill (la loma). Their only
use is as decoration. That’s the truth. Helen: So the police are part of the problem? Charlotte: Do you feel that the government is taking care of you? Vanessa: Are you kidding, no! Just imagine, to me I am more ... and I live more ... Yes, it frightens me more and makes me more nervous to see a police officer than
knowing someone is ‘‘bad’’ because of the abuse of power. Really the state does not
give protection in any way, ever. When people come to visit I tell them to be careful,
and to be even more careful with the police. There are thousands of cases of people
who are in jail for things that never happened ... Or at least things that they passed. And that is what I fear most, because the narcos ... Well, as a woman I fear being
raped, I guess ... But if they kill me, well at least then that’s it. Living in a society afflicted by violent crime is one thing; having to also cope with state institutions
that neither deliver nor are transparent, nor can be minimally trusted, aggravates people’s
predicaments to such an extent that they avoid protection from the state rather than seek reliable
information or protection from it. In Cazucá the territory is contested by gangs associated with actors in the broader conflict and
the security apparatus of the state. This creates severe insecurity for occupants (which oftentimes
repeats the insecurity and violence from which they have fled). The police maintain a presence in
the community via a van that is parked low on the hillside. Many occupants of Cazucá are deeply
sceptical about police willingness to intervene, as indicated above. When the state does intervene,
it is often sudden and violent, most often typified by night raids on suspected gang members’
houses that often spill over to violence on the street. In addition to the organised threat of gangs,
violence against the property of people is common through robberies at gunpoint or, more
commonly, knifepoint, both on the street and in the house, as well as through acts of vandalism. Specific manifestations of violence in an everyday context are a consequence of the ‘pervasive
indifference, endemic oppression and sense of abjection that can make a person feel as though he
or she is a mere object ... of no account ...’ (Jackson 2002: 44). Other research in Colombia notes
that the police were identified as the main source of mistrust. Helen: So the police are part of the problem? Laura: Sometimes ... Because if you go to them with a problem they just say ‘so
what do you want me to do about it?’ and do nothing. Rosa: (15): It is because the police now, are busy being thieves. They are just
thieves dressed like police. Because they also rob you or say you need to pay for
help from somewhere else. Luis, in San Luis Potosí made a similar observation: ‘When they become too old to be gang
members and are not dead or in jail yet, they become police officers so they can legally fuck with
people’. In San Luis Potosí, as in cities in other countries in the region, few crimes are reported to state
officials as a result of the high levels of impunity (Briceño‐León and Zubillaga 2002). In general,
only homicides and those crimes that require a judicial report to avoid accusation or to collect
private insurance indemnities – as in the case of vehicle theft – are reported. The thousands of
other crimes committed against persons are often not reported and do not find their way into the
statistics. Crimes are not reported because people consider it useless. It will not help them
recover their goods and they do not expect the guilty to be punished (Briceño‐León and Zubillaga
2002). An important side effect of this is that, when official statistics are released, they only
represent a fraction of the crimes that are committed and leave out the ones that are not reported. The assumption that crime statistics become totally unreliable in countries where law 109 IJCJ&SD
© 2017 6(1) Online version via www.crimejusticejournal.com Online version via www.crimejusticejournal.com elen Berents, Charlotte ten Have: Navigating Violence: Fear and Everyday Life in Colombia and Mex enforcement is corrupt, inept or is itself prone to criminal acts is demonstrated here in an
unmistakable way. The distrust of police and the state in San Luis Potosí is evident in this
exchange with Vanessa: enforcement is corrupt, inept or is itself prone to criminal acts is demonstrated here in an
unmistakable way. The distrust of police and the state in San Luis Potosí is evident in this
exchange with Vanessa: Charlotte: Do you feel that the government is taking care of you? Charlotte: Do you feel that the government is taking care of you? Not only would they not provide
‘formal’ security (that they were meant to do as part of their job) but they would also extort
people for bribes, cut deals with local gangs, and become complicit with violent occurrences
(McIlwaine and Moser 2007: 131). An individual’s survival depends on their ‘ability to
comprehend and manage the logics of bureaucratic processes and evade the official norm’
because the community runs via a ‘chain of legalities and illegalities’ that involve local authority
figures and agents of the state (Picon, Arciniegas and Becerra 2006: 14). Such knowledge blurs
the distinctions between Galtung’s (1990) forms of violence, with direct violence being enabled
by the structural violence due to the abnegation of communities and corruption of officials. For
occupants of both researched communities, violence is complex and this knowledge and
management of the intertwined manifestations of violence is an aspect of the successful
navigation of life by occupants in these communities. Expected, but never normal(ised) violence and it’s that, there are so many problems and people can’t realise what is normal,
like they kill someone and it is ‘normal’. It is because of this we need to solve these
problems, the actual situation, because it can change but we need to realise what
is wrong to change it. Thus violence, both structural and direct, also has an emotional, fearful component. Occupants of
both Cazucá and San Luis Potosí explain how those elements of insecurity and violence that they
cannot control deeply affect their interactions in their daily lives and their understandings of self
and community. It is crucial to recognise that violence and insecurity in both these sites is not normal or
normalised. Narratives that tend towards an explanation of place as inherently violent or
predisposed to insecurity create and perpetuate an illusion that violence can and should come to
be expected as a normal part of life. We strongly reject this reading; while many of the participants
in both Colombia and Mexico spoke about the everyday violence and how it is an expected part of
life, none felt that this was something normalised or which should be accepted as normal within
their everyday lives. Expected, but never normal(ised) violence These experiences of violence and insecurity prompt a sense of fear with which people live. As
Green argues, fear becomes a ‘chronic condition’ (1994: 230). In Mexico Vanessa observes how
violence comes to be expected but never normalised: There is a lot of indifference right now when it comes to violence, violence has
become a part of us. One more corpse or murder, or to see the police on the street,
it has become normal. I knew many friends who are now missing; many. But we IJCJ&SD 110
© 2017 6(1) IJCJ&SD
© 2017 6(1) 110 Online version via www.crimejusticejournal.com Helen Berents, Charlotte ten Have: Navigating Violence: Fear and Everyday Life in Colombia and Mexico have become indifferent and have learned to live with it. Initially there was a lot of
paranoia. Two years ago, for example, there was a motorcycle that made a noise
and someone said that it was a shooting. Within seconds it was a mess, people were
living in paranoia. We all have experiences, bad experiences. It has happened to us
all directly or indirectly. The kind of situation that we are in is very ugly and I think
that San Luis Potosí has changed a lot. Fear breaks the bonds of community and thus violence, which ‘occurs in the contested space of
intersubjectivity, [and thus] its most devastating effects are not on individuals per se but on the
fields of interrelationships that constitute their lifeworlds’ (Jackson 2002: 39, emphasis in
original). This breakage of bonds is also translated in a telling Mexican proverb, which states:
‘trust is good but distrust is better’. Proverbs in this sense are the type of cultural products that
are also reflected in Galtung’s three pillars of violence, in which direct and structural violence
translate into cultural violence. Within a conflict area the experience of these multiple violences
are both structural and symbolic, and have profound effects on the respondent’s negotiation of
their environment, their relationships and their ‘self’. In Cazucá, Felipe (aged 16 years) feels the daily experience of violence impacts his ability to see
himself as capable of effecting change: ‘The violence, well, I think it affects whoever it wants, no? ... I can’t do anything because the violence never leaves us’, while Brayan Alexander (aged 15
years) highlights how violence becomes normalised and thus difficult to change, despite its
damaging consequences: ... Navigating everyday life So you leave anyway
and you hope that you return home’. Such navigation requires knowing about the community;
about why people might accuse you of doing something; about who, what and which locations to
avoid; and how to present yourself. Activities might temporarily be suspended in the case of violent events that directly affect a
community but daily life often resumes again promptly. This occurs not as a negation of risk or a
lack of fear but rather the opposite; namely, the importance of the routines of everyday life. When
Alejandro in los Altos de Cazucá was asked if he ever felt scared walking to or from school, he
explained slowly and clearly – as if it should be self‐evident – that he ‘can’t just stop going to
school; how else will things ever change if [he] gives up?’. As a result, young people and their
parents used information from neighbours as well as their own expertise about their community
to mitigate risk and attempt safe navigation. As Claudia in San Luis Potosí explained: You can’t just stop living your life, you have needs. You have to go to work, you
have to continue your life. You have to ... I mean you can learn to live with it ... You
have to accept that it can happen to anyone at any time, you can always be at the
wrong place at the wrong time. Despite the threat of violence, Claudia recognises that individuals must continue to move through
their everyday environments, and that requires certain recognition that even a skilled navigator
can fail to escape violence; the oppressive and unpredictable nature of structural and direct
violences in these places can frustrate individual efforts. In this way occupants of both locations sometimes restricted their own movements, avoiding
going out. This seemed one of the most used navigation strategies and one of which respondents
were consciously aware. Many of the respondents in San Luis Potosí, for example, argued that
their lives had not changed, until they discussed precaution‐taking. However, avoidance of
leaving the home seemingly had an emotional impact that led to awareness about the strategy
adapted, and many of the respondents had their own reflections about this. Carlos, for example,
stated: ‘San Luis is like the world upside down. Navigating everyday life We have discussed the concept of navigation as a counter to merely ‘coping’ or ‘normalising’
violence. This idea, developed from Vigh’s work (2009) affirms that individuals are not fixed
points on a landscape battered by violences but, rather, individuals in situations of insecurity who
navigate topographically and temporally to minimise harm, avoid violence and mitigate fear. Furthermore, we argue that the occupants of los Altos de Cazucá and San Luis Potosí are in fact
skilled navigators, and have expertise in these topographical and temporal navigations of
violence. Occupants must ‘involve themselves in their social worlds’ (Guyer 2007) and thus must
embody ‘motion within motion’ to respond to situations as they arise. In los Altos de Cazucá, decisions by young people and their parents to take alternate paths to
school because of information received by neighbours is an example of the skilled navigator at 111 IJCJ&SD
© 2017 6(1) Online version via www.crimejusticejournal.com Helen Berents, Charlotte ten Have: Navigating Violence: Fear and Everyday Life in Colombia and Mexico work. Equally, the networks of information passed on from neighbour to neighbour in Colombia
are evidence of sharing expertise to assist other’s navigation: ‘motion within motion’. While
violence and fear can be oppressive, everyday life cannot cease. For Paola Andrea and Alejandro
the most difficult thing about living in Cazucá is simply ‘to survive [sobrevivir]’. In San Luis Potosí
respondents similarly identified adapting many daily activities in attempts to better navigate and
predict violence. These included trading expensive cars for cheaper models so as not to draw
attention; asking family members to pick up respondents rather than taking a bus or a taxi;
avoiding eye contact with police or strangers; avoiding talking publicly about the problems or
salary, whether high or low; prompted by fear of extortions or kidnappings, changing social media
names into fictional names; and women starting to dress more conservatively. In doing so,
respondents skilfully navigate their environment through informed, rationalised and weighted
decision‐making. Adaptations to navigate violence so that the vital parts of daily life can continue
thus occur in many small steps rather than a few big ones. Martha, in San Luis Potosí, explained: ‘you take precautions, right. Because you know that the
violence is present but you cannot leave your routine because of fear. So you take your
precautions and continue your life, even though something might happen. Navigating everyday life The murderers, kidnappers and criminals walk
freely in the streets and the citizens are locked in the prisons made up by their own houses’. In
Colombia, participants also spoke of feeling ‘stuck’ in their houses, at times unable to leave. In
these stories they are navigating dangerous waters by avoiding being on the streets at times when
it is potentially risky. But navigation has consequences: people can feel trapped by the violence. Navigation, then, is not a straightforward ‘solution’ to violence or fear, but rather merely a way of
responding to the best of an individual’s ability. 112 IJCJ&SD
© 2017 6(1) Online version via www.crimejusticejournal.com Helen Berents, Charlotte ten Have: Navigating Violence: Fear and Everyday Life in Colombia and Mexico Although a large part of the responses by participants in Colombia were about the difficulties and
dangers they had become experts in navigating, there was a concurrent series of stories about
resilience and hope that challenged a totalising narrative. These efforts stemmed from frustration
with the duration of the civil war and the absence of state assistance. One woman in Cazucá
described how, since being in this community, she had become more aware of her rights as a
person, and noted that, ‘because we are all in this [the uncertain situation of daily life] together,’
she has built networks of advocacy and resilience with her neighbours and friends. The
community of Cazucá, and other communities in Soacha have a history of social organising despite
(or sometimes because of) threats of violence (Rodriguez et al. 2009), in particular the Madres de
Soacha who protested the kidnap and killing of their sons by government forces at great personal
risk (Mateo Medina 2013). This is a more fraught form of navigation because it exposes
individuals to risk and retribution but it is also one that these individuals are uniquely skilled in
and able to navigate. In the case of San Luis Potosí, where the respondents had been experiencing
a steady and shocking increase in violence rather than the long duration of insecurity of those in
Cazucá, the sentiments were less optimistic. However, a growing resistance accompanied by a
desire to counter the violence through breaking the self‐censorship was observed. Claudia, for
instance, stated: Violence is also something that has kept us quiet, I think it is time to talk, to say:
you know what, I do not agree. Navigating everyday life I do not agree with these people who have kept us
living in fear; living in fear is an ugly thing. I have certain rules. I do not answer
phone numbers that I don’t know on the phone or caller ID because they might
extort you and sometimes when they ask my name, I give them another name
because I have become very suspicious of people. It’s like if you go somewhere cool
and someone wants to talk to you and you just almost say nothing, things like that. Well it is true that Potosínos are a bit closed, but due to this we have become more
hermetic. But I think it is time to talk. Fear of violence can shut down individual’s capacity to respond. However, in both these
communities individuals identify and navigate towards visions of life that affirm capacity for
change, although to very different degrees. Such examples demonstrate that navigation can be
more than avoidance strategies and can, in some cases, result in organised resistance to violence
and insecurity. While navigation can allow an amelioration or avoidance of violence, it is undertaken because of
pervasive and shifting waters that hold the promise of violence and involves risk to the individual. There are many ways in which occupants of both communities have become skilled at the
topographical and temporal navigation of their daily lives. While some of these techniques allow
for the mitigation of risk so that the vital aspects of daily life can continue to be carried out, at the
same time individuals often feel trapped by fear, and avoid leaving the house or limit their
movements. Navigation as a form of expression of resilience or hope was more evident in Cazucá
because the duration of violence had meant occupants had built networks of capacity building in
response to structural violence and state abnegation. In San Luis Potosí, however, respondents
spoke less of resilience than of a fear that a new generation was growing up without ever having
known peace and therefore not feeling a need to restore it. Fear and violence are not totalising
narratives of either community, and skilled navigators wayfind with varying degrees of success
on a day‐to‐day basis. Navigation enables individuals and communities to find ways of moving
through and between the insecurity and fear. Conclusion Occupants of San Luis Potosí and Cazucá live in contexts of insecurity and risk. Their daily lives
are impacted by experiences of insecurity and violence in different forms. Life in communities
affected by violence is fraught and challenging but those who live daily in such places are not 113 IJCJ&SD
© 2017 6(1) Online version via www.crimejusticejournal.com Helen Berents, Charlotte ten Have: Navigating Violence: Fear and Everyday Life in Colombia and Mexico inherently powerless victims. Instead they acquire knowledge from many sources – their own
experiences; experiences of their neighbours, family and friends; news sources; and stories and
rumours – and their ability to judge, ‘read’ the situation and use this information determines how
they navigate their daily lives. This knowledge, vital in navigating the unpredictable waters of
their communities, is a learned skill which enables manoeuvring in ways that minimise, avoid and
mitigate risk and fear. For those outside these places, the environment may seem totalising and
impossible to read. However, those who live there have learnt about the local topography and
temporality of violence and insecurity through storytelling and experience. This has made them
into skilled navigators who are able to read the currents, predict the weather, and set course to
sail in these choppy waters. We suggested previously that such violence and fear is unable to be
‘cured’ but rather has to be managed like a chronic condition. It is this management that takes the
form of temporal and spatial navigation through, at times, shifting unpredictable waters that
makes the skilled navigator. Violence is complex; it can appear in direct, structural and cultural forms (Galtung 1990), all of
which impinge on the lifeworlds of individuals. However, to render an area inherently violent or
assumptively predisposed to violence and insecurity flattens and limits understandings of such
communities. We have argued through this paper, drawing on Vigh’s (2009) concept of a social
navigator, that attention to people’s everyday lives and navigation strategies crucially resists
totalising narratives and instead opens new entry points to theorising and responding to violence
and fear. Complex violences, including direct violence by organised criminal groups, structural
violence of the abnegation of communities or the loss of monopoly of force by the state, and the
cultural violence in which assumptions and stereotypes exist about communities and individuals,
are reoccurring experiences throughout the global South. 2 Research that critiques the issues of academic work driven from the global North include Boadventura de Sousa
Santos’s (2014) Epistemologies of the South, which argues Western understandings of the world are limited and
limiting and forwards an epistemology of the South which rejects Northern epistemologies and grounds knowing in
emancipatory action within the South; and Arturo Escobar’s (1995) Encountering Development, which encourages
ethnographic approaches to explore ways of moving beyond neoliberal frameworks without claiming universality.
3 The contributors to the edited volume by McGee and Pearce (2009) explore in varied detail the complexities of
researching violence and violence‐affected communities. In the introduction Pearce (2009: 7‐8) uses the term
‘navigate’ to describe the actions of the researcher in learning the ‘rules’ or practices of violence in the communities g
p
pp
p
y
g
y
g
y
3 The contributors to the edited volume by McGee and Pearce (2009) explore in varied detail the complexities of
researching violence and violence‐affected communities. In the introduction Pearce (2009: 7‐8) uses the term
‘navigate’ to describe the actions of the researcher in learning the ‘rules’ or practices of violence in the communities
in which they work. We see our own work reflected in this discussion of navigation and becoming skilled; however,
in neither situation did we ever fully overcome these obstacles and our navigation was always conditional and
choppy. It is useful to include this reflexivity here, and acknowledge the work done by Pearce (2009), Baird (2009)
and Wheeler (2009) in drawing it out explicitly in the aforementioned edited volume. 2 Research that critiques the issues of academic work driven from the global North include Boadventura de Sousa
Santos’s (2014) Epistemologies of the South, which argues Western understandings of the world are limited and
limiting and forwards an epistemology of the South which rejects Northern epistemologies and grounds knowing in
emancipatory action within the South; and Arturo Escobar’s (1995) Encountering Development, which encourages
ethnographic approaches to explore ways of moving beyond neoliberal frameworks without claiming universality. p
pp
p
y
y
2 Research that critiques the issues of academic work driven from the global North include Boadventura de Sousa
Santos’s (2014) Epistemologies of the South, which argues Western understandings of the world are limited and
limiting and forwards an epistemology of the South which rejects Northern epistemologies and grounds knowing in
emancipatory action within the South; and Arturo Escobar’s (1995) Encountering Development, which encourages
ethnographic approaches to explore ways of moving beyond neoliberal frameworks without claiming universality.
3 The contributors to the edited volume by McGee and Pearce (2009) explore in varied detail the complexities of
researching violence and violence‐affected communities. In the introduction Pearce (2009: 7‐8) uses the term
‘navigate’ to describe the actions of the researcher in learning the ‘rules’ or practices of violence in the communities
in which they work. We see our own work reflected in this discussion of navigation and becoming skilled; however,
in neither situation did we ever fully overcome these obstacles and our navigation was always conditional and
choppy. It is useful to include this reflexivity here, and acknowledge the work done by Pearce (2009), Baird (2009)
and Wheeler (2009) in drawing it out explicitly in the aforementioned edited volume 1 Approval for research in Colombia was granted by the University of Queensland’s Behavioural and Social Science
Review Board on 1 September 2010. Support for research in Mexico was provided by the VU University Amsterdam Conclusion We propose that the theoretical
intervention we make here, illustrated by our work in Colombia and Mexico, is a starting point to
think in more productive ways about violence, fear and the skilled navigation of everyday life. Recognising individuals as skilled navigators is crucial for understanding complex live
experiences amidst violence. Correspondence: Dr Helen Berents, Lecturer, School of Justice, Faculty of Law, Queensland
University of Technology, 2 George Street, Brisbane 4000 QLD, Australia. Email:
helen.berents@qut.edu.au 3 The contributors to the edited volume by McGee and Pearce (2009) explore in varied detail the complexities of
researching violence and violence‐affected communities. In the introduction Pearce (2009: 7‐8) uses the term
‘navigate’ to describe the actions of the researcher in learning the ‘rules’ or practices of violence in the communities
in which they work. We see our own work reflected in this discussion of navigation and becoming skilled; however,
in neither situation did we ever fully overcome these obstacles and our navigation was always conditional and
choppy. It is useful to include this reflexivity here, and acknowledge the work done by Pearce (2009), Baird (2009)
and Wheeler (2009) in drawing it out explicitly in the aforementioned edited volume. IJCJ&SD 114
© 2017 6(1) IJCJ&SD
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A ranking framework based on interval self and cross-efficiencies in a two-stage DEA system
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Kremantzis, M. D., Beullens, P., & Klein, J. (2022). A ranking
framework based on interval self and cross-efficiencies in a two-stage
DEA system. RAIRO - Operations Research, 56(3), 1293-1319. https://doi.org/10.1051/ro/2022056 Publisher's PDF, also known as Version of record
License (if available):
CC BY
Link to published version (if available):
10.1051/ro/2022056 Link to publication record on the Bristol Research Portal
PDF-document This is the final published version of the article (version of record). It first appeared online via EDP Sciences at
https://doi.org/10.1051/ro/2022056 .Please refer to any applicable terms of use of the publisher. Kremantzis, M. D., Beullens, P., & Klein, J. (2022). A ranking
framework based on interval self and cross-efficiencies in a two-stage
DEA system. RAIRO - Operations Research, 56(3), 1293-1319.
https://doi.org/10.1051/ro/2022056 Kremantzis, M. D., Beullens, P., & Klein, J. (2022). A ranking
framework based on interval self and cross-efficiencies in a two-stage
DEA system. RAIRO - Operations Research, 56(3), 1293-1319. https://doi.org/10.1051/ro/2022056 A RANKING FRAMEWORK BASED ON INTERVAL SELF AND
CROSS-EFFICIENCIES IN A TWO-STAGE DEA SYSTEM os Dominikos Kremantzis1,* , Patrick Beullens2 and Jonathan s Kremantzis1,* , Patrick Beullens2 and Jonathan Klein3 Marios Dominikos Kremantzis1,* , Patrick Beullens2 an Abstract. The evaluation of the performance of a decision-making unit (DMU) can be measured by
its own optimistic and pessimistic multipliers, leading to an interval self-efficiency score. While this
concept has been thoroughly studied with regard to single-stage systems, there is still a gap when it
is extended to two-stage tandem structures, which better correspond to a real-world scenario. In this
paper, we argue that in this context, a meaningful ranking of the DMUs is obtained; this outcome
simultaneously considers the optimistic and pessimistic viewpoints within the self-appraisal context,
and the most favourable and unfavourable weight sets of each of the other DMUs in a peer-appraisal
setting. We initially extend the optimistic-pessimistic Data Envelopment Analysis (DEA) models to
the specifications of such a two-stage structure. The two opposing self-efficiency measures are merged
to a combined self-efficiency measure via the geometric average. Under this framework, the DMUs
are further evaluated in a peer setting via the interval cross-efficiency (CE). This methodological tool
is applied to evaluate the target DMU in relation to the most favourable and unfavourable weight
profiles of each of the other DMUs, while maintaining the combined self-efficiency measure. We, thus,
determine an interval individual CE score for each DMU and flow. By treating the interval CE matrix
as a multi-criteria decision making problem and by utilising several well-established approaches from
the literature, we delineate its remaining elements; we show how these lead us to a meaningful ultimate
ranking of the DMUs. A numerical example about the efficiency evaluation of ten bank branches in
China illustrates the applicability of our modelling approaches. Mathematics Subject Classification. 90B10, 90C05, 90C90, 91B06. Mathematics Subject Classification. 90B10, 90C05, 90C90, 91B06. Received July 1, 2021. Accepted April 20, 2022. University of Bristol – Bristol Research Portal
General rights This document is made available in accordance with publisher policies. Please cite only the
published version using the reference above. Full terms of use are available:
http://www.bristol.ac.uk/red/research-policy/pure/user-guides/brp-terms/ RAIRO Operations Research
www.rairo-ro.org RAIRO-Oper. Res. 56 (2022) 1293–1319
https://doi.org/10.1051/ro/2022056 RAIRO-Oper. Res. 56 (2022) 1293–1319
https://doi.org/10.1051/ro/2022056 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. eywords. Data envelopment analysis, network, interval self-efficiency, interval cross-efficiency, ranking. 3 Southampton Business School and CORMSIS, University of Southampton, Southampton, UK.
*C
di
th
@ 1 School of Management, University of Bristol, Bristol, UK. Mathematical Sciences, Southampton Business School and CORMSIS, University of Southampton, Sou uthampton Business School and CORMSIS, University of Southampton, Southampton, UK.
responding author: marios.kremantzis@bristol.ac.uk *Corresponding author: marios.kremantzis@bristol.ac.uk *Corresponding author: marios.kremantzis@bristol.ac.uk Received July 1, 2021. Accepted April 20, 2022. 1. Introduction The two separate efficiencies were combined into the Relative Closeness (RC) index to obtain a
unique ranking order. Wu [51] identified a weakness in Wang and Luo’s [48] paper dealing with the ADMU for
DEA modelling. Wu argued that it is inconsistent to aggregate an input-oriented IDMU and an output-oriented
ADMU into the RC index. Wang and Yang [49] proposed an alternative way of measuring the performance of DMUs. The efficiencies of
DMUs are measured within the range of an interval, in which the upper bound is 1 and the lower bound equals to
the performance of a virtual ADMU, which is the worst among all DMUs. This approach, which only considers
the performance of the lower bound, was extended by Azizi and Jahed [4], who suggested a pair of improved
bounded models for the target DMU. Wang et al. [50] combined optimistic and pessimistic efficiencies into a
geometric average efficiency to measure the overall performance of a DMU. The geometric average efficiency
was deemed effective, as it was simultaneously an efficiency measure and a ranking index. Toloo and Tichy [45]
proposed a multiplier model to identify the maximum efficiency scores and applied the envelopment model to
attain the maximum discrimination among efficient DMUs . Khodabakhshi and Aryavash [23] used a double
frontier DEA procedure to introduce a new cross-efficiency method; the merit of their approach lied on the non-
use of any alternative secondary goal. Based on the ideal and anti-ideal DMUs, Liu and Wang [29] developed
the normalised efficiency metric and then formulated two DEA models to obtain its lower and upper bounds. ¨Orkc¨u et al. [35] proposed a non-cooperative game like iterative optimistic-pessimistic DEA approach to fully
rank the DMUs. Badiezadeh et al. [5] were, to our knowledge, the first to conceive the idea of considering
optimistic-pessimistic DEA models under a network DEA context to evaluate the performance of a sustainable
supply-chain management. With the exception of Badiezadeh et al. [5], the majority of the existing studies on the double frontier DEA
models are concerned with a system handled as a whole unit, ignoring its internal structure. Several studies
illustrate that this condition might produce misleading results [22]. In reality, systems can be composed of
two sub-stages operating interdependently. 1. Introduction Data Envelopment Analysis (DEA) is a benchmarking technique for comparing the relative efficiency of a
Decision Making Unit (DMU) with the best observed efficiency [7]. The evaluation of a DMU is based on the
comparison between the amount of input(s) consumed and the amount of output(s) produced [8] by DMUs. comparison between the amount of input(s) consumed and the amount of output(s) produced [8] by DMUs. One of the undeniably attractive features of DEA is its weight flexibility. This allows each DMU to be allocated
its most favourable set of weights to be assigned to inputs and outputs for determining its relative efficiency. One of the undeniably attractive features of DEA is its weight flexibility. This allows each DMU to be allocated
its most favourable set of weights to be assigned to inputs and outputs for determining its relative efficiency. *Corresponding author: marios.kremantzis@bristol.ac.uk c○The authors. Published by EDP Sciences, ROADEF, SMAI 2022 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 1294 M.D. KREMANTZIS ET AL. Hence, in the conventional DEA, the overall assessment of a DMU is based on the optimistic viewpoint and
on the concept of the efficiency frontier [56]. On the other hand, if the performance of a DMU is based on the
pessimistic viewpoint, then an inefficiency frontier is defined. Hence, in the conventional DEA, the overall assessment of a DMU is based on the optimistic viewpoint and
on the concept of the efficiency frontier [56]. On the other hand, if the performance of a DMU is based on the
pessimistic viewpoint, then an inefficiency frontier is defined. Optimistic and pessimistic perspectives illustrate two extreme cases for each DMU. Taking only one scenario
into account limits the examination of the performance of a unit. The obtained results might be unreasonable
[3]. Therefore, it is thought to be valuable to consider the two distinctive efficiencies together. [ ]
Research on exploring both aspects of viewing the efficiency of a DMU within a single-stage structure is
relatively extensive. Wang and Luo [48] evaluated each DMU in terms of the optimistic and pessimistic viewpoint,
by introducing an input-oriented virtual Ideal DMU (IDMU) and an output-oriented virtual Anti-ideal DMU
(ADMU). 1. Introduction There is also no confirmation that these formulations will result in the same
ranking or that their optimal set of multipliers are unique [46]. To alleviate these deficiencies, Yang et al. [53] suggested the “interval CE” for the exploration of the cross-
efficiencies in a weight space considering all the weight profiles, within the single-stage DEA structure. In such a
peer-appraisal setting, the base DMU is assessed regarding the most unfavourable and favourable weight profiles
of each of the other DMUs. The aggressive and benevolent models of this process were, however, keeping only
the optimistic self-efficiency value of each DMU fixed. In summary, this paper adapts an optimistic-pessimistic DEA approach in the light of the two-stage tandem
system, in order to then support the interval CE method in such a network system. Using the proposed framework
as shown in Figure 1, a meaningful evaluation and ranking of the considered DMUs is attained. Decision makers
will be enabled to simultaneously consider: (i) both the optimistic and the pessimistic viewpoints within the
self-appraisal context, and (ii) the most favourable and unfavourable weight sets of each of the other DMUs in
a peer-appraisal setting. We believe that the combination of the methods that compose our framework has not
been considered before in the literature; in our view, this could lead to a meaningful ranking in addition to it
being adjusted to a two-stage tandem DEA structure. The procedures implemented in the first three steps of our proposed framework (Fig. 1) have been applied
in several studies (e.g., [50]) that focus on double frontier DEA models to evaluate DMUs in a self-appraisal
context in a single-stage structure. As for these steps, our study differs in that our optimistic-pessimistic DEA
models, which are inspired by the studies of Wang and Luo [48] and Wu [51], are built towards the two-stage
tandem (network) system. The remaining steps of the proposed framework pursue to support the peer-evaluation of the considered
DMUs via the customisation of the interval CE method to the specifications of the two-stage tandem structure
while embedding the respective combined self-efficiency measure (that considers the effects of both opposing
standpoints). To rank the DMUs in the interval CE matrix of the corresponding flow, this paper views this
matrix as a multi-criteria decision-making problem. 1. Introduction In this paper, we will extend our selected optimistic-pessimistic
ranking procedure to a two-stage tandem system to not only measure the efficiency of the overall system and
its individual stages’ efficiencies; thus, the stage that causes inefficiencies can be identified. The optimistic and pessimistic self-efficiency scores can be unified via the geometric average efficiency. As
shown in Wang et al. [50], this score has a better discriminating power than either of the opposing efficiencies. Yet, this feature has not been explored in a network environment, implying the possible existence of a non-unique
ranking. It also considers the effects of the optimistic and pessimistic standpoints only within the self-appraisal
context. The integration of the geometric average score in a peer-appraisal context would contribute to the
assessment of a DMU in terms of the weight sets of other players, leading to a more logical ranking. These
points make us infer that this framework could be further extended by the use of the cross-efficiency (CE) to
ensure more fairness in the evaluation outcomes. The CE concept is based on the peer-evaluation notion [42]. As stressed by Anderson et al. [1], CE improves
the probability of obtaining a unique ranking. A shortcoming of the CE is the non-uniqueness of DEA optimal
weights, leading to the non-uniqueness of cross-efficiencies. Remedial actions have been suggested towards the
adoption of secondary goals in an aim to select unique optimal multipliers [10,26–28,52,57]. The non-uniqueness
issue is also critical in a two-stage (network) system [18,22,32–34]. Kao and Liu [22], for instance, developed an
aggressive CE model to measure the efficiency in two basic network structures. ¨Orkc¨u et al. [34] came up with 1295 A RANKING FRAMEWORK BASED ON INTERVAL SELF AND CROSS-EFFICIENCIES a neutral CE model in a two-stage system, which is indifferent to the preference choice between the aggressive
and benevolent formulations. Doyle and Green [10] introduced an aggressive and a benevolent secondary goal model to remedy the non-
uniqueness of the optimal weights. The former ensures the minimisation and the latter the maximisation of the
cross-efficiencies of all other DMUs, whilst both maintaining the optimistic self-efficiency of the target DMU. The use of any formulation of the two may be subject to an individual judgement, possibly leading to an
irrational selection of either model. 1. Introduction To solve this problem, we implement the goal programming
method of Wang and Elhag [47] to obtain the interval local weight of each criterion. To delineate the interval
global weight of each alternative, we suggest a pair of linear programming models, introduced by Entani and
Tanaka [13]. Finally, we apply the grey relational analysis [25] for ranking the interval global weights. To our
knowledge, the aforementioned well-established approaches have not been previously considered for extracting
valuable information from an interval CE matrix. We have also shown that our proposed framework offers
a more informative assessment of the units under consideration than particular existing methods in network
DEA-relevant literature. The remainder of the paper is organised as follows. Section 2 shortly describes the preliminaries and the
methodological background. Section 3 proposes the framework to meaningfully rank DMUs. Section 4 illustrates
the methods with a numerical example. Section 5 presents conclusions and further research. 2. Methodological background We assume that each DMU𝑗(𝑗= 1, 2, . . ., 𝑛) uses 𝑚inputs (𝑖= 1, 2, . . ., 𝑚) to produce 𝑠outputs (𝑟=
1, 2, . . ., 𝑠). Let 𝑋𝑖𝑗be the input value of 𝑖∈𝑀for DMU 𝑗∈𝑁and 𝑌𝑟𝑗be the output value of 𝑟∈𝑆for DMU
𝑗∈𝑁. We estimate the optimistic self-efficiency for each DMU, based on determining an optimal set of the
most favourable input and output weights. The conventional input-oriented CCR DEA model [7], that assesses 1296 M.D. KREMANTZIS ET AL. Figure 1 The proposed framework Table 1. Cross-efficiency matrix [10]. Table 1. Cross-efficiency matrix [10]. Target
DMU𝑗
Evaluator DMU𝑘
1
2
. . . 𝑛
1
𝐸11
𝐸12
. . . 𝐸1𝑛
2
𝐸21
𝐸22
. . . 𝐸2𝑛
. . . . . . . . . . . . . . . 𝑛
𝐸𝑛1
𝐸𝑛2
. . . 𝐸𝑛𝑛 Figure 1. The proposed framework. Figure 1. The proposed framework. the efficiency of the target DMU𝑘, is illustrated as follows: 𝐸𝑘𝑘= Max
𝑠
∑︁
𝑟=1
𝜇𝑟𝑘𝑌𝑟𝑘
subject to
𝑚
∑︁
𝑖=1
𝜈𝑖𝑘𝑋𝑖𝑘= 1,
𝑠
∑︁
𝑟=1
𝜇𝑟𝑘𝑌𝑟𝑗−
𝑚
∑︁
𝑖=1
𝜈𝑖𝑘𝑋𝑖𝑗≤0,
∀𝑗,
𝜇𝑟𝑘, 𝜈𝑖𝑘≥0,
∀𝑟, 𝑖,
(2.1) 𝐸𝑘𝑘= Max
𝑠
∑︁
𝑟=1
𝜇𝑟𝑘𝑌𝑟𝑘
subject to
𝑚
∑︁
𝑖=1
𝜈𝑖𝑘𝑋𝑖𝑘= 1,
𝑠
∑︁
𝑟=1
𝜇𝑟𝑘𝑌𝑟𝑗−
𝑚
∑︁
𝑖=1
𝜈𝑖𝑘𝑋𝑖𝑗≤0,
∀𝑗,
𝜇𝑟𝑘, 𝜈𝑖𝑘≥0,
∀𝑟, 𝑖,
(2.1) ︁
𝑟
1︁
𝑖
1
𝜇𝑟𝑘, 𝜈𝑖𝑘≥0,
∀𝑟, 𝑖,
(2.1) (2.1) where 𝜇𝑟𝑘, 𝜈𝑖𝑘are the 𝑟th output and the 𝑖th input weights for DMU𝑘, respectively. If the optimal (optimistic)
self-efficiency 𝐸*
𝑘𝑘= 1, then DMU𝑘is called DEA efficient; otherwise it is said to be DEA inefficient. where 𝜇𝑟𝑘, 𝜈𝑖𝑘are the 𝑟th output and the 𝑖th input weights for DMU𝑘, respectively. If the optimal (optimistic)
self-efficiency 𝐸*
𝑘𝑘= 1, then DMU𝑘is called DEA efficient; otherwise it is said to be DEA inefficient. 1297 A RANKING FRAMEWORK BASED ON INTERVAL SELF AND CROSS-EFFICIENCIES Table 2. Interval cross-efficiency matrix [53]. Table 2. Interval cross-efficiency matrix [53]. Target
DMU𝑗
Evaluator DMU𝑘
1
2
. . . 𝑛
1
[𝐸11, 𝐸11]
[︀
𝐸L
12, 𝐸U
12
]︀
. . . [︀
𝐸L
1𝑛, 𝐸U
1𝑛
]︀
2
[︀
𝐸L
21, 𝐸U
21
]︀
[𝐸22, 𝐸22]
. . . [︀
𝐸L
2𝑛, 𝐸U
2𝑛
]︀
. . . . . . . . . . . . . . . 𝑛
[︀
𝐸L
𝑛1, 𝐸U
𝑛1
]︀
[︀
𝐸L
𝑛2, 𝐸U
𝑛2
]︀
. . . [𝐸𝑛𝑛, 𝐸𝑛𝑛] Model (2.3) is a benevolent-based model to obtain an optimal set of weights and thus to determine the maximum
individual cross-efficiency of DMU𝑗based on DMU𝑘. Max
𝐸U
𝑘𝑗=
𝑠
∑︁
𝑟=1
𝜇𝑟𝑘𝑌𝑟𝑗
(2.3) (2.3) ︁
subject to the same constraints as in model (2.2). In the above two models, the optimistic self-efficiency score 𝐸*
𝑘𝑘, derived from model (2.1), remains fixed;
this keeps one of the basic properties of the traditional CE concept intact. Overall, in this peer-evaluation
procedure an interval individual cross-efficiency score of DMU𝑗in terms of DMU𝑘is formed and lies in the
range
[︁
𝐸L
𝑘𝑗, 𝐸U
𝑘𝑗
]︁
. 𝐸L
𝑘𝑗is the lower bound and is found from model (2.2), whereas 𝐸U
𝑘𝑗is the upper bound
obtained from model (2.3). Table 2 depicts the individual cross-efficiencies as interval numbers, boosting the
DM’s uncertainty. The elements in the diagonal column of Table 2 show the special case of the self-efficiency
scores, for which 𝐸𝑘𝑘= 𝐸L
𝑘𝑘= 𝐸U
𝑘𝑘, ∀𝑘∈{1, 2, . . ., 𝑛}. Models (2.2) and (2.3) make use of unfavourable and favourable multipliers, respectively, to identify the
individual cross-efficiencies towards the single-stage structure. In either case, only the optimistic self-efficiency
measure is involved to accommodate their purpose. In Section 3.1, a combined self-efficiency score is obtained indicating the merger of the optimistic and pes-
simistic self-efficiencies. That score is embedded to the adjusted cross-efficiency models (Sect. 3.2) to explore the
effect of both opposing viewpoints. The above-mentioned processes are part of a broader framework presented
herein to reasonably rank DMUs towards the two-stage tandem structure. 2.1. Cross-efficiency & interval cross-efficiency in single-stage structures A significant challenge of the conventional single-stage DEA model, is to distinguish the efficient DMUs
and thus to acquire a unique ranking of the DMUs. A potential remedy to overcome this inability is the
implementation of the CE concept [42]. Let 𝜇*
𝑟𝑘and 𝜈*
𝑖𝑘be the optimal set of multipliers of model (2.1). Then,
𝐸*
𝑘𝑘= ∑︀𝑠
𝑟=1 𝜇*
𝑟𝑘𝑌𝑟𝑘is the optimal self-efficiency score of DMU𝑘and reflects its desire to be assessed only on the
basis of its own most favourable weights. On the other hand, CE, in which peer-appraisal is the main notion,
evaluates each DMU, considering the weight profiles of all DMUs. The ratio 𝐸𝑘𝑗= ∑︀𝑠
𝑟=1 𝜇*
𝑟𝑘𝑌𝑟𝑗/ ∑︀𝑚
𝑖=1 𝜈*
𝑖𝑘𝑋𝑖𝑗
denotes the individual cross-efficiency of DMU𝑗, based on the optimal weight scheme of DMU𝑘. A CE matrix
(Tab. 1) can be a valuable tool to integrate both the peer-efficiency scores 𝐸𝑘𝑗(𝑘, 𝑗= 1, 2, . . ., 𝑛) and the self-
efficiency scores 𝐸𝑘𝑘(in the leading diagonal column). The ultimate cross-efficiency can be defined by averaging
all individual cross-efficiencies of the corresponding DMU being evaluated. The ultimate score in this case is
ˆ𝑒𝑗= 1
𝑛· ∑︀𝑛
𝑘=1 𝐸𝑘𝑗, ∀𝑗[2]. ︀
The existence of multiple optimal weights from model (2.1) can deteriorate the theoretical usefulness of the
results obtained via the cross-efficiency concept. To tackle this issue, Doyle and Green [10] proposed two opposed
secondary goals to choose their weights, favourable or unfavourable, among the optimal solutions. Considering the DEA-related literature, there is not a well-established methodological approach to guide the
decision-maker in reasonably selecting either the benevolent or the aggressive strategy. In addition, the selection
of either the former or the latter model might not provide the same ranking or a unique optimal set of weights. To overcome these obstacles, Yang et al. [53] suggested the simultaneous use of the two extreme cases in the
context of a single-stage structure. Model (2.2) is an aggressive-based model to obtain an optimal set of multipliers and thus to identify the
minimum individual cross-efficiency value of DMU𝑗based on DMU𝑘. Min 𝐸L
𝑘𝑗=
𝑠
∑︁
𝑟=1
𝜇𝑟𝑘𝑌𝑟𝑗
subject to
𝑚
∑︁
𝑖=1
𝜈𝑖𝑘𝑋𝑖𝑗= 1,
𝑠
∑︁
𝑟=1
𝜇𝑟𝑘𝑌𝑟𝑘−𝐸*
𝑘𝑘
𝑚
∑︁
𝑖=1
𝜈𝑖𝑘𝑋𝑖𝑘= 0,
𝑠
∑︁
𝑟=1
𝜇𝑟𝑘𝑌𝑟𝑗−
𝑚
∑︁
𝑖=1
𝜈𝑖𝑘𝑋𝑖𝑗≤0,
∀𝑗; 𝑗̸= 𝑘,
𝜇𝑟𝑘, 𝜈𝑖𝑘≥0,
∀𝑟, 𝑖. (2.2) (2.2) 1298 M.D. KREMANTZIS ET AL. Table 2. Interval cross-efficiency matrix [53]. 3. Models development At optimality of model (3.1), the system efficiency is estimated as 𝐸𝑠
𝑘= (∑︀𝑠
𝑟=1 𝜇*
𝑟𝑘𝑌𝑟𝑘)/(∑︀𝑚
𝑖=1 𝜈*
𝑖𝑘𝑋𝑖𝑘), the effi-
ciency of stage 1 as 𝐸1
𝑘=
(︁∑︀𝐷
𝑑=1 𝜂*
𝑑𝑘𝑍𝑑𝑘
)︁
/(∑︀𝑚
𝑖=1 𝜈*
𝑖𝑘𝑋𝑖𝑘), and the efficiency of stage 2 as 𝐸2
𝑘= (∑︀𝑠
𝑟=1 𝜇*
𝑟𝑘𝑌𝑟𝑘)/
(︁∑︀𝐷
𝑑=1 𝜂*
𝑑𝑘𝑍𝑑𝑘
)︁
. It is obvious that the overall efficiency is the product of the efficiencies of the stage efficiencies. ︁︀︁︀︀
(︁∑︀𝐷
𝑑=1 𝜂*
𝑑𝑘𝑍𝑑𝑘
)︁
. It is obvious that the overall efficiency is the product of the efficiencies of the stage efficiencies. 3. Models development The exploration of the internal processes taking place in the core of a DMU sets the foundation for the
transition from a single-stage to a two-stage DEA structure. Each DMU𝑗(𝑗= 1, 2, . . ., 𝑛) consumes 𝑚inputs (𝑖=
1, 2, . . ., 𝑚) in the first stage to generate 𝐷intermediate products (𝑑= 1, 2, . . ., 𝐷). The outputs (intermediate
measures) of the first stage are converted into inputs in the second stage to produce 𝑠final outputs (𝑟=
1, 2, . . ., 𝑠). Let 𝑋𝑖𝑗be the input value of 𝑖∈𝑀, 𝑍𝑑𝑗be the intermediate product of 𝑑∈𝐷, and 𝑌𝑟𝑗be the
output value of 𝑟∈𝑆, for DMU 𝑗∈𝑁[21]. The above process is illustrated in the exploratory Figure 2. According to the relational model of Kao and Hwang [21], to measure the performance of the overall system
it is necessary to consider not only its operations, but also the operations of its individual sub-stages. In model
(3.1) these operations are described by the constraints, which indicate that the aggregate output can not exceed
the aggregate input. 1299 A RANKING FRAMEWORK BASED ON INTERVAL SELF AND CROSS-EFFICIENCIES Figure 2. Typical two-stage tandem DEA structure. Figure 2. Typical two-stage tandem DEA structure. Figure 2. Typical two-stage tandem DEA structure. 𝐸𝑠
𝑘= Max
𝑠
∑︁
𝑟=1
𝜇𝑟𝑘𝑌𝑟𝑘
subject to
𝑚
∑︁
𝑖=1
𝜈𝑖𝑘𝑋𝑖𝑘= 1,
𝐷
∑︁
𝑑=1
𝜂𝑑𝑘𝑍𝑑𝑗−
𝑚
∑︁
𝑖=1
𝜈𝑖𝑘𝑋𝑖𝑗≤0,
∀𝑗,
𝑠
∑︁
𝑟=1
𝜇𝑟𝑘𝑌𝑟𝑗−
𝐷
∑︁
𝑑=1
𝜂𝑑𝑘𝑍𝑑𝑗≤0,
∀𝑗,
𝜇𝑟𝑘, 𝜈𝑖𝑘, 𝜂𝑑𝑘≥0,
∀𝑟, 𝑖, 𝑑. (3.1) ∀𝑟, 𝑖, 𝑑. (3.1) (3.1) ︁︁
𝜇𝑟𝑘, 𝜈𝑖𝑘, 𝜂𝑑𝑘≥0, At optimality of model (3.1), the system efficiency is estimated as 𝐸𝑠
𝑘= (∑︀𝑠
𝑟=1 𝜇*
𝑟𝑘𝑌𝑟𝑘)/(∑︀𝑚
𝑖=1 𝜈*
𝑖𝑘𝑋𝑖𝑘), the effi-
ciency of stage 1 as 𝐸1
𝑘=
(︁∑︀𝐷
𝑑=1 𝜂*
𝑑𝑘𝑍𝑑𝑘
)︁
/(∑︀𝑚
𝑖=1 𝜈*
𝑖𝑘𝑋𝑖𝑘), and the efficiency of stage 2 as 𝐸2
𝑘= (∑︀𝑠
𝑟=1 𝜇*
𝑟𝑘𝑌𝑟𝑘)/︁︀︁ At optimality of model (3.1), the system efficiency is estimated as 𝐸𝑠
𝑘= (∑︀𝑠
𝑟=1 𝜇*
𝑟𝑘𝑌𝑟𝑘)/(∑︀𝑚
𝑖=1 𝜈*
𝑖𝑘𝑋𝑖𝑘), the effi-
ciency of stage 1 as 𝐸1
𝑘=
(︁∑︀𝐷
𝑑=1 𝜂*
𝑑𝑘𝑍𝑑𝑘
)︁
/(∑︀𝑚
𝑖=1 𝜈*
𝑖𝑘𝑋𝑖𝑘), and the efficiency of stage 2 as 𝐸2
𝑘= (∑︀𝑠
𝑟=1 𝜇*
𝑟𝑘𝑌𝑟𝑘)/
(︁∑︀𝐷
𝑑=1 𝜂*
𝑑𝑘𝑍𝑑𝑘
)︁
. It is obvious that the overall efficiency is the product of the efficiencies of the stage efficiencies. 3.1. Optimistic & pessimistic models in basic two-stage structure The above model can set the basis for the exploration of the optimistic and pessimistic self-efficiencies and,
in turn, their integration into a geometric average efficiency score within the two-stage tandem system. Sub-stage 1 consumes inputs to generate intermediate products. The following input-oriented CCR model
(3.2) [21] examines the performance of sub-stage 1: The above model can set the basis for the exploration of the optimistic and pessimistic self-efficiencies and,
in turn, their integration into a geometric average efficiency score within the two-stage tandem system. Sub-stage 1 consumes inputs to generate intermediate products. The following input-oriented CCR model
(3.2) [21] examines the performance of sub-stage 1: 𝐸1
𝑘= Max
𝐷
∑︁
𝑑=1
𝜂𝑑𝑘𝑍𝑑𝑘
subject to
𝑚
∑︁
𝑖=1
𝜈𝑖𝑘𝑋𝑖𝑘= 1,
𝐷
∑︁
𝑑=1
𝜂𝑑𝑘𝑍𝑑𝑗−
𝑚
∑︁
𝑖=1
𝜈𝑖𝑘𝑋𝑖𝑗≤0,
∀𝑗,
𝜈𝑖𝑘, 𝜂𝑑𝑘≥0,
∀𝑖, 𝑑. (3.2) ∀𝑖, 𝑑. (3.2) (3.2) ∀𝑖, 𝑑. ︁︁
𝜈𝑖𝑘, 𝜂𝑑𝑘≥0,
∀𝑖, 𝑑. (3.2) ︁
𝜈𝑖𝑘, 𝜂𝑑𝑘≥0, ︁
𝜈𝑖𝑘, 𝜂𝑑𝑘≥0, With reference to sub-stage 1 of a basic two-stage DEA structure, two fundamental concepts, the IDMU and
the ADMU, are introduced, following the principles of Wang and Luo [48]. IDMU is a hypothetical DMU
that utilises the least amount of inputs to generate the most intermediate products. An ADMU, on the other
side, uses the most inputs to produce the least intermediate products. The IDMU can be expressed with the
vectors (𝑋min, 𝑍max), where 𝑋min
𝑖
= min𝑘{𝑋𝑖𝑘} and 𝑍max
𝑑
= max𝑘{𝑍𝑑𝑘}, ∀𝑖, 𝑑. The ADMU can be determined
with the vectors (𝑋max, 𝑍min), where 𝑋max
𝑖
= max𝑘{𝑋𝑖𝑘} and 𝑍min
𝑑
= min𝑘{𝑍𝑑𝑘}, ∀𝑖, 𝑑. As stressed in 1300 M.D. KREMANTZIS ET AL. Hatami-Marbini et al. [16], the performance of the IDMU cannot be worse than any of the actual DMUs, and
the performance of the ADMU cannot be better than that of the worst performing actual DMU. Hatami-Marbini et al. [16], the performance of the IDMU cannot be worse than any of the actual DMUs, and
the performance of the ADMU cannot be better than that of the worst performing actual DMU. 3.1. Optimistic & pessimistic models in basic two-stage structure (3.5) (3.5) 1301 A RANKING FRAMEWORK BASED ON INTERVAL SELF AND CROSS-EFFICIENCIES The efficiency of the ADMU for the entire system can be illustrated as 𝐸ADMU(𝑠) =
(︀∑︀𝑠
𝑟=1 𝜇𝑟𝑌min
𝑟
)︀
/
(∑︀𝑚
𝑖=1 𝜈𝑖𝑋max
𝑖
). The associated optimisation model is formulated as follows: 𝐸ADMU(𝑠) = Min
𝑠
∑︁
𝑟=1
𝜇𝑟𝑌min
𝑟
subject to
𝑚
∑︁
𝑖=1
𝜈𝑖𝑋max
𝑖
= 1,
𝐷
∑︁
𝑑=1
𝜂𝑑𝑍𝑑𝑗−
𝑚
∑︁
𝑖=1
𝜈𝑖𝑋𝑖𝑗≤0,
∀𝑗,
𝑠
∑︁
𝑟=1
𝜇𝑟𝑌𝑟𝑗−
𝐷
∑︁
𝑑=1
𝜂𝑑𝑍𝑑𝑗≤0,
∀𝑗,
𝑠
∑︁
𝑟=1
𝜇𝑟𝑌max
𝑟
−𝐸IDMU(𝑠)*
𝑚
∑︁
𝑖=1
𝜈𝑖𝑋min
𝑖
≥0,
𝜇𝑟, 𝜈𝑖, 𝜂𝑑≥0,
∀𝑟, 𝑖, 𝑑. (3.6) ∀𝑟, 𝑖, 𝑑. (3.6) (3.6) ︁︁
𝜇𝑟, 𝜈𝑖, 𝜂𝑑≥0,
∀𝑟, 𝑖, 𝑑. (3.6) ︁
𝜇𝑟, 𝜈𝑖, 𝜂𝑑≥0, Model (3.6) aims to minimise the pessimistic efficiency measure of the ADMU, while keeping the optimistic
efficiency of the IDMU for the overall system no less than 𝐸IDMU(𝑠)*. It should be noted that the second and
third sets of constraints in both models imply that the overall efficiency of DMU cannot exceed 1. Model (3.6) aims to minimise the pessimistic efficiency measure of the ADMU, while keeping the optimistic
efficiency of the IDMU for the overall system no less than 𝐸IDMU(𝑠)*. It should be noted that the second and
third sets of constraints in both models imply that the overall efficiency of DMU cannot exceed 1. The next point to focus on in this paper is the examination of the highest and the lowest relative efficiency
of each DMU, considering their self-evaluation. In model (3.7), the optimistic relative efficiency of DMU𝑘for
the sub-stage 1 is examined while 𝐸IDMU(1)* is kept fixed; it is related to Wang and Luo’s [48] framework: 𝐸IDMU(1)
𝑘
= Max
𝐷
∑︁
𝑑=1
𝜂𝑑𝑘𝑍𝑑𝑘
subject to
𝑚
∑︁
𝑖=1
𝜈𝑖𝑘𝑋𝑖𝑘= 1,
𝐷
∑︁
𝑑=1
𝜂𝑑𝑘𝑍𝑑𝑗−
𝑚
∑︁
𝑖=1
𝜈𝑖𝑘𝑋𝑖𝑗≤0,
∀𝑗,
𝐷
∑︁
𝑑=1
𝜂𝑑𝑘𝑍max
𝑑
−𝐸IDMU(1)*
𝑚
∑︁
𝑖=1
𝜈𝑖𝑘𝑋min
𝑖
= 0,
𝜈𝑖𝑘, 𝜂𝑑𝑘≥0,
∀𝑖, 𝑑. (3.7) ∀𝑖, 𝑑. (3.7) ︁
𝑑
1︁
𝜈𝑖𝑘, 𝜂𝑑𝑘≥0,
∀𝑖, 𝑑. (3.7) (3.7) ︁
𝜈𝑖𝑘, 𝜂𝑑𝑘≥0, ︁
𝜈𝑖𝑘, 𝜂𝑑𝑘≥0, In the same manner, we construct the counterpart model for measuring the highest relative efficiency of DMU𝑘
for the sub-stage 2, considering 𝐸IDMU(2)* as the fixed parameter. IDMU( )︀ The overall optimistic efficiency score of DMU𝑘can be determined as 𝐸IDMU(𝑠)
𝑘
= (∑︀𝑠
𝑟=1 𝜇𝑟𝑘𝑌𝑟𝑘)/
(∑︀𝑚
𝑖=1 𝜈𝑖𝑘𝑋𝑖𝑘). 3.1. Optimistic & pessimistic models in basic two-stage structure The best and worst relative efficiency scores in terms of sub-stage 1 can be defined by the following two CCR
models, respective to the IDMU and the ADMU; they are related to Wang and Luo [48] and Wu’s [51] models: 𝐸IDMU(1) = Max
𝐷
∑︁
𝑑=1
𝜂𝑑𝑍max
𝑑
subject to
𝑚
∑︁
𝑖=1
𝜈𝑖𝑋min
𝑖
= 1,
𝐷
∑︁
𝑑=1
𝜂𝑑𝑍𝑑𝑗−
𝑚
∑︁
𝑖=1
𝜈𝑖𝑋𝑖𝑗≤0,
∀𝑗, ∀𝑗, (3.3) ︁
𝜈𝑖, 𝜂𝑑≥0, 𝐸ADMU(1) = Min
𝐷
∑︁
𝑑=1
𝜂𝑑𝑍min
𝑑
subject to
𝑚
∑︁
𝑖=1
𝜈𝑖𝑋max
𝑖
= 1,
𝐷
∑︁
𝑑=1
𝜂𝑑𝑍𝑑𝑗−
𝑚
∑︁
𝑖=1
𝜈𝑖𝑋𝑖𝑗≤0,
∀𝑗,
𝐷
∑︁
𝑑=1
𝜂𝑑𝑍max
𝑑
−𝐸IDMU(1)*
𝑚
∑︁
𝑖=1
𝜈𝑖𝑋min
𝑖
≥0,
𝜈𝑖, 𝜂𝑑≥0,
∀𝑖, 𝑑,
(3.4) (3.4) ︁
𝜈𝑖, 𝜂𝑑≥0, where 𝐸IDMU(1)* is the optimal optimistic score of IDMU in terms of sub-stage 1, obtained in model (3.3). Model (3.4) ensures that the best relative efficiency of sub-stage 1 is fixed at a value greater than or equal to
𝐸IDMU(1)*. where 𝐸IDMU(1)* is the optimal optimistic score of IDMU in terms of sub-stage 1, obtained in model (3.3). Model (3.4) ensures that the best relative efficiency of sub-stage 1 is fixed at a value greater than or equal to
𝐸IDMU(1)*. By the same token, we establish the definitions as well as formulate the appropriate optimisation models for
the IDMU and the ADMU, regarding sub-stage 2 of the basic two-stage structure. Note that sub-stage 2 focuses
on the consumption of intermediate products for the generation of the final outputs. The next stage concerns the determination of the optimistic and pessimistic efficiency scores of the IDMU
and the ADMU, respectively, in terms of the overall system. The reference model is the relational two-
stage DEA model (3.1). The efficiency of the IDMU for the entire system can be defined as 𝐸IDMU(𝑠) =
(∑︀𝑠
𝑟=1 𝜇𝑟𝑌max
𝑟
)/
(︀∑︀𝑚
𝑖=1 𝜈𝑖𝑋min
𝑖
)︀
. The factor weights 𝜇𝑟and 𝜈𝑖are assigned to the 𝑟th output and the 𝑖th input,
respectively. We thus construct the following LP model that aims to maximise the efficiency of the IDMU. 𝐸IDMU(𝑠) = Max
𝑠
∑︁
𝑟=1
𝜇𝑟𝑌max
𝑟
subject to
𝑚
∑︁
𝑖=1
𝜈𝑖𝑋min
𝑖
= 1,
𝐷
∑︁
𝑑=1
𝜂𝑑𝑍𝑑𝑗−
𝑚
∑︁
𝑖=1
𝜈𝑖𝑋𝑖𝑗≤0,
∀𝑗,
𝑠
∑︁
𝑟=1
𝜇𝑟𝑌𝑟𝑗−
𝐷
∑︁
𝑑=1
𝜂𝑑𝑍𝑑𝑗≤0,
∀𝑗,
𝜇𝑟, 𝜈𝑖, 𝜂𝑑≥0,
∀𝑟, 𝑖, 𝑑. (3.5 ︁︁
𝜇𝑟, 𝜈𝑖, 𝜂𝑑≥0,
∀𝑟, 𝑖, 𝑑. 3.1. Optimistic & pessimistic models in basic two-stage structure It is clear that this measure is the product of the optimistic efficiencies of the DMU𝑘of the
two sub-stages, adopting the principle of the multiplicative efficiency decomposition approach [21]. Thus, we
propose model (3.8), that maximises the above ratio. 𝐸IDMU(𝑠)
𝑘
= Max
𝑠
∑︁
𝑟=1
𝜇𝑟𝑘𝑌𝑟𝑘
subject to
𝑚
∑︁
𝑖=1
𝜈𝑖𝑘𝑋𝑖𝑘= 1, 1302 M.D. KREMANTZIS ET AL. 𝐷
∑︁
𝑑=1
𝜂𝑑𝑘𝑍𝑑𝑗−
𝑚
∑︁
𝑖=1
𝜈𝑖𝑘𝑋𝑖𝑗≤0,
∀𝑗,
𝑠
∑︁
𝑟=1
𝜇𝑟𝑘𝑌𝑟𝑗−
𝐷
∑︁
𝑑=1
𝜂𝑑𝑘𝑍𝑑𝑗≤0,
∀𝑗,
𝐷
∑︁
𝑑=1
𝜂𝑑𝑘𝑍max
𝑑
−𝐸IDMU(1)*
𝑚
∑︁
𝑖=1
𝜈𝑖𝑘𝑋min
𝑖
= 0,
𝑠
∑︁
𝑟=1
𝜇𝑟𝑘𝑌max
𝑟
−𝐸IDMU(2)*
𝐷
∑︁
𝑑=1
𝜂𝑑𝑘𝑍min
𝑑
= 0,
𝜇𝑟𝑘, 𝜈𝑖𝑘, 𝜂𝑑𝑘≥0,
∀𝑟, 𝑖, 𝑑. 𝐷
∑︁
𝑑=1
𝜂𝑑𝑘𝑍𝑑𝑗−
𝑚
∑︁
𝑖=1
𝜈𝑖𝑘𝑋𝑖𝑗≤0,
∀𝑗,
𝑠
∑︁
𝑟=1
𝜇𝑟𝑘𝑌𝑟𝑗−
𝐷
∑︁
𝑑=1
𝜂𝑑𝑘𝑍𝑑𝑗≤0,
∀𝑗,
𝐷
∑︁
𝑑=1
𝜂𝑑𝑘𝑍max
𝑑
−𝐸IDMU(1)*
𝑚
∑︁
𝑖=1
𝜈𝑖𝑘𝑋min
𝑖
= 0,
𝑠
∑︁
𝑟=1
𝜇𝑟𝑘𝑌max
𝑟
−𝐸IDMU(2)*
𝐷
∑︁
𝑑=1
𝜂𝑑𝑘𝑍min
𝑑
= 0,
𝜇𝑟𝑘, 𝜈𝑖𝑘, 𝜂𝑑𝑘≥0,
∀𝑟, 𝑖, 𝑑. (3.8) ∀𝑟, 𝑖, 𝑑. (3.8) (3.8) ︁︁
𝜇𝑟𝑘, 𝜈𝑖𝑘, 𝜂𝑑𝑘≥0,
∀𝑟, 𝑖, 𝑑. (3.8) ︁
𝜇𝑟𝑘, 𝜈𝑖𝑘, 𝜂𝑑𝑘≥0, ︁
𝜇𝑟𝑘, 𝜈𝑖𝑘, 𝜂𝑑𝑘≥0, The fourth and fifth constraints indicate that 𝐸IDMU(1)* and 𝐸IDMU(2)*, respectively, remain unchanged. Let
𝜈*
𝑘= (𝜈*
1𝑘, 𝜈*
2𝑘, . . ., 𝜈*
𝑚𝑘), 𝜂*
𝑘= (𝜂*
1𝑘, 𝜂*
2𝑘, . . ., 𝜂*
𝐷𝑘), 𝜇*
𝑘= (𝜇*
1𝑘, 𝜇*
2𝑘, . . ., 𝜇*
𝑠𝑘), be an optimal solution to model
(3.8). For DMU𝑘, 𝐸IDMU(𝑠)
𝑘
= (∑︀𝑠
𝑟=1 𝜇*
𝑟𝑘𝑌𝑟𝑘)/(∑︀𝑚
𝑖=1 𝜈*
𝑖𝑘𝑋𝑖𝑘), 𝐸IDMU(1)
𝑘
=
(︁∑︀𝐷
𝑑=1 𝜂*
𝑑𝑘𝑍𝑑𝑘
)︁
/(∑︀𝑚
𝑖=1 𝜈*
𝑖𝑘𝑋𝑖𝑘), and
𝐸IDMU(2)
𝑘
= (∑︀𝑠
𝑟=1 𝜇*
𝑟𝑘𝑌𝑟𝑘)/
(︁∑︀𝐷
𝑑=1 𝜂*
𝑑𝑘𝑍𝑑𝑘
)︁
, which are referred to as optimistic self-efficiency measures with
respect to the overall system and its sub-stages, respectively. The fourth and fifth constraints indicate that 𝐸IDMU(1)* and 𝐸IDMU(2)*, respectively, remain unchanged. Let
𝜈*
𝑘= (𝜈*
1𝑘, 𝜈*
2𝑘, . . ., 𝜈*
𝑚𝑘), 𝜂*
𝑘= (𝜂*
1𝑘, 𝜂*
2𝑘, . . ., 𝜂*
𝐷𝑘), 𝜇*
𝑘= (𝜇*
1𝑘, 𝜇*
2𝑘, . . ., 𝜇*
𝑠𝑘), be an optimal solution to model
(3.8). 3.1. Optimistic & pessimistic models in basic two-stage structure For DMU𝑘, 𝐸IDMU(𝑠)
𝑘
= (∑︀𝑠
𝑟=1 𝜇*
𝑟𝑘𝑌𝑟𝑘)/(∑︀𝑚
𝑖=1 𝜈*
𝑖𝑘𝑋𝑖𝑘), 𝐸IDMU(1)
𝑘
=
(︁∑︀𝐷
𝑑=1 𝜂*
𝑑𝑘𝑍𝑑𝑘
)︁
/(∑︀𝑚
𝑖=1 𝜈*
𝑖𝑘𝑋𝑖𝑘), and
𝐸IDMU(2)
𝑘
= (∑︀𝑠
𝑟=1 𝜇*
𝑟𝑘𝑌𝑟𝑘)/
(︁∑︀𝐷
𝑑=1 𝜂*
𝑑𝑘𝑍𝑑𝑘
)︁
, which are referred to as optimistic self-efficiency measures with
respect to the overall system and its sub-stages, respectively. The fourth and fifth constraints indicate that 𝐸IDMU(1)* and 𝐸 The fourth and fifth constraints indicate that 𝐸IDMU(1)* and 𝐸IDMU(2)*, respectively, remain unchanged. Let
𝜈*
𝑘= (𝜈*
1𝑘, 𝜈*
2𝑘, . . ., 𝜈*
𝑚𝑘), 𝜂*
𝑘= (𝜂*
1𝑘, 𝜂*
2𝑘, . . ., 𝜂*
𝐷𝑘), 𝜇*
𝑘= (𝜇*
1𝑘, 𝜇*
2𝑘, . . ., 𝜇*
𝑠𝑘), be an optimal solution to model
(3.8). For DMU𝑘, 𝐸IDMU(𝑠)
𝑘
= (∑︀𝑠
𝑟=1 𝜇*
𝑟𝑘𝑌𝑟𝑘)/(∑︀𝑚
𝑖=1 𝜈*
𝑖𝑘𝑋𝑖𝑘), 𝐸IDMU(1)
𝑘
=
(︁∑︀𝐷
𝑑=1 𝜂*
𝑑𝑘𝑍𝑑𝑘
)︁
/(∑︀𝑚
𝑖=1 𝜈*
𝑖𝑘𝑋𝑖𝑘), and
𝐸IDMU(2)
𝑘
= (∑︀𝑠
𝑟=1 𝜇*
𝑟𝑘𝑌𝑟𝑘)/
(︁∑︀𝐷
𝑑=1 𝜂*
𝑑𝑘𝑍𝑑𝑘
)︁
, which are referred to as optimistic self-efficiency measures with
respect to the overall system and its sub-stages, respectively. ︀︁︀︁
Then, model (3.9) evaluates the worst relative efficiency of DMU𝑘, in terms of sub-stage 1, while the parameter
𝐸ADMU(1)* takes the value as determined previously from model (3.4). This model is related to Wu’s [51]
framework. 𝐸ADMU(1)
𝑘
= Min
𝐷
∑︁
𝑑=1
𝜂𝑑𝑘𝑍𝑑𝑘
subject to
𝑚
∑︁
𝑖=1
𝜈𝑖𝑘𝑋𝑖𝑘= 1,
𝐷
∑︁
𝑑=1
𝜂𝑑𝑘𝑍𝑑𝑗−
𝑚
∑︁
𝑖=1
𝜈𝑖𝑘𝑋𝑖𝑗≤0,
∀𝑗,
𝐷
∑︁
𝑑=1
𝜂𝑑𝑘𝑍min
𝑑
−𝐸ADMU(1)*
𝑚
∑︁
𝑖=1
𝜈𝑖𝑘𝑋max
𝑖
= 0,
𝜈𝑖𝑘, 𝜂𝑑𝑘≥0,
∀𝑖, 𝑑. (3.9) ∀𝑖, 𝑑. (3.9) (3.9) ︁
𝜈𝑖𝑘, 𝜂𝑑𝑘≥0, ︁
𝜈𝑖𝑘, 𝜂𝑑𝑘≥0, Similarly, we formulate the counterpart model for measuring the lowest relative efficiency of DMU𝑘for the
sub-stage 2, considering 𝐸ADMU(2)* as the unchanged parameter. ADMU( )︀︀ Similarly, we formulate the counterpart model for measuring the lowest relative efficiency of DMU𝑘for the
sub-stage 2, considering 𝐸ADMU(2)* as the unchanged parameter. ADMU( )︀︀ The overall pessimistic score of DMU𝑘can be determined as 𝐸ADMU(𝑠)
𝑘
= (∑︀𝑠
𝑟=1 𝜇𝑟𝑘𝑌𝑟𝑘)/(∑︀𝑚
𝑖=1 𝜈𝑖𝑘𝑋𝑖𝑘) and
denotes the product of the pessimistic efficiencies of the DMU𝑘of the two sub-stages. Thus, we suggest model
(3.10), whose purpose is to minimise the above ratio. 𝐸ADMU(1)* and 𝐸ADMU(2)* are maintained. 3.1. Optimistic & pessimistic models in basic two-stage structure 𝐸ADMU(𝑠)
𝑘
= Min
𝑠
∑︁
𝑟=1
𝜇𝑟𝑘𝑌𝑟𝑘
subject to
𝑚
∑︁
𝑖=1
𝜈𝑖𝑘𝑋𝑖𝑘= 1,
𝐷
∑︁
𝑑=1
𝜂𝑑𝑘𝑍𝑑𝑗−
𝑚
∑︁
𝑖=1
𝜈𝑖𝑘𝑋𝑖𝑗≤0,
∀𝑗, ∀𝑗, 1303 A RANKING FRAMEWORK BASED ON INTERVAL SELF AND CROSS-EFFICIENCIES 𝑠
∑︁
𝑟=1
𝜇𝑟𝑘𝑌𝑟𝑗−
𝐷
∑︁
𝑑=1
𝜂𝑑𝑘𝑍𝑑𝑗≤0,
∀𝑗,
𝐷
∑︁
𝑑=1
𝜂𝑑𝑘𝑍min
𝑑
−𝐸ADMU(1)*
𝑚
∑︁
𝑖=1
𝜈𝑖𝑘𝑋max
𝑖
= 0,
𝑠
∑︁
𝑟=1
𝜇𝑟𝑘𝑌min
𝑟
−𝐸ADMU(2)*
𝐷
∑︁
𝑑=1
𝜂𝑑𝑘𝑍max
𝑑
= 0,
𝜇𝑟𝑘, 𝜈𝑖𝑘, 𝜂𝑑𝑘≥0,
∀𝑟, 𝑖, 𝑑. (3.10) ∀𝑗, ∀𝑟, 𝑖, 𝑑. (3.10) ︁︁
𝜇𝑟𝑘, 𝜈𝑖𝑘, 𝜂𝑑𝑘≥0,
∀𝑟, 𝑖, 𝑑. (3.10) (3.10) ︁
𝜇𝑟𝑘, 𝜈𝑖𝑘, 𝜂𝑑𝑘≥0, Let 𝜈∼
𝑘= (𝜈∼
1𝑘, 𝜈∼
2𝑘, . . ., 𝜈∼
𝑚𝑘), 𝜂∼
𝑘= (𝜂∼
1𝑘, 𝜂∼
2𝑘, . . ., 𝜂∼
𝐷𝑘), 𝜇∼
𝑘= (𝜇∼
1𝑘, 𝜇∼
2𝑘, . . ., 𝜇∼
𝑠𝑘), be an optimal solution to model
(3.10). For DMU𝑘, 𝐸ADMU(𝑠)
𝑘
= (∑︀𝑠
𝑟=1 𝜇∼
𝑟𝑘𝑌𝑟𝑘)/(∑︀𝑚
𝑖=1 𝜈∼
𝑖𝑘𝑋𝑖𝑘), 𝐸ADMU(1)
𝑘
=
(︁∑︀𝐷
𝑑=1 𝜂∼
𝑑𝑘𝑍𝑑𝑘
)︁
/(∑︀𝑚
𝑖=1 𝜈∼
𝑖𝑘𝑋𝑖𝑘),
and 𝐸ADMU(2)
𝑘
= (∑︀𝑠
𝑟=1 𝜇∼
𝑟𝑘𝑌𝑟𝑘)/
(︁∑︀𝐷
𝑑=1 𝜂∼
𝑑𝑘𝑍𝑑𝑘
)︁
, which are referred to as pessimistic self-efficiency measures
with respect to the overall system and its constituent parts, respectively. Let 𝜈∼
𝑘= (𝜈∼
1𝑘, 𝜈∼
2𝑘, . . ., 𝜈∼
𝑚𝑘), 𝜂∼
𝑘= (𝜂∼
1𝑘, 𝜂∼
2𝑘, . . ., 𝜂∼
𝐷𝑘), 𝜇∼
𝑘= (𝜇∼
1𝑘, 𝜇∼
2𝑘, . . ., 𝜇∼
𝑠𝑘), be an optimal solution to model
(3.10). For DMU𝑘, 𝐸ADMU(𝑠)
𝑘
= (∑︀𝑠
𝑟=1 𝜇∼
𝑟𝑘𝑌𝑟𝑘)/(∑︀𝑚
𝑖=1 𝜈∼
𝑖𝑘𝑋𝑖𝑘), 𝐸ADMU(1)
𝑘
=
(︁∑︀𝐷
𝑑=1 𝜂∼
𝑑𝑘𝑍𝑑𝑘
)︁
/(∑︀𝑚
𝑖=1 𝜈∼
𝑖𝑘𝑋𝑖𝑘),
and 𝐸ADMU(2)
𝑘
= (∑︀𝑠
𝑟=1 𝜇∼
𝑟𝑘𝑌𝑟𝑘)/
(︁∑︀𝐷
𝑑=1 𝜂∼
𝑑𝑘𝑍𝑑𝑘
)︁
, which are referred to as pessimistic self-efficiency measures
with respect to the overall system and its constituent parts, respectively. ︀︀
Consequently, in a two-stage DEA structure, a self-efficiency interval is formulated for each DMU under
consideration, both for the overall system and its constituent stages. For instance, considering the overall system,
an efficiency interval denoted by
[︁
𝐸ADMU(𝑠)*
𝑘
, 𝐸IDMU(𝑠)*
𝑘
]︁
is shaped, where 𝐸ADMU(𝑠)*
𝑘
(lower bound) represents
the worst relative efficiency of DMU𝑘and 𝐸IDMU(𝑠)*
𝑘
(upper bound) illustrates the best relative efficiency of
DMU𝑘, obtained via models (3.10) and (3.8), respectively. There is a clear need to integrate both optimistic and pessimistic self-efficiency measures to provide an
overall assessment of the performance of each DMU in a two-stage DEA process. 3.1. Optimistic & pessimistic models in basic two-stage structure This study adopts the ge-
ometric average efficiency measure, proposed and verified by Wang et al. [50], to meet this requirement. Let
𝐸comb(𝜖)*
𝑘
=
√︁
𝐸ADMU(𝜖)*
𝑘
· 𝐸IDMU(𝜖)*
𝑘
be the combined self-efficiency measure of DMU𝑘, where 𝜖= 𝑠(over-
all system) or 1 (sub-stage 1) or 2 (sub-stage 2). We easily prove that the combined self-efficiency score
of DMU𝑘for the overall system is the product of the combined self-efficiency measures of DMU𝑘for the
two sub-stages: 𝐸𝑐𝑜𝑚𝑏(𝑠)*
𝑘
=
√︁
𝐸ADMU(𝑠)*
𝑘
· 𝐸IDMU(𝑠)*
𝑘
=
√︁
𝐸ADMU(1)*
𝑘
· 𝐸ADMU(2)*
𝑘
· 𝐸IDMU(1)*
𝑘
· 𝐸IDMU(2)*
𝑘
=
√︁
𝐸ADMU(1)*
𝑘
· 𝐸IDMU(1)*
𝑘
·
√︁
𝐸ADMU(2)*
𝑘
· 𝐸IDMU(2)*
𝑘
= 𝐸𝑐𝑜𝑚𝑏(1)*
𝑘
· 𝐸𝑐𝑜𝑚𝑏(2)*
𝑘
. √︁
√︁
The geometric average efficiency is an approachable efficiency measure that leads to a fairer ranking index
[50]. However, we should consider that it sheds light on the effects of the optimistic and pessimistic standpoints
only within the self-appraisal context. In other words, each DMU is assessed, based on its own most favourable
and unfavourable weights, without considering the weight scheme of each of the other DMUs. This score also
ensures a better discriminating power than either of the optimistic and pessimistic efficiencies [50]. Yet, this
feature has not been explored in a more complex network structure. To this end, in the next section, the double
frontier DEA models are further extended by the use of the interval CE within a two-stage tandem system, to
ensure a more logical ranking order. 3.2. Interval cross-efficiencies in basic two-stage structure Model (3.11) pursues to minimise the cross-efficiency value
of DMU𝑗under the condition that the combined self-efficiency scores for the overall system and its constituent In model (3.11), 𝐸comb(𝑠)*
𝑘
and 𝐸comb(2)*
𝑘
are the crisp combined self-efficiency measures of the system and
the sub-stage 2 for DMU𝑘, respectively, obtained from Section 3.1. The second and third constraint maintain
combined system and sub-stage efficiencies for DMUs. Model (3.11) pursues to minimise the cross-efficiency value
of DMU𝑗under the condition that the combined self-efficiency scores for the overall system and its constituent
parts remain unchanged. At optimality, the minimum individual cross-efficiencies of DMU𝑗based on DMU𝑘(𝑗̸=
𝑘) for the overall system, the stage 1, and the stage 2, are determined as 𝐸𝐿(𝑠)
𝑘𝑗
= (∑︀𝑠
𝑟=1 𝜇*
𝑟𝑘𝑌𝑟𝑗)/(∑︀𝑚
𝑖=1 𝜈*
𝑖𝑘𝑋𝑖𝑗),
𝐸𝐿(1)
𝑘𝑗
=
(︁∑︀𝐷
𝑑=1 𝜂*
𝑑𝑘𝑍𝑑𝑗
)︁
/(∑︀𝑚
𝑖=1 𝜈*
𝑖𝑘𝑋𝑖𝑗), and 𝐸𝐿(2)
𝑘𝑗
= (∑︀𝑠
𝑟=1 𝜇*
𝑟𝑘𝑌𝑟𝑗)/
(︁∑︀𝐷
𝑑=1 𝜂*
𝑑𝑘𝑍𝑑𝑗
)︁
, respectively. By the same In model (3.11), 𝐸comb(𝑠)*
𝑘
and 𝐸comb(2)*
𝑘
are the crisp combined self-efficiency measures of the system and
the sub-stage 2 for DMU𝑘, respectively, obtained from Section 3.1. The second and third constraint maintain
combined system and sub-stage efficiencies for DMUs. Model (3.11) pursues to minimise the cross-efficiency value
of DMU𝑗under the condition that the combined self-efficiency scores for the overall system and its constituent
parts remain unchanged. At optimality, the minimum individual cross-efficiencies of DMU𝑗based on DMU𝑘(𝑗̸=
𝑘) for the overall system, the stage 1, and the stage 2, are determined as 𝐸𝐿(𝑠)
𝑘𝑗
= (∑︀𝑠
𝑟=1 𝜇*
𝑟𝑘𝑌𝑟𝑗)/(∑︀𝑚
𝑖=1 𝜈*
𝑖𝑘𝑋𝑖𝑗),
𝐸𝐿(1)
𝑘𝑗
=
(︁∑︀𝐷
𝑑=1 𝜂*
𝑑𝑘𝑍𝑑𝑗
)︁
/(∑︀𝑚
𝑖=1 𝜈*
𝑖𝑘𝑋𝑖𝑗), and 𝐸𝐿(2)
𝑘𝑗
= (∑︀𝑠
𝑟=1 𝜇*
𝑟𝑘𝑌𝑟𝑗)/
(︁∑︀𝐷
𝑑=1 𝜂*
𝑑𝑘𝑍𝑑𝑗
)︁
, respectively. By the same
token, a benevolent strategy is implemented to construct the following maximisation model: 𝐸𝑈(𝑠)
𝑘𝑗
= Max
𝑠
∑︁
𝑟=1
𝜇𝑟𝑘· 𝑌𝑟𝑗 𝐸𝑈(𝑠)
𝑘𝑗
= Max
𝑠
∑︁
𝑟=1
𝜇𝑟𝑘· 𝑌𝑟𝑗
(3.12) (3.12) ︁
subject to the same constraints as in model (3.11). This model seeks to maximise the cross-efficiency of DMU𝑗given that the combined self-efficiency measures are
kept fixed for the overall system and its sub-stages. Similarly, we define the maximum individual cross-efficiencies
of DMU𝑗for the system and its stages. 3.2. Interval cross-efficiencies in basic two-stage structure In terms of 𝜖, where 𝜖= 𝑠(overall system), 1 (stage 1) or 2 (stage 2), for DMU𝑗, its cross-efficiency rated by
DMU𝑘lies in
[︁
𝐸L(𝜖)
𝑘𝑗, 𝐸U(𝜖)
𝑘𝑗
]︁
, where 𝐸L(𝜖)
𝑘𝑗
is the lower bound and 𝐸U(𝜖)
𝑘𝑗
is the upper bound. Therefore, three
generalised interval CE matrices (based on the concept of Tab. 2) are shaped for the 𝑛DMUs, in regard to
the overall system, the stage 1, and the stage 2, respectively. The diagonal column in each of these matrices
demonstrates the special case in which 𝐸L(𝜖)*
𝑗𝑗
= 𝐸U(𝜖)*
𝑗𝑗
= 𝐸comb(𝜖)*
𝑗
∀𝑗, where 𝜖= 𝑠, 1 or 2. 𝑗𝑗
𝑗𝑗
𝑗
The recently created interval CE matrices can be viewed as MCDM problems. Taking that into consideration,
we will set the scene for the determination of the interval local weights of criteria and the interval global weights
of alternatives (ultimate cross-efficiencies) to fully rank the DMUs, in a basic two-stage DEA structure. 3.2. Interval cross-efficiencies in basic two-stage structure In this section, we will propose the customisation and simultaneous use of the traditional aggressive and
benevolent secondary models in the context of the basic two-stage DEA structure with combined self-efficiencies,
obtained in Section 3.1. Their purpose is the determination of the minimum and maximum individual cross-
efficiencies of DMU𝑗, with respect to the optimal weight scheme of DMU𝑘(𝑘, 𝑗= 1, 2, . . ., 𝑛), respectively. A fruitful aspect we believe, is the integration of the combined self-efficiency score for the corresponding sys-
tem/stage within the CE process. This is irrespective of the type of multipliers, favourable for a benevolent or
unfavourable for an aggressive strategy, that are used to capture the cross-efficiencies. 1304 M.D. KREMANTZIS ET AL. We initially adopt an aggressive strategy to establish the following minimisation model:︁ We initially adopt an aggressive strategy to establish the following minimisation model:︁ 𝐸𝐿(𝑠)
𝑘𝑗
= Min
𝑠
∑︁
𝑟=1
𝜇𝑟𝑘𝑌𝑟𝑗
subject to
𝑚
∑︁
𝑖=1
𝜈𝑖𝑘𝑋𝑖𝑗= 1,
𝑠
∑︁
𝑟=1
𝜇𝑟𝑘𝑌𝑟𝑘−𝐸comb(𝑠)*
𝑘
𝑚
∑︁
𝑖=1
𝜈𝑖𝑘𝑋𝑖𝑘= 0,
𝑠
∑︁
𝑟=1
𝜇𝑟𝑘𝑌𝑟𝑘−𝐸comb(2)*
𝑘
𝐷
∑︁
𝑑=1
𝜂𝑑𝑘𝑍𝑑𝑘= 0,
𝐷
∑︁
𝑑=1
𝜂𝑑𝑘𝑍𝑑𝑗−
𝑚
∑︁
𝑖=1
𝜈𝑖𝑘𝑋𝑖𝑗≤0,
∀𝑗,
𝑠
∑︁
𝑟=1
𝜇𝑟𝑘𝑌𝑟𝑗−
𝐷
∑︁
𝑑=1
𝜂𝑑𝑘𝑍𝑑𝑗≤0,
∀𝑗,
𝜇𝑟𝑘, 𝜈𝑖𝑘, 𝜂𝑑𝑘≥0,
∀𝑟, 𝑖, 𝑑. (3. 𝐸𝐿(𝑠)
𝑘𝑗
= Min
𝑠
∑︁
𝑟=1
𝜇𝑟𝑘𝑌𝑟𝑗
subject to
𝑚
∑︁
𝑖=1
𝜈𝑖𝑘𝑋𝑖𝑗= 1,
𝑠
∑︁
𝑟=1
𝜇𝑟𝑘𝑌𝑟𝑘−𝐸comb(𝑠)*
𝑘
𝑚
∑︁
𝑖=1
𝜈𝑖𝑘𝑋𝑖𝑘= 0,
𝑠
∑︁
𝑟=1
𝜇𝑟𝑘𝑌𝑟𝑘−𝐸comb(2)*
𝑘
𝐷
∑︁
𝑑=1
𝜂𝑑𝑘𝑍𝑑𝑘= 0,
𝐷
∑︁
𝑑=1
𝜂𝑑𝑘𝑍𝑑𝑗−
𝑚
∑︁
𝑖=1
𝜈𝑖𝑘𝑋𝑖𝑗≤0,
𝑠
∑︁
𝑟=1
𝜇𝑟𝑘𝑌𝑟𝑗−
𝐷
∑︁
𝑑=1
𝜂𝑑𝑘𝑍𝑑𝑗≤0,
𝜇𝑟𝑘, 𝜈𝑖𝑘, 𝜂𝑑𝑘≥0, ∀𝑟, 𝑖, 𝑑. (3.11) (3.11) ︁︁
𝜇𝑟𝑘, 𝜈𝑖𝑘, 𝜂𝑑𝑘≥0,
∀𝑟, 𝑖, 𝑑. (3.11) ︁︁
𝜇𝑟𝑘, 𝜈𝑖𝑘, 𝜂𝑑𝑘≥0, In model (3.11), 𝐸comb(𝑠)*
𝑘
and 𝐸comb(2)*
𝑘
are the crisp combined self-efficiency measures of the system and
h
b
2 f
DMU
i
l
b
i
d f
S
i
3 1 Th
d
d hi d
i
i
i In model (3.11), 𝐸comb(𝑠)*
𝑘
and 𝐸comb(2)*
𝑘
are the crisp combined self-efficiency measures of the system and
the sub-stage 2 for DMU𝑘, respectively, obtained from Section 3.1. The second and third constraint maintain
combined system and sub-stage efficiencies for DMUs. 3.3. Interval cross-efficiencies and MCDM context We will provide their optimisation model as we would apply this within the basic 2-stage series
structure: Ω= Min
𝑛
∑︁
𝑘=1
(︀
𝛿+
𝑘+ 𝛿−
𝑘+ 𝛾+
𝑘+ 𝛾−
𝑘
)︀
subject to (𝐸L −𝐼)𝑊U −(𝑛−1)𝑊L −∆+ + ∆−= 0,
(𝐸U −𝐼)𝑊L −(𝑛−1)𝑊U −Γ+ + Γ−= 0,
𝑤L
𝑘+
𝑛
∑︁
𝜔=1,𝜔̸=𝑘
𝑤U
𝜔≥1,
∀𝑘,
𝑤U
𝑘+
𝑛
∑︁
𝜔=1,𝜔̸=𝑘
𝑤L
𝜔≤1,
∀𝑘,
𝑊U −𝑊L ≥0,
𝑊U 𝑊L ∆+ ∆−Γ+ Γ−≥0 Ω= Min
𝑛
∑︁
𝑘=1
(︀
𝛿+
𝑘+ 𝛿−
𝑘+ 𝛾+
𝑘+ 𝛾−
𝑘
)︀
subject to (𝐸L −𝐼)𝑊U −(𝑛−1)𝑊L −∆+ + ∆−= 0,
(𝐸U −𝐼)𝑊L −(𝑛−1)𝑊U −Γ+ + Γ−= 0, 𝑤L
𝑘+
𝑛
∑︁
𝜔=1,𝜔̸=𝑘
𝑤U
𝜔≥1,
∀𝑘, ︁
𝑤U
𝑘+
𝑛
∑︁
𝜔=1,𝜔̸=𝑘
𝑤L
𝜔≤1,
∀𝑘, ︁
𝑊U −𝑊L ≥0,
𝑊U, 𝑊L, ∆+, ∆−, Γ+, Γ−≥0, ︁
𝑊U −𝑊L ≥0,
𝑊U, 𝑊L, ∆+, ∆−, Γ+, Γ−≥0,
(3.13) 𝑊U
𝑊L ≥0,
𝑊U, 𝑊L, ∆+, ∆−, Γ+, Γ−≥0,
(3.13)︀︀︀︀︀︀︀ U
L ≥,
𝑊U, 𝑊L, ∆+, ∆−, Γ+, Γ−≥0, (3.13) here ∆+ =
(︀
𝛿+
1 , . . ., 𝛿+
𝑛
)︀𝑇, ∆−=
(︀
𝛿−
1 , . . ., 𝛿−
𝑛
)︀𝑇, Γ+ =
(︀
𝛾+
1 , . . ., 𝛾+
𝑛
)︀𝑇, Γ−=
(︀
𝛾−
1 , . . ., 𝛾−
𝑛
)︀𝑇, 𝑊U =︀
U
U)︀𝑇
(︀
L
L)︀𝑇 where ∆+ =
(︀
𝛿+
1 , . . ., 𝛿+
𝑛
)︀𝑇, ∆−=
(︀
𝛿−
1 , . . ., 𝛿−
𝑛
)︀𝑇, Γ+ =
(︀
𝛾+
1 , . . ., 𝛾+
𝑛
)︀𝑇, Γ−=
(︀
𝛾−
1 , . . ., 𝛾−
𝑛
)︀𝑇, 𝑊U =
(︀
𝑤U
1 , . . ., 𝑤U
𝑛
)︀𝑇, 𝑊L =
(︀
𝑤L
1 , . . ., 𝑤L
𝑛
)︀𝑇, 𝐼is a 𝑛⊗𝑛unit matrix whose elements on the diagonal are 1, and
𝐸L and 𝐸U are the minimum and maximum individual cross-efficiency matrices, whose elements are in the form
of 𝐸L(𝜖)
𝑘𝑗
and 𝐸U(𝜖)
𝑘𝑗
respectively. The deviation vectors ∆+, ∆−, Γ+, Γ−, that appear in the first two constraint
sets, pursue to eliminate the uncertainty and connect the lower level criteria 𝑊L with the upper level criteria 𝑊U. The third and fourth sets of constraints ensure the normalisation of the local interval weights, whereas the fifth
constraint set determines their lower and upper bounds. 3.3. Interval cross-efficiencies and MCDM context Each generalised interval CE matrix (see Sect. 3.2) can be treated as a multi-criteria decision making (MCDM)
problem with 𝑗= 1, 2, . . ., 𝑛DMUs that act as alternatives. Each DMU𝑗is assessed considering the weight profile A RANKING FRAMEWORK BASED ON INTERVAL SELF AND CROSS-EFFICIENCIES 1305 of 𝑘= 1, 2, . . ., 𝑛DMUs that act as criteria. Interestingly, the former intuition is attributed to the novel study
of Cook et al. [8], according to which each DEA-related problem could be viewed as a multi-criteria evaluation
problem. This has also been consolidated by Rakhshan [37], who argues that the combination of the MCDM
and the DEA tools could mitigate their drawbacks when applied as stand-alone techniques. g
pp
q
Our primary target is to estimate the interval ultimate cross-efficiency scores, which are the interval global
weights for the evaluated DMUs. To this end, our approach is twofold as it requires not only the local weights
of alternatives with respect to a certain criterion, but also the local weights of criteria. The former are the
elements 𝐸L(𝜖)
𝑘𝑗
and 𝐸U(𝜖)
𝑘𝑗
, which act as lower-level and upper-level local weights of alternative 𝑗in reference
to criterion 𝑘for 𝜖= 𝑠, 1 or 2, respectively, and overall compose
[︁
𝐸L(𝜖)
𝑘𝑗, 𝐸U(𝜖)
𝑘𝑗
]︁
. These elements have been
obtained in Section 3.2. The latter illustrates the local weight of criterion 𝑘, that is manifested as an interval
value with lower bound 𝑤L
𝑘and upper bound 𝑤U
𝑘. The existence of this interval value is due to dealing with two
diametrically opposed strategies for the overall system and its constituent stages. Wang and Elhag [47] suggest a goal programming (GP) method to elicit normalised interval local weights
from an interval comparison matrix. In our scenario, the interval CE matrix is committed to undertaking the
role of the interval comparison matrix. Their method captures the lower and upper limits of the local weight of
criterion 𝑘(𝑘= 1, 2, . . ., 𝑛) without ignoring the interval individual cross-efficiencies and the potential existence
of uncertainty. Table 3. Synthesis of interval cross-efficiencies. Target
DMU𝑗
Evaluator DMU𝑘
1
2
. . . 𝑛
1
[︁
𝑤L(𝜖)
1
, 𝑤U(𝜖)
1
]︁
[︁
𝐸L(𝜖)
11 , 𝐸U(𝜖)
11
]︁
[︁
𝐸L(𝜖)
12 , 𝐸U(𝜖)
12
]︁
. . . [︁
𝐸L(𝜖)
1𝑛, 𝐸U(𝜖)
1𝑛
]︁
2
[︁
𝑤L(𝜖)
2
, 𝑤U(𝜖)
2
]︁
[︁
𝐸L(𝜖)
21 , 𝐸U(𝜖)
21
]︁
[︁
𝐸L(𝜖)
22 , 𝐸U(𝜖)
22
]︁
. . . [︁
𝐸L(𝜖)
2𝑛, 𝐸U(𝜖)
2𝑛
]︁
. . . . . . . . . . . . . . . 𝑛
[︁
𝑤L(𝜖)
𝑛
, 𝑤U(𝜖)
𝑛
]︁
[︁
𝐸L(𝜖)
𝑛1 , 𝐸U(𝜖)
𝑛1
]︁
[︁
𝐸L(𝜖)
𝑛2 , 𝐸U(𝜖)
𝑛2
]︁
. . . [︁
𝐸L(𝜖)
𝑛𝑛, 𝐸U(𝜖)
𝑛𝑛
]︁
Ultimate cross-efficiencies
[︁
𝐸L.B.(𝜖)
1
, 𝐸U.B.(𝜖)
1
]︁
[︁
𝐸L.B.(𝜖)
2
, 𝐸U.B.(𝜖)
2
]︁
. . . [︁
𝐸L.B.(𝜖)
𝑛
, 𝐸U.B.(𝜖)
𝑛
]︁ Taking the interval local weight for each criterion 𝑘and the interval local weight of each alternative 𝑗with
respect to criterion 𝑘into account, we determine the interval ultimate cross-efficiencies for the alternatives. We
recommend using the practical method of Entani and Tanaka [13] that is based on a pair of linear programming
(LP) models. Their approach treats the local weights of criteria as decision variables to be optimised and intends
to determine the global weights for each DMU. The pair of LP models is described as follows: 𝐸L.B.(𝜖)
𝑗
= Min
𝑛
∑︁
𝑘=1
𝑤(𝜖)
𝑘𝐸L(𝜖)
𝑘𝑗
subject to
𝑛
∑︁
𝑘=1
𝑤(𝜖)
𝑘
= 1,
𝑤L(𝜖)
𝑘
≤𝑤(𝜖)
𝑘
≤𝑤U(𝜖)
𝑘
,
∀𝑘,
(3.14) (3.14) and 𝐸U.B.(𝜖)
𝑗
= Max
𝑛
∑︁
𝑘=1
𝑤(𝜖)
𝑘𝐸U(𝜖)
𝑘𝑗 𝐸U.B.(𝜖)
𝑗
= Max
𝑛
∑︁
𝑘=1
𝑤(𝜖)
𝑘𝐸U(𝜖)
𝑘𝑗
(3.15) (3.15) ︁
subject to the same constraints as in model (3.14), ︁
subject to the same constraints as in model (3.14), where 𝑤(𝜖)
𝑘
is the decision variable of the 𝑘th local criterion weight (𝑘= 1, 2, . . ., 𝑛) for 𝜖= 𝑠(overall system),
1 (stage 1) or 2 (stage 2). The above pair of LP models (3.14) and (3.15) results in the interval global weight
for each alternative 𝑗(𝑗= 1, 2, . . ., 𝑛), denoted by
[︁
𝐸L.B.(𝜖)
𝑗
, 𝐸U.B.(𝜖)
𝑗
]︁
for the entire system and its sub-stages. Table 3 illustrates the synthesis of the interval cross-efficiencies. where 𝑤(𝜖)
𝑘
is the decision variable of the 𝑘th local criterion weight (𝑘= 1, 2, . Table 3. Synthesis of interval cross-efficiencies. . ., 𝑛) for 𝜖= 𝑠(overall system),
1 (stage 1) or 2 (stage 2). The above pair of LP models (3.14) and (3.15) results in the interval global weight
for each alternative 𝑗(𝑗= 1, 2, . . ., 𝑛), denoted by
[︁
𝐸L.B.(𝜖)
𝑗
, 𝐸U.B.(𝜖)
𝑗
]︁
for the entire system and its sub-stages. Table 3 illustrates the synthesis of the interval cross-efficiencies. 3.3. Interval cross-efficiencies and MCDM context Model (3.13) should, in effect, run three times, based
on the investigation of the interval CE matrix of the respective system and stage to compose
[︁
𝑤L(𝜖)
𝑘
, 𝑤U(𝜖)
𝑘
]︁
. [︁
]︁
Their approach might make sense in our study for two reasons. It has a greater scope for action due to
its compatibility with any interval comparison matrix, and involves less constraints than other methods such
as that of Sugihara et al. [43]. This enables it as an easier-to-use method for the DM. The fewer number of
constraints was owed to its practice, putting more emphasis on the matrix as a whole rather than on each
element individually. Wang and Elhag’s [47] technique has, to our knowledge, not received attention on eliciting
interval local weights from an interval CE matrix. Therefore, this section intends to use their approach to achieve
this goal. 1306 M.D. KREMANTZIS ET AL. Table 4. Interval ultimate cross-efficiencies. DMU𝑗
1 (L.B.)
2 (U.B.)
1
𝐸L.B.(𝜖)
1
𝐸U.B.(𝜖)
1
2
𝐸L.B.(𝜖)
2
𝐸U.B.(𝜖)
2
. . . . . . . . . 𝑛
𝐸L.B.(𝜖)
𝑛
𝐸U.B.(𝜖)
𝑛 performance of all alternatives into comparability sequence. According to the comparability sequence, an ideal
target sequence (reference sequence) is defined in the reference sequence definition (second step). In a third step,
a grey relational coefficient is calculated to illustrate the distance between the comparability and the reference
sequence. In a final step, the GRG between the reference and every comparability sequence is calculated, based
on the grey relational coefficient. If the comparability sequence of an alternative has the highest grey relational
grade, then this alternative is deemed as the most desirable one [25]. Below, we will provide an overview of the
GRA as we would apply this to ranking interval ultimate cross-efficiencies. performance of all alternatives into comparability sequence. According to the comparability sequence, an ideal
target sequence (reference sequence) is defined in the reference sequence definition (second step). In a third step,
a grey relational coefficient is calculated to illustrate the distance between the comparability and the reference
sequence. In a final step, the GRG between the reference and every comparability sequence is calculated, based
on the grey relational coefficient. If the comparability sequence of an alternative has the highest grey relational
grade, then this alternative is deemed as the most desirable one [25]. Below, we will provide an overview of the
GRA as we would apply this to ranking interval ultimate cross-efficiencies. To start with, we collect the data to be evaluated from the mathematical viewpoint. The interval ultimate
cross-efficiency scores, defined in Section 3.3, are gathered into a 𝑛⊗2 matrix, setting out the appropriate
conditions for translating the DMUs into alternatives and the two extreme cases (lower bound, upper bound)
into criteria. Hence, we form another MCDM problem with 𝑗= 1, 2, . . ., 𝑛alternatives that are assessed by
𝑖= 1, 2 attributes. Table 4 depicts what we described above.︁︁ The 𝑗th alternative can be expressed as 𝐸(𝜖)
𝑗
=
(︁
𝐸L.B.(𝜖)
𝑗
, 𝐸U.B.(𝜖)
𝑗
)︁
, where 𝐸𝑖(𝜖)
𝑗
is the ultimate cross-efficiency
of attribute 𝑖of alternative 𝑗and where 𝜖= 𝑠(overall system), 1 (stage 1) or 2 (stage 2). 3.4. Grey relational analysis for ranking DMUs In Section 3.3, we obtained an interval ultimate cross-efficiency score for DMU𝑗(𝑗= 1, 2, . . ., 𝑛). It is apparent
that there is a significant need to identify a simple yet efficient ranking approach for comparing and ranking
different DMUs, whose performance is expressed in the form of interval values. In this study, the Grey Relational
Analysis (GRA) is applied to obtain a unique ranking order for the DMUs, whose ultimate cross-efficiencies
are illustrated within certain boundaries, and thus to determine the most desirable alternative. GRA is based
on the grey system theory proposed by Julong [20]. It has proved to be a worthy methodological tool when
uncertain information emerges. GRA has fruitfully examined complex interconnections among several factors
[6] as well as obtained the optimal alternative among several alternatives [25,41]. GRA consists of four main steps: grey relational generating, reference sequence definition, grey relational
coefficient calculation, and grey relational grade (GRG) calculation. In a first step, GRA translates the existing 1307 A RANKING FRAMEWORK BASED ON INTERVAL SELF AND CROSS-EFFICIENCIES Table 4. Interval ultimate cross-efficiencies. The term 𝐸(𝜖)
𝑗
is
translated into the comparability sequence
¯
𝐸(𝜖)
𝑗
=
(︁
¯
𝐸L.B.(𝜖)
𝑗
,
¯
𝐸U.B.(𝜖)
𝑗
)︁
by use of one of the following equations: ︁︁
¯
𝐸𝑖(𝜖)
𝑗
=
𝐸𝑖(𝜖)
𝑗
−min
{︁
𝐸𝑖(𝜖)
𝑗
, ∀𝑗
}︁
max
{︁
𝐸𝑖(𝜖)
𝑗
, 𝑗
}︁
−min
{︁
𝐸𝑖(𝜖)
𝑗
, ∀𝑗
}︁,
∀𝑗, 𝑖,
(3.16)
¯
𝐸𝑖(𝜖)
𝑗
=
max
{︁
𝐸𝑖(𝜖)
𝑗
, ∀𝑗
}︁
−𝐸𝑖(𝜖)
𝑗
max
{︁
𝐸𝑖(𝜖)
𝑗
, ∀𝑗
}︁
−min
{︁
𝐸𝑖(𝜖)
𝑗
, ∀𝑗
}︁,
∀𝑗, 𝑖,
(3.17)
¯
𝐸𝑖(𝜖)
𝑗
=
⃒⃒⃒𝐸𝑖(𝜖)
𝑗
−𝐸𝑖(𝜖)
𝑑𝑒𝑠
⃒⃒⃒
max
{︁
𝐸𝑖(𝜖)
𝑗
, ∀𝑗
}︁
−min
{︁
𝐸𝑖(𝜖)
𝑗
, ∀𝑗
}︁,
∀𝑗, 𝑖. (3.18) (3.16) (3.17) (3.18) ︁︁︁︁
Equation (3.16) is used for the greater-the-better attributes, equation (3.17) is used for the smaller-the-better
attributes, and equation (3.18) is used for the closer-to-the-desired-value- 𝐸𝑖(𝜖)
𝑑𝑒𝑠-the-better. (︁
( ))︁ We proceed to calculating the grey relation distance between the reference sequence
(︁
𝐸𝑖(𝜖)
𝑗
)︁
and the com-
parability sequence
(︁¯
𝐸𝑖(𝜖)
𝑗
)︁
, which is ∆𝑖(𝜖)
𝑗
=
⃒⃒⃒𝐸𝑖(𝜖)
𝑗
−
¯
𝐸𝑖(𝜖)
𝑗
⃒⃒⃒, ∀𝑗, 𝑖. As stressed in Kuo et al. [25], the reference
sequence 𝐸𝑖(𝜖)
𝑗
=
(︁
𝐸1(𝜖)
𝑗
, 𝐸2(𝜖)
𝑗
)︁
= (1, 1). (︁
¯ )︁ ︁︁
Then, we compute the grey relational coefficient 𝛾
(︁
𝐸𝑖(𝜖)
𝑗
,
¯
𝐸𝑖(𝜖)
𝑗
)︁
. It is used to determine how close 𝐸𝑖(𝜖)
𝑗
is to
¯
𝐸𝑖(𝜖)
𝑗
. The larger the coefficient, the closer 𝐸𝑖(𝜖)
𝑗
and
¯
𝐸𝑖(𝜖)
𝑗
are. Let 𝛾
(︁
𝐸𝑖(𝜖)
𝑗
,
¯
𝐸𝑖(𝜖)
𝑗
)︁
= Δ(𝜖)
min +𝜁·Δ(𝜖)
max
Δ𝑖(𝜖)
𝑗
+𝜁·Δ(𝜖)
max , ∀𝑗, 𝑖where ︁︁
Then, we compute the grey relational coefficient 𝛾
(︁
𝐸𝑖(𝜖)
𝑗
, 𝐸𝑖(𝜖)
𝑗
)︁
. It is used to determine how close 𝐸𝑖(𝜖)
𝑗
is to
¯
𝐸𝑖(𝜖)
𝑗
. The larger the coefficient, the closer 𝐸𝑖(𝜖)
𝑗
and
¯
𝐸𝑖(𝜖)
𝑗
are. Let 𝛾
(︁
𝐸𝑖(𝜖)
𝑗
,
¯
𝐸𝑖(𝜖)
𝑗
)︁
= Δ(𝜖)
min +𝜁·Δ(𝜖)
max
Δ𝑖(𝜖)
𝑗
+𝜁·Δ(𝜖)
max , ∀𝑗, 𝑖where 1308 M.D. KREMANTZIS ET AL. Table 5. The numerical application of Zhou et al. [55]. Table 5. The numerical application of Zhou et al. [55]. Table 4. Interval ultimate cross-efficiencies. DMU
𝑋1
𝑋2
𝑋3
𝑍1
𝑍2
𝑌1
𝑌2
1
0.526
0.478
0.385
49.917
5.461
34.990
0.843
2
0.713
1.236
0.555
37.495
4.083
20.601
0.486
3
0.443
0.446
0.342
20.985
0.690
8.633
0.129
4
0.638
1.248
0.457
45.051
1.724
9.235
0.302
5
0.575
0.705
0.404
38.163
2.249
12.017
0.314
6
0.432
0.645
0.401
30.168
2.335
13.813
0.377
7
0.510
0.724
0.371
26.539
1.342
5.096
0.145
8
0.322
0.336
0.233
16.124
0.489
5.980
0.093
9
0.423
0.668
0.347
22.185
1.177
9.235
0.200
10
0.256
0.342
0.159
13.436
0.406
2.533
0.006 ∆(𝜖)
min = min
{︁
∆𝑖(𝜖)
𝑗
, ∀𝑗, 𝑖
}︁
, ∆(𝜖)
max = max
{︁
∆𝑖(𝜖)
𝑗
, ∀𝑗, 𝑖
}︁
, and 𝜁denotes the distinguishing coefficient, 𝜁∈[0, 1]. 𝜁is used to expand or squeeze the range of the grey relational coefficient. ( ) ∆(𝜖)
min = min
{︁
∆𝑖(𝜖)
𝑗
, ∀𝑗, 𝑖
}︁
, ∆(𝜖)
max = max
{︁
∆𝑖(𝜖)
𝑗
, ∀𝑗, 𝑖
}︁
, and 𝜁denotes the distinguishing coefficient, 𝜁∈[0, 1]. 𝜁is used to expand or squeeze the range of the grey relational coefficient. ( ) ︁︁︁︁
Finally, the GRG Γ(𝜖)
𝑗, which is the weighted average of the grey relational coefficients, is estimated as
Γ(𝜖)
𝑗
= ∑︀2
𝑖=1 𝑤𝑖· 𝛾𝑖(𝜖)
𝑗
, ∀𝑗, where 𝑤𝑖is the weight of the criterion 𝑖and can be more prone to subjective
modifications by a DM. Nevertheless, it is possible to delineate it with the use of an objective method [19]. Besides, ∑︀2
𝑖=1 𝑤𝑖= 1. We should emphasise that GRG only ranks the alternatives; thus, it is not an efficiency
measure. The DMU with the highest GRG is placed first. Finally, the GRG Γ(𝜖)
𝑗, which is the weighted average of the grey relational coefficients, is estimated as
Γ(𝜖)
𝑗
= ∑︀2
𝑖=1 𝑤𝑖· 𝛾𝑖(𝜖)
𝑗
, ∀𝑗, where 𝑤𝑖is the weight of the criterion 𝑖and can be more prone to subjective
modifications by a DM. Nevertheless, it is possible to delineate it with the use of an objective method [19]. Besides, ∑︀2
𝑖=1 𝑤𝑖= 1. We should emphasise that GRG only ranks the alternatives; thus, it is not an efficiency
measure. The DMU with the highest GRG is placed first. ︀
To conclude, GRA is considered as an efficient ranking tool not only for traditional MCDM problems [25],
but also for efficiency evaluation DEA problems as a MCDM context in disguise [41]. Table 4. Interval ultimate cross-efficiencies. Nevertheless, GRA has, to
our knowledge, not yet received explicit attention on ranking interval values and, in particular, interval ultimate
cross-efficiencies within an interval CE matrix. Hence, this section has aspired to attain this target, in the light
of a meaningful prioritisation of the DMUs. ︀
To conclude, GRA is considered as an efficient ranking tool not only for traditional MCDM problems [25],
but also for efficiency evaluation DEA problems as a MCDM context in disguise [41]. Nevertheless, GRA has, to
our knowledge, not yet received explicit attention on ranking interval values and, in particular, interval ultimate
cross-efficiencies within an interval CE matrix. Hence, this section has aspired to attain this target, in the light
of a meaningful prioritisation of the DMUs. Table 6. Highest and lowest relative efficiency scores for the overall system, stage 1, and stage 2. Table 6. Highest and lowest relative efficiency scores for the overall system, stage 1, and stage 2. 𝐸IDMU(1)
2.41405
𝐸ADMU(1)
0.05162
𝐸IDMU(2)
10.92813
𝐸ADMU(2)
0.00550
𝐸IDMU(s)
2.41405
𝐸ADMU(s)
0.00469 presented in Table 6. These scores are also accompanied by the combined self-efficiency ratings for each DMU
and system/stage. The numbers in parentheses illustrate the rankings of the corresponding bank branches for
each type of efficiency measure. presented in Table 6. These scores are also accompanied by the combined self-efficiency ratings for each DMU
and system/stage. The numbers in parentheses illustrate the rankings of the corresponding bank branches for
each type of efficiency measure. In Table 7, no matter what point of view efficiency is measured from, DMU 1 is certainly the best unit and
DMU 10 is the worst unit, in terms of the entire system (second expanded column). Considering stage 1, regard-
less of the viewpoint, DMU 1 and DMU 3 are the most and least desirable units, respectively (third expanded
column). In stage 2 (fourth expanded column), DMU 10 is deemed as the least promising unit. However, there
is no correspondence between the optimistic and pessimistic perspectives regarding the best unit. Notably, none
of the 10 bank branches perform efficiently in both stages and viewpoints. This is, for instance, seen in the
non-efficient overall optimistic self-efficiency scores
(︁
𝐸IDMU(𝑠)
𝑘
)︁
, where the highest score is 0.8132 occurring at
DMU 1, followed by 0.3490 occurring at DMU 6. ︁︁
The next focal point of the framework is the geometric aggregation of the optimistic and pessimistic perspec-
tives, to build a combined self-efficiency measure for each DMU, with respect to the system
(︁
𝐸comb(𝑠)
𝑘
)︁
, the
stage 1
(︁
𝐸comb(1)
𝑘
)︁
, and the stage 2
(︁
𝐸comb(2)
𝑘
)︁
. In Table 7, DMU 1 has the best performance among all units,
reflecting the two opposed standpoints. This is completely verified by the consistent results of the overall system
and the stage 1. Nevertheless, regarding stage 2, there is a significant inconsistency between the optimistic and
the pessimistic efficiency. In detail, DMU 1 receives a moderate rating (0.8132) with respect to the optimistic
aspect. This rating is compensated by its exceptional pessimistic performance (0.0760). The overall performance
of bank branch 1 is also grievously higher than the corresponding performance of all others. A RANKING FRAMEWORK BASED ON INTERVAL SELF AND CROSS-EFFICIENCIES A RANKING FRAMEWORK BASED ON INTERVAL SELF AND CROSS-EFFICIENCIES 1309 4. Numerical application This section illustrates the use of the mathematical models presented in Section 3 to meaningfully evaluate
and rank the DMUs. There are two salient factors that evaluate each DMU within the two-stage tandem
structure herein: (i) the optimistic and pessimistic efficiency scores within a self-evaluation context, and (ii)
the most favourable and unfavourable weight sets of each of the other DMUs, in a peer-appraisal setting that
integrates the combined self-efficiency measure. The numerical example drawn from Zhou et al. [55] is used for illustrative purposes. In Table 5, ten bank
branches of China Construction Bank in Anhui are assessed within the two-stage tandem structure (see Fig. 2). The employee (𝑋1), the fixed assets (𝑋2), and the expenses (𝑋3) are the input resources of the first stage to be
consumed to produce the intermediate products; the credit (𝑍1) and the inter-bank loans (𝑍2). The latter are
used as inputs in the second stage to generate the final outputs; the loan (𝑌1) and the profit (𝑌2). For modelling,
running, and analysing our data, we have utilised the programming language Python 3.7.6 and in particular
the version 2.1 of PuLP as the free linear programming library. The experiment ran on a computer with 16GB
RAM. In our framework, we first consider determining the best and worst relative efficiencies of the IDMU and the
ADMU, respectively, for the overall system and its individual stages. Table 6 exhibits the corresponding scores
from solving models (3.3)–(3.6), introduced in Section 3.1. Then, models (3.7)–(3.10) are used to obtain the highest and the lowest relative efficiency scores of the target
DMU𝑘in terms of the overall system, the stage 1, and the stage 2. These scores are given in Table 7. Recall that
these relative self-efficiency scores indicate their distance from the respective IDMU and ADMU efficiencies, Table 6. Highest and lowest relative efficiency scores for the overall system, stage 1, and stage 2. For instance, in
stage 1 the combined self-efficiency score of DMU 1 approximates 0.51, whereas the corresponding rating of
DMU 2 (in the second place) equals to 0.2733. The geometric average efficiency also indicates that DMU 10 has
the worst performance in terms of the overall system and the stage 2. p
y
g
The combined self-efficiencies calculated for every DMU, satisfy the sound mathematical property that the
overall system combined self-efficiency score is the product of the two sub-stages, as discussed in Section 3.1. As an example, the combined self-efficiencies calculated for DMU 1 satisfy 0.1267 = 0.5099 * 0.2486. Since this
property is satisfied, every 𝐸comb(𝑠)
𝑘
is no greater than its corresponding 𝐸comb(1)
𝑘
and 𝐸comb(2)
𝑘
. Another point
to be noted is that most bank branches have a smaller 𝐸comb(2)
𝑘
than 𝐸comb(1)
𝑘
. Only DMUs 3, 8, and 9 have a
smaller 𝐸comb(1)
𝑘
than 𝐸comb(2)
𝑘
. However, after implementing the Wilcoxon’s matched-pairs signed-ranks test [9]
we found that there is not sufficient evidence to say that the average efficiency measures of these two sub-stages
are not equal. This may be due to the limited sample under examination. In addition, it is noteworthy that the
difference between ratings and ranks of the combined self-efficiency measures in all stages is quite significant
for several bank branches. For instance, the rank of DMU 3 for the overall system, the stage 1, and the stage
2, is 8, 10, and 2, respectively, indicating that at least 6 ranks difference exists. A large difference may reveal
the source that causes the inefficiency of the overall system. For example, DMUs 2 and 5 perform satisfactorily
in stage 1 (as compared to stage 2) whereas DMUs 3 and 8 perform satisfactorily in stage 2 (as compared to
stage 1). Decomposing the overall system combined self-efficiency score into the product of its two sub-stages,
may assist the respective bank branch in identifying the sub-stage that triggers inefficiency. 1310 M.D. KREMANTZIS ET AL. The combined self-efficiency measures obtained with our proposed method (see the respective columns of
Tab. 7) are also compared with the respective scores (Tab. 8) obtained with Kao and Hwang’s [21] approach. As mentioned in Section 3, the latter approach aims to explore the efficiency decomposition in a two-stage
production process by taking into consideration the series relationship of the two sub-stages. Table 6. Highest and lowest relative efficiency scores for the overall system, stage 1, and stage 2. As a matter of fact, there is no absolute discrimination of some inefficient
DMUs considering the combined self-evaluation results at each stage, presented in Table 7. In particular, in the
overall system the DMUs 2 and 6 tied (0.0528) in the second place. Similarly, at stage 2 the DMUs 3 and 6 also
tied (0.2094), sharing the second place. In such results, each DMU is self-assessed ignoring the weight profile of
each of the other DMUs. Embedding the geometric average score into a peer context, would possibly contribute
to a more comprehensive ranking. To this end, the proposed framework is further extended by the use of the
interval CE. The next step in our proposed approach concerns the implementation of the interval CE towards the evaluated
network structure, as discussed in Section 3.2. Tables 9–11 showcase the interval individual cross-efficiencies of
DMU𝑗based on the optimal weight scheme of DMU𝑘for the overall system, the stage 1, and the stage 2,
respectively. In this case, each DMU is evaluated considering simultaneously an aggressive (model (3.11)) and
a benevolent (model (3.12)) strategy; this originally creates an atmosphere of neutrality. (
(
))
gy;
g
y
p
y
To make the content of Tables 9–11 comprehensible to the reader, it should be ideal to present a few examples. In the second column of Table 9, DMU 1 is assessed based on the weight profile of all other DMUs, except its
own weight set. The minimum and maximum individual cross-efficiencies of DMU 1 based on the optimal weight
scheme of DMU 2 are 0.1216 and 0.2371, respectively, for the overall system. In the fifth column of Table 10,
DMU 4 is also peer-appraised with respect to the weight profile of all other DMUs. The minimum and maximum
individual cross-efficiencies of DMU 4 based on the weight profile of DMU 10 are 0.1475 and 0.2281, respectively,
for sub-stage 1. Table 11 determines in a similar manner the individual cross-efficiencies for each DMU, for the
sub-stage 2. The diagonal leading column in each of these three matrices demonstrates the special case in which
𝐸L(𝜖)*
𝑗𝑗
= 𝐸U(𝜖)*
𝑗𝑗
= 𝐸comb(𝜖)*
𝑗
∀𝑗, where 𝜖= 𝑠(overall system), 1 (stage 1) or 2 (stage 2). These are the combined
self-efficiency scores. Table 6. Highest and lowest relative efficiency scores for the overall system, stage 1, and stage 2. Their relational
model (see model (3.1)) was found to be reliable in terms of measuring overall and division efficiencies along
with the better identification of the causes of inefficiency. Our study has applied their relational model to further
analyse and validate the dataset provided in Table 5, by measuring the efficiencies of the whole process and
its constituent sub-stages for the ten DMUs. In Table 8, the self-efficiency scores along with their ranks of the
overall system, the stage 1, and the stage 2, are depicted in the second, third, and fourth column, respectively. The rankings of the two models with respect to the overall system are quite similar, showing that the largest
difference is 1 occurring at the bank branches 2, 3, and 8. The rankings of the two models with respect to
sub-stage 1 are also quite close to each other. In the latter case, the largest difference occurs at DMU 7 with a
rank difference of 4. The second largest difference occurs at DMUs 9 and 10 with a rank difference of 2. For the
remaining 7 bank branches, their rank differences are less than 2. The rank differences look very similar even
with the case of sub-stage 2. Correlation analysis suggests that there is a highly strong association between the
ranks of these two approaches, as indicated by the Spearman coefficients [9] of 0.985 (overall system), 0.806
(stage 1), and 0.841 (stage 2), which are significant at the 0.01 level (two-tailed). This can be demonstrated
even by the fact that both our method and Kao and Hwang’s method identify DMU 1 as the best performer. However, our approach is more informative within the self-appraisal context, in that it not only considers the
most favourable self-efficiency scores (as in [21]), but also the most unfavourable ones to obtain a more accurate
and less misleading overall assessment for each DMU and flow. As a result, it puts emphasis on both sides of
the same coin simultaneously. The above points further validate the rationale of our approach. As discussed in Section 1, the geometric average efficiency is an easy-to-use measure with a good discriminating
power amongst the evaluated DMUs. However, it may not be sufficiently strong in terms of leading to a unique
ranking in this two-stage process. Table 6. Highest and lowest relative efficiency scores for the overall system, stage 1, and stage 2. DMU
𝐸𝑠
𝑘(Rank)
𝐸1
𝑘(Rank)
𝐸2
𝑘(Rank)
1
1.00 (1)
1.00 (1)
1.00 (1)
2
0.43 (3)
0.55 (2)
0.79 (8)
3
0.29 (7)
0.29 (9)
1.00 (1)
4
0.30 (6)
0.32 (7)
0.94 (4)
5
0.35 (4)
0.43 (4)
0.83 (7)
6
0.54 (2)
0.54 (3)
1.00 (1)
7
0.18 (9)
0.28 (10)
0.62 (9)
8
0.28 (8)
0.31 (8)
0.92 (5)
9
0.33 (5)
0.38 (5)
0.85 (6)
10
0.17 (10)
0.37 (6)
0.47 (10) Table 9. Interval cross-efficiencies for the overall system. Table 9. Interval cross-efficiencies for the overall system. DMU
1
2
3
4
5
6
7
8
9
10
1
0.1267 0.1267
0.0282 0.0550
0.0207 0.0371
0.0127 0.0381
0.0294 0.0449
0.0370 0.0690
0.0121 0.0226
0.0198 0.0357
0.0215 0.0415
0.0011 0.0221
2
0.1216 0.2371
0.0528 0.0528
0.0225 0.0613
0.0234 0.0397
0.0382 0.0597
0.0489 0.0785
0.0182 0.0269
0.0223 0.0563
0.0347 0.0438
0.0017 0.0234
3
0.0970 0.1738
0.0244 0.0667
0.0284 0.0284
0.0108 0.0498
0.0250 0.0587
0.0314 0.0854
0.0103 0.0295
0.0261 0.0300
0.0203 0.0463
0.0016 0.0178
4
0.1018 0.3039
0.0407 0.0691
0.0175 0.0802
0.0307 0.0307
0.0354 0.0707
0.0437 0.0888
0.0182 0.0292
0.0184 0.0737
0.0268 0.0573
0.0014 0.0307
5
0.1192 0.1819
0.0374 0.0585
0.0205 0.0480
0.0183 0.0366
0.0423 0.0423
0.0489 0.0677
0.0174 0.0220
0.0216 0.0441
0.0285 0.0442
0.0015 0.0225
6
0.0966 0.1801
0.0354 0.0568
0.0175 0.0475
0.0181 0.0369
0.0329 0.0455
0.0528 0.0528
0.0164 0.0219
0.0173 0.0437
0.0269 0.0407
0.0013 0.0243
7
0.1161 0.2161
0.0407 0.0600
0.0199 0.0571
0.0218 0.0349
0.0397 0.0502
0.0498 0.0664
0.0208 0.0208
0.0210 0.0524
0.0305 0.0453
0.0016 0.0240
8
0.1012 0.1825
0.0267 0.0675
0.0270 0.0310
0.0118 0.0473
0.0273 0.0557
0.0343 0.0864
0.0112 0.0282
0.0285 0.0285
0.0221 0.0468
0.0017 0.0176
9
0.1105 0.2136
0.0437 0.0551
0.0222 0.0507
0.0194 0.0415
0.0347 0.0538
0.0469 0.0708
0.0166 0.0246
0.0220 0.0466
0.0363 0.0363
0.0017 0.0216
10
0.0357 0.6610
0.014 0.1913
0.0099 0.1081
0.0062 0.1326
0.0117 0.1666
0.0135 0.2448
0.0053 0.0798
0.0100 0.1033
0.0104 0.1327
0.0062 0.0062
ultim. CE
0.1000 0.2611
0.0323 0.0755
0.0197 0.0579
0.0158 0.0508
0.0305 0.0673
0.0394 0.0953
0.0140 0.0316
0.0198 0.0540
0.0246 0.0554
0.0015 0.0227 Recalling the discussion in Section 3.3, we view each interval CE matrix as a MCDM problem. In Tables 9–11,
the ten DMUs (alternatives) located in their last 11 columns, are evaluated by the weight profiles of the ten
DMUs (criteria) presented in their first column. Table 6. Highest and lowest relative efficiency scores for the overall system, stage 1, and stage 2. Clearly, the property of maintaining the combined self-efficiency measure for each DMU
is satisfied both for the overall system and its individual stages; this accomplishes our efforts towards a more
reasoned peer-appraisal setting that entails the effects of the optimistic and pessimistic viewpoints. A RANKING FRAMEWORK BASED ON INTERVAL SELF AND CROSS-EFFICIENCIES 1311 Table 7. Self-efficiency ratings and ranks of the overall system, the stage 1, and the stage 2,
with the proposed method. DMU 𝐸IDMU(s)
𝑘
𝐸ADMU(s)
𝑘
𝐸comb(s)
𝑘
𝐸IDMU(1)
𝑘
𝐸ADMU(1)
𝑘
𝐸comb(1)
𝑘
𝐸IDMU(2)
𝑘
𝐸ADMU(2)
𝑘
𝐸comb(2)
𝑘
1
0.8132 (1)
0.0197 (1)
0.1267 (1)
1.0000 (1)
0.2600 (1)
0.5099 (1)
0.8132 (6)
0.0760 (1)
0.2486 (1)
2
0.3255 (3)
0.0086 (2)
0.0528 (2) 0.5186 (2)
0.1441 (5)
0.2733 (2)
0.6277 (8)
0.0595 (2)
0.1933 (6)
3
0.1398 (9)
0.0058 (6)
0.0284 (8)
0.1421 (10) 0.1298 (10) 0.1358 (10) 0.9838 (2)
0.0446 (5)
0.2094 (2)
4
0.2450 (5)
0.0038 (8)
0.0307 (6)
0.2655 (5)
0.1731 (3)
0.2144 (5)
0.9227 (3)
0.0222 (8)
0.1432 (8)
5
0.2886 (4)
0.0062 (4)
0.0423 (4)
0.3927 (4)
0.1818 (2)
0.2672 (3)
0.7350 (7)
0.0341 (7)
0.1584 (7)
6
0.3490 (2)
0.0080 (3)
0.0528 (2) 0.4101 (3)
0.1549 (4)
0.2521 (4)
0.8509 (5)
0.0515 (3)
0.2094 (2)
7
0.1454 (8)
0.0030 (9)
0.0208 (9)
0.2549 (6)
0.1425 (7)
0.1906 (6)
0.5706 (9)
0.0208 (9)
0.1090 (9)
8
0.1476 (7)
0.0055 (7)
0.0285 (7)
0.1476 (9)
0.1372 (8)
0.1423 (9)
1.0000 (1)
0.0402 (6)
0.2005 (5)
9
0.2141 (6)
0.0061 (5)
0.0363 (5)
0.2389 (7)
0.1362 (9)
0.1804 (7)
0.8963 (4)
0.0451 (4)
0.2011 (4)
10
0.0133 (10) 0.0029 (10) 0.0062 (10) 0.1796 (8)
0.1438 (6)
0.1607 (8)
0.0739 (10) 0.0204 (10) 0.0389 (10)
Table 8. Self-efficiency ratings and ranks of the overall system, the stage 1, and the stage 2,
with Kao and Hwang’s [21] method. Table 8. Self-efficiency ratings and ranks of the overall system, the stage 1, and the stage 2,
with Kao and Hwang’s [21] method. Table 10. Interval cross-efficiencies for the stage 1. Table 11. Interval cross-efficiencies for the stage 2. and stage 2, respectively. The interval weights are obtained via the GP model (3.13), and the interval global
weights, according to the pair of optimisation models (3.14) and (3.15), as stated in Section 3.3. For instance, in the second column of the last row of Table 9, we obtain the interval ultimate CE of DMU 1:
[0.1000, 0.2611], where 0.1000 is the minimum and 0.2611 is the maximum CE score. The minimum score of
DMU 1 for the overall system is estimated via solving model (3.14). The basic prerequisites of this model are to
recognise the minimum individual cross-efficiencies of DMU 1 based on the weight profile of all ten DMUs (left
side of column 2 of Tab. 9) and the interval weights per criterion for the overall system (column 2 of Tab. 12). The maximum ultimate cross-efficiency of DMU 1 for the overall system is estimated via solving model (3.17). The basic prerequisites of this model are to identify the maximum individual cross-efficiencies of DMU 1 based
on the weight profile of all 10 DMUs (right side of column 2 of Tab. 9) and the interval weights per criterion
for the overall system (column 2 of Tab. 12). The final step of our methodological approach seeks for a unique and reasonable prioritisation of the interval
ultimate cross-efficiencies via the established GRA, as discussed in Section 3.4. This step continues to allow
the DMUs, located in the columns of the interval CE matrices mentioned above, to act as alternatives and
to be assessed by two attributes; the first attribute concerns the minimum (worst condition) and the second
attribute is pertinent to the maximum (best condition) ultimate CE of each DMU, towards the corresponding
system/stage. The interval ultimate cross-efficiencies (last row of each of the Tabs. 9–11) form the appropriate
matrix, as shown in Table 4. The data of performance values of the two attributes are subsequently normalised through the greater-the-
better equation (3.16); this choice reflects the necessity of pushing up the peer-efficiency of each DMU. The
results are depicted in the second column of Appendix A for the overall system, of Appendix B for stage 1,
and of Appendix C for stage 2. Table 6. Highest and lowest relative efficiency scores for the overall system, stage 1, and stage 2. To designate the interval global weights (interval ultimate cross-
efficiencies) in the last row of each of these matrices, it is required to determine the interval weight per criterion
except the known interval individual cross-efficiencies. To start with, the interval weight of each criterion is
determined in the second, third, and fourth column of Table 11, with respect to the overall system, stage 1, 1312 M.D. KREMANTZIS ET AL. Table 10. Interval cross-efficiencies for the stage 1. DMU
1
2
3
4
5
6
7
8
9
10
1
0.5099 0.5099
0.1472 0.2811
0.0689 0.0764
0.0615 0.1353
0.1422 0.2002
0.1615 0.2654
0.0826 0.1299
0.0648 0.0752
0.0785 0.1366
0.0530 0.0915
2
0.4956 0.9466
0.2733 0.2733
0.0743 0.1279
0.1143 0.1399
0.1867 0.2640
0.2161 0.2998
0.1255 0.1533
0.0724 0.1203
0.1258 0.1458
0.0757 0.0984
3
0.9056 0.9999
0.2887 0.4994
0.1358 0.1358
0.1211 0.2537
0.2793 0.3751
0.3170 0.4715
0.1623 0.2435
0.1277 0.1410
0.1543 0.2426
0.1044 0.1716
4
0.8073 1.0000
0.4186 0.2893
0.1147 0.2665
0.2144 0.2144
0.3103 0.3785
0.3311 0.3713
0.2057 0.2407
0.1191 0.2653
0.1928 0.2223
0.1259 0.2174
5
0.6804 0.9578
0.2765 0.3911
0.0967 0.1295
0.1156 0.1845
0.2672 0.2672
0.2790 0.3692
0.1552 0.1796
0.1004 0.1217
0.1475 0.1900
0.0995 0.1222
6
0.4923 0.8093
0.2336 0.3241
0.0738 0.1094
0.0977 0.1659
0.1855 0.2454
0.2521 0.2521
0.1247 0.1593
0.0720 0.1028
0.1246 0.1493
0.0752 0.1122
7
0.7478 1.0000
0.2889 0.4148
0.1062 0.1828
0.1547 0.1985
0.2834 0.3151
0.3067 0.3917
0.1906 0.1906
0.1100 0.1773
0.1786 0.2015
0.1150 0.1343
8
0.9640 0.9999
0.2887 0.5365
0.1370 0.1496
0.1310 0.2559
0.2898 0.3785
0.3227 0.5066
0.1706 0.2465
0.1423 0.1423
0.1615 0.2606
0.1164 0.1731
9
0.6732 1.0000
0.2889 0.3915
0.1009 0.1858
0.1568 0.2005
0.2536 0.3173
0.3097 0.3378
0.1705 0.1924
0.0984 0.1803
0.1804 0.1804
0.1029 0.1356
10
0.2473 0.6610
0.1239 0.2484
0.1170 0.1958
0.1475 0.2281
0.1802 0.2651
0.1433 0.2834
0.1364 0.1816
0.1317 0.1961
0.1218 0.1858
0.1607 0.1607
ultim. CE
0.6083 0.7509
0.2172 0.3407
0.0892 0.1257
0.1018 0.1754
0.2013 0.2661
0.2260 0.3277
0.1245 0.1705
0.0880 0.1230
0.1195 0.1731
0.0808 0.1240
Table 11. Interval cross-efficiencies for the stage 2. Table 6. Highest and lowest relative efficiency scores for the overall system, stage 1, and stage 2. DMU
1
2
3
4
5
6
7
8
9
10
1
0.2486 0.2486
0.1918 0.1957
0.3007 0.4856
0.2078 0.2820
0.2072 0.2246
0.2294 0.2600
0.1473 0.1743
0.3056 0.4745
0.2739 0.3044
0.0225 0.2417
2
0.2454 0.2504
0.1933 0.1933
0.3030 0.4795
0.2052 0.2842
0.2046 0.2263
0.2265 0.2620
0.1455 0.1757
0.3080 0.4686
0.2760 0.3006
0.0227 0.2387
3
0.1072 0.1738
0.0848 0.1336
0.2094 0.2094
0.0897 0.1964
0.0896 0.1564
0.0993 0.1811
0.0636 0.1214
0.2045 0.2128
0.1316 0.1908
0.0157 0.1042
4
0.1261 0.3039
0.0973 0.2389
0.1526 0.3012
0.1432 0.1432
0.1140 0.1867
0.1320 0.2393
0.0885 0.1213
0.1551 0.2779
0.1390 0.2580
0.0114 0.1413
5
0.1752 0.1899
0.1352 0.1495
0.2119 0.3710
0.1588 0.1988
0.1584 0.1584
0.1753 0.1833
0.1126 0.1229
0.2154 0.3626
0.1931 0.2326
0.0159 0.1847
6
0.1963 0.2225
0.1515 0.1753
0.2375 0.4348
0.1861 0.2228
0.1775 0.1856
0.2094 0.2094
0.1319 0.1377
0.2414 0.4249
0.2164 0.2726
0.0178 0.2165
7
0.1553 0.2161
0.1409 0.1447
0.1878 0.3122
0.1410 0.1762
0.1403 0.1595
0.1625 0.1696
0.1090 0.1090
0.1909 0.2959
0.1711 0.2251
0.0141 0.1787
8
0.1050 0.1825
0.0925 0.1258
0.1972 0.2075
0.0905 0.185
0.0943 0.1473
0.1065 0.1706
0.0661 0.1143
0.2005 0.2005
0.1373 0.1797
0.0148 0.1021
9
0.1641 0.2136
0.1512 0.1408
0.2207 0.2732
0.1237 0.2070
0.1368 0.1697
0.1515 0.2095
0.0973 0.1280
0.2243 0.2585
0.2011 0.2011
0.0165 0.1596
10
0.1444 1.0000
0.1132 0.7699
0.0847 0.5521
0.0422 0.5815
0.0649 0.6285
0.0943 0.8637
0.0395 0.4397
0.0764 0.5268
0.0858 0.7140
0.0389 0.0389
ultim. CE
0.1606 0.2970
0.1310 0.2247
0.2055 0.3782
0.1354 0.2522
0.1349 0.2235
0.1540 0.2722
0.0977 0.1644
0.2071 0.3656
0.1770 0.2904
0.0168 0.1773 Table 10. Interval cross-efficiencies for the stage 1. Table 10. Interval cross-efficiencies for the stage 1. The unique final rank in Table 13 reflects the improvement of the discriminating power, as compared
to the original rank derived from the combined self-efficiency measures in Table 7. This practically means that
the non-dominated bank branches, which cannot be fully discriminated by the self-evaluation notion, can be
discriminated by the methodologies followed in peer notion. In detail, DMU 10 is without a doubt the least
desirable unit in all three cases. DMU 1 is also considered to be the most promising bank branch for the overall
system and stage 1, while DMU 3 is the best unit according to stage 2. Generally, one can deduce that the ranking
results for all branches (except DMU 10) are not consistent and may show a higher degree of uncertainty and
inefficiency in specific stages. The GRG grades obtained with our proposed framework (see Tab. 13) are also compared with the respective
ultimate cross-efficiency ratings (Tab. 14) obtained via the Kao and Liu’s [21] approach. In their study, they
applied the concept of cross-evaluation to measure the efficiency of basic (parallel & series) network structures. Their proposed aggressive-based secondary goal model was particularly able to decompose the cross-efficiency
score of the overall system into the product of those of the internal sub-stages for the series structure. Our study
has applied their aggressive-based model under the two-stage tandem series structure and the peer-appraisal
setting to further analyse the dataset provided in Table 5. In Table 14, the peer-efficiency scores along with
their ranks of the overall system, the stage 1, and the stage 2, are respectively presented in the second, third,
and fourth column. Firstly, we have noticed that the multiplicative mathematical relationship between the
overall system and its sub-stage efficiencies is indeed satisfied. For example, the ultimate cross-efficiency score
of DMU 6 (0.446) is equal to the product of its sub-stage 1 (0.574) and sub-stage 2 (0.778) efficiencies. Secondly,
the rankings of the two methods with respect to the overall system and the stage 1 are not significantly different
based on a Spearman rank order correlation test with statistics of 0.964 and 0.830, respectively. These are
significant at the 0.01 level (two-tailed). However, it is worthwhile to mention that DMU 10 has a difference of
3 ranks in terms of the evaluation of stage 1. Thirdly, as for the stage 2, the rankings from the two methods are
not so close. Table 10. Interval cross-efficiencies for the stage 1. The grey relational distance calculation is also utilised to measure the distance
between the reference sequence and the comparability sequence (normalised values), see the third column of
each of the appendices. In addition, we compute the grey relational coefficient to explore how close the reference
and the comparability sequences are. In this formula, the value of 𝜁may affect the size of the correlation degree
distribution interval, thereby affecting the results of the correlation analysis. The value of 𝜁can be determined
considering the DMU’s tendency towards optimism-pessimism. Following [21], we have set 𝜁= 0.5 implying 1313 A RANKING FRAMEWORK BASED ON INTERVAL SELF AND CROSS-EFFICIENCIES Table 12. Interval weights per criterion for the overall system, the stage 1, and the stage 2. Criteria
(DMUs)
Interval weights
per criterion
(overall system)
Interval weights per
criterion (stage 1)
Interval weights per
criterion (stage 2)
1
0.0000
0.1111
0.4502
0.4726
0.0134
0.1198
2
0.0000
0.1111
0.0286
0.0510
0.0133
0.1197
3
0.0000
0.1111
0.0506
0.0730
0.0016
0.1080
4
0.0000
0.1111
0.0479
0.0703
0.0023
0.1088
5
0.0000
0.1111
0.0390
0.0613
0.0068
0.1132
6
0.0000
0.1111
0.0283
0.0507
0.0091
0.1156
7
0.0000
0.1111
0.0433
0.0657
0.0052
0.1117
8
0.0000
0.1111
0.0538
0.0761
0.0017
0.1081
9
0.0000
0.1111
0.0394
0.0618
0.0060
0.1125
10
0.0000
0.1111
0.0170
0.0394
0.0000
0.0887 12. Interval weights per criterion for the overall system, the stage 1, and the stage 2. that the DMU has neither an optimistic nor a conservative attitude. The respective results are portrayed in the
last column of each of the appendices. that the DMU has neither an optimistic nor a conservative attitude. The respective results are portrayed in the
last column of each of the appendices. that the DMU has neither an optimistic nor a conservative attitude. The respective results are portrayed in the
last column of each of the appendices. The GRG and the rank for each DMU with respect to the overall system, the stage 1, and the stage 2,
are illustrated in the second, third, and fourth column of Table 13, respectively. It is important to make two
remarks about the process of obtaining the GRG: firstly, the relative importance weights of the two performance
attributes were assumed to be equal (𝑤1 = 𝑤2 = 0.5) illustrating that the two extremes are of the same
importance, and secondly, the GRG is just an index that only captures the rank rather than an efficiency
measure. Table 10. Interval cross-efficiencies for the stage 1. The bank branch 2 is the extreme case with a rank difference of 6. The second largest difference
occurs at DMU 8 with a rank difference of 4. All the remaining bank branches have a rank difference of no 1314 M.D. KREMANTZIS ET AL. Table 13. Grey Relational Grade and ranks of the overall system, the stage 1, and the stage 2. DMU
GRG
Γ(𝑠)
𝑗
Rank over-
all system
GRG
Γ(1)
𝑗
Rank
stage 1
GRG
Γ(2)
𝑗
Rank
stage 2
1
1.0000
1
1.0000
1
0.6199
4
2
0.4062
3
0.4181
2
0.4830
8
3
0.3750
6
0.3356
8
0.9916
1
4
0.3655
8
0.3477
7
0.5145
6
5
0.3978
4
0.3931
4
0.4886
7
6
0.4333
2
0.4171
3
0.5719
5
7
0.3530
9
0.3519
5
0.3992
9
8
0.3729
7
0.3349
9
0.9471
2
9
0.3811
5
0.3512
6
0.6543
3
10
0.3333
10
0.3335
10
0.3403
10
Table 14. Peer-efficiency ratings and ranks of the overall system, the stage 1, and the stage
2, with Kao and Liu’s [22] method. Table 13. Grey Relational Grade and ranks of the overall system, the stage 1, and the stage 2. 3. Grey Relational Grade and ranks of the overall system, the stage 1, and the stage 2. Table 14. Peer-efficiency ratings and ranks of the overall system, the stage 1, and the stage
2, with Kao and Liu’s [22] method. Table 14. Peer-efficiency ratings and ranks of the overall system, the stage 1, and the stage
2, with Kao and Liu’s [22] method. DMU
System CE (Rank)
Stage 1 CE (Rank)
Stage 2 CE (Rank)
1
1.000 (1)
1.000 (1)
1.000 (1)
2
0.416 (3)
0.534 (4)
0.780 (2)
3
0.239 (7)
0.315 (10)
0.760 (4)
4
0.251 (6)
0.488 (5)
0.513 (8)
5
0.330 (4)
0.573 (3)
0.576 (7)
6
0.446 (2)
0.574 (2)
0.778 (3)
7
0.160 (9)
0.420 (6)
0.381 (9)
8
0.238 (8)
0.336 (9)
0.710 (6)
9
0.297 (5)
0.392 (8)
0.756 (5)
10
0.072 (10)
0.397 (7)
0.182 (10) DMU
System CE (Rank)
Stage 1 CE (Rank)
Stage 2 CE (Rank) more than 3. Statistically, this situation is even further validated by the Spearman coefficient of 0.503, which
implies a moderate association between the rankings of the two methods. 5. Conclusions & future research This paper has provided new insight into the attainment of a meaningful and unique ranking of DMUs under a
two-stage tandem (network) structure. In particular, it extends the selected optimistic-pessimistic DEA models
into the two-stage tandem system, to then complement the interval CE method within such a system. Decision
makers are offered with the chance of evaluating the performance of the DMUs by considering: (i) the optimistic
and pessimistic self-efficiency scores, and (ii) the most favourable and unfavourable weight profiles of each of the
other DMUs in a peer-appraisal setting. In this study, we have introduced a 7-step methodological approach, as
shown in Figure 1, which combines existing methods from the literature in a novel way. This approach supports
the aforementioned conditions and ensures more multi-dimensional evaluation outcomes. The procedures implemented in the first three steps of our framework indicate how the optimistic and
pessimistic DEA models, which are inspired by the studies of Wang and Luo [48] and Wu [51], are built
towards the more realistic two-stage tandem system that better reflects the complex interconnections among its
internal sub-systems. The DMUs are initially evaluated, based on their own most favourable (optimistic) and
unfavourable (pessimistic) optimal multipliers, and then are aggregated into a combined self-efficiency measure
via the geometric average. The remaining steps of our framework ensure the peer-evaluation of the DMUs via the customisation of the
interval CE method to the specifications of the two-stage tandem structure while keeping the combined self-
efficiency measure unchanged. To rank all DMUs in the interval CE matrix of the corresponding flow, the study
introduces an alternative novel use of the GP method of Wang and Elhag [47], the LP models by Entani and
Tanaka [13], and the GRA of Kuo et al. [25]. The combination of such well-established techniques for extracting
valuable insights from an interval CE matrix has not been considered before. This combination underpins the
wider MCDM context to which the elements of the interval CE matrix belong. We envisage that our study could be applicable in several areas. In the non-life insurance industry [21],
for example, operations consist of the insurance service and the capital investment. Customers pay direct
written and reinsurance premiums, which are then invested in a portfolio to earn underwriting profit. A RANKING FRAMEWORK BASED ON INTERVAL SELF AND CROSS-EFFICIENCIES 1315 A RANKING FRAMEWORK BASED ON INTERVAL SELF AND CROSS-EFFICIENCIES Table 10. Interval cross-efficiencies for the stage 1. Finally, Kao and Liu’s [22] approach
only considers the most unfavourable weight sets of each of the other DMUs, while keeping the optimistic
self-efficiency score constant. However, our study is more multi-dimensional since it simultaneously takes into
account the most favourable and unfavourable weight sets of each of the other players, while integrating the
respective combined self-efficiency measure. Finally, it can be statistically inferred that the rankings of the DMUs obtained from the combined self-
efficiency measures (self-appraisal), and the grey relational grades after showing peer-appraisal, are similar
with respect to the overall system and its sub-stages. As an example, for the overall system, according to the
Spearman correlation test [9], the 𝑟𝑠= 0.948. This indicates that under the significance level of 0.01, there is
a strong positive association between the ranking values of the DMUs obtained by the two separate conditions
(self-appraisal & peer-appraisal), confirming the validity of our framework. Exceptions are considered the DMUs
1, 6, and 8 within the evaluation of the second sub-stage, where there is a larger rank difference of 3. This could
be justified by the nature of the self-appraisal setting to let each bank branch to be evaluated based only on its
own (favourable and unfavourable) standpoint, while the peer-appraisal setting expects the bank branches to
be evaluated from the (favourable and unfavourable) standpoint of all branches. 5. Conclusions & future research Finally, further study could focus on
the testing of the proposed models and frameworks with empirical data. In the shipping industry, for example,
it could be deployed to compare the efficiency of potential designs of a particular type of vessel, including the
selection of the right mixture of maintenance policies. interval CE matrix and, thus, our paper has worked towards this direction. Finally, further study could focus on
the testing of the proposed models and frameworks with empirical data. In the shipping industry, for example,
it could be deployed to compare the efficiency of potential designs of a particular type of vessel, including the
selection of the right mixture of maintenance policies. g
p
The models in this study were developed under the assumption of the constant returns-to-scale. A direction
for future research could be their advancement to variable returns-to-scale DEA models. In addition, current
research studies the evaluation of the performance of several DMUs with a two-stage tandem structure in a
self and in a peer-appraisal setting, only when the data (i.e., the input and output factors) are accurate and
unambiguous, and the DEA models are based on this condition. Future research could relax this assumption
by allowing the data points to be imprecise (e.g., to be expressed as linguistic terms) and lie in an interval. Other cases to be investigated concern missing data or intervals, where some values are more likely to occur
over other values. In the latter case, since there is no information of the probability distributions, fuzzy numbers
and mathematical operations [58] could be used as an ideal alternative option. For example, there is a growing
body of literature [11,17,36,40] surrounding the development of novel fuzzy DEA approaches and models char-
acterised by intuitionistic fuzzy data, applied possibility, necessity, credibility, general fuzzy measures, and/or
trapezoidal fuzzy numbers. Some of these models were solved with the aid of either a linear programming with
an intuitionistic fuzzy objective function and an alphabetical technique, a chance-constrained programming, a
lexicographic multi-objective linear programming, or a fuzzy linear programming. The network double-frontier
DEA models introduced in this study could be adjusted to the specifications of such an uncertain (fuzzy)
environment adopting the most suitable formulation and solution techniques. Finally, it would be worthwhile to adjust the modelling approaches, introduced in our study, ensuring that
they will be taking into consideration the decision maker’s preferences. 5. Conclusions & future research Another
promising area would be the evaluation of the performance of the high-technology industry that is decomposed
into the technology development and the economic application [54]. In this two-stage tandem network, raw
data and knowledge are processed into technological achievements, which are then transformed into economic
development. A third application connects our study’s methodological framework with the operational activities
of the international shipping industry; these could be divided into the supervision of the ship dispatching
management and the control of the working time in the port [15]. Finally, the efficiency evaluation of two-stage
(food) supply chains of different factories or farming communities [24] could also serve the goals of our paper. For instance, the process of the refinement of selected cocoa beans into milk/dark chocolate and the production
of black tea through withering, fermentation, drying, and sieving across a number of specialised factory branches
could further highlight the importance of our evaluation and ranking framework. This paper treats the two sub-stages of a DMU equally. In reality, however, there might be a certain degree
of leader-follower relationship between the upstream and downstream of a particular DMU. We acknowledge
this as a limitation of our study and we believe that the introduction of relative weights for the different stages
when calculating overall efficiency could accommodate such an issue. In addition, one of the main steps of the
grey relational analysis methodology, used to rank the interval ultimate cross-efficiencies within an interval
cross-efficiency matrix, is the calculation of the grey relational grade. It is defined as the weighted average of
the grey relational coefficients, where the weight of the respective criterion is subjectively determined by the
decision maker. To better reflect the reality, we would have taken advantage of an existing powerful multi-criteria
decision-making method, such as the analytic network process [39] or the best-worst method [38], to identify in
an objective manner the weights. We have also recognised that the grey relational grade is just an index that can
only capture the rank rather than an efficiency measure. In other words, there is no sufficient information that
would allow the identification of the DEA-efficient DMUs that constitute the best-practice frontier. However, we
acknowledge that the GRA technique has not received attention on ranking interval cross-efficiencies within an 1316 M.D. KREMANTZIS ET AL. interval CE matrix and, thus, our paper has worked towards this direction. 5. Conclusions & future research Relevant literature has already focused
on this aspect by combining DEA and multiple-objective linear programming [12,30,31,44]. Appendix B. Stage 1 and grey relational analysis Table B.1. Normalisation of data, calculation of grey relational distance and grey relational
coefficient for the stage 1. DMU
Normalisation of data
Grey relational distance
Grey relational coefficient
C1
C2
C1
C2
C1
C2
1
1.0000
1.0000
0.0000
0.0000
1.0000
1.0000
2
0.2585
0.3467
0.7415
0.6533
0.4028
0.4335
3
0.0159
0.0043
0.9841
0.9957
0.3369
0.3343
4
0.0397
0.0834
0.9603
0.9166
0.3424
0.3529
5
0.2284
0.2278
0.7716
0.7722
0.3932
0.3930
6
0.2753
0.3259
0.7247
0.6741
0.4083
0.4259
7
0.0827
0.0756
0.9173
0.9244
0.3528
0.3510
8
0.0135
0.0000
0.9865
1.0000
0.3364
0.3333
9
0.0732
0.0797
0.9268
0.9203
0.3504
0.3520
10
0.0000
0.0015
1.0000
0.9985
0.3333
0.3337
Reference value
1.0000
1.0000
–
–
–
– Appendix C. Stage 2 and grey relational analysis Table C.1. Normalisation of data, calculation of grey relational distance and grey relational
coefficient for the stage 2. DMU
Normalisation of data
Grey relational distance
Grey relational coefficient
C1
C2
C1
C2
C1
C2
1
0.7556
0.6199
0.2444
0.3801
0.6717
0.5681
2
0.6000
0.2820
0.4000
0.7180
0.5555
0.4105
3
0.9915
1.0000
0.0085
0.0000
0.9833
1.0000
4
0.6230
0.4104
0.3770
0.5896
0.5701
0.4589
5
0.6204
0.2765
0.3796
0.7235
0.5685
0.4087
6
0.7209
0.5041
0.2791
0.4959
0.6418
0.5021
7
0.4250
0.0000
0.5750
1.0000
0.4651
0.3333
8
1.0000
0.9409
0.0000
0.0591
1.0000
0.8942
9
0.8417
0.5893
0.1583
0.4107
0.7596
0.5491
10
0.0000
0.0602
1.0000
0.9398
0.3333
0.3473
Reference value
1.0000
1.0000
–
–
–
– Acknowledgements. The work was supported by the Engineering and Physical Sciences Research Council (Industrial
CASE award) under grant 5162031105656, and BAE Systems. Appendix A. Overall system and grey relational analysis Table A.1. Normalisation of data, calculation of grey relational distance and grey relational
coefficient for the overall system. le A.1. Normalisation of data, calculation of grey relational distance and grey relationa
fficient for the overall system. DMU
Normalisation of data
Grey relational distance
Grey relational coefficient
C1
C2
C1
C2
C1
C2
1
1.0000
1.0000
0.0000
0.0000
1.0000
1.0000
2
0.3132
0.2217
0.6868
0.7783
0.4213
0.3911
3
0.1851
0.1477
0.8149
0.8523
0.3803
0.3697
4
0.1456
0.1181
0.8544
0.8819
0.3692
0.3618
5
0.2941
0.1872
0.7059
0.8128
0.4146
0.3809
6
0.3847
0.3046
0.6153
0.6954
0.4483
0.4183
7
0.1267
0.0376
0.8733
0.9624
0.3641
0.3419
8
0.1861
0.1313
0.8139
0.8687
0.3806
0.3653
9
0.2349
0.1372
0.7651
0.8628
0.3952
0.3669
10
0.0000
0.0000
1.0000
1.0000
0.3333
0.3333
Reference value
1.0000
1.0000
–
–
–
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2014 (2014) 8. [33] F. Meng and B. Xiong, Logical efficiency decomposition for general two-stage systems in view of cross efficiency. Eur. J. Oper. Res. 294 (2022) 622–632. [34] H.H. ¨Orkc¨u, V.S. ¨Ozsoy, M. ¨Orkc¨u and H. Bal, A neutral cross efficiency approach for basic two stage production systems. Expert Syst. App. 125 (2019) 333–344. [35] H.H. ¨Orkc¨u, V.S. ¨Ozsoy, M. ¨Orkc¨u and H. Bal, An optimistic-pessimistic DEA model based on game cross efficiency approach. RAIRO: Oper. Res. 54 (2020) 1215–1230. [36] P. Peykani, F. Hosseinzadeh Lotfi, S.J. Sadjadi, A. Ebrahimnejad and E. Mohammadi, Fuzzy chance-constrained data envelop-
ment analysis: a structured literature review, current trends, and future directions. Fuzzy Optim. Decision Making 21 (2022)
197–261 [37] S.A. Rakhshan, Efficiency ranking of decision making units in data envelopment analysis by using TOPSIS-DEA method. J. Oper. Res. Soc. 68 (2017) 906–918. 1319 A RANKING FRAMEWORK BASED ON INTERVAL SELF AND CROSS-EFFICIENCIES Please help to maintain this journal in open access! Check that your library subscribes to the journal, or make a personal donation to the S2O programme, by contacting
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https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0014065&type=printable
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English
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Taking Root: Enduring Effect of Rhizosphere Bacterial Colonization in Mangroves
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PloS one
| 2,010
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cc-by
| 9,085
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Abstract Background: Mangrove forests are of global ecological and economic importance, but are also one of the world’s most
threatened ecosystems. Here we present a case study examining the influence of the rhizosphere on the structural
composition and diversity of mangrove bacterial communities and the implications for mangrove reforestation approaches
using nursery-raised plants. Methodology/Principal Findings: A barcoded pyrosequencing approach was used to assess bacterial diversity in the
rhizosphere of plants in a nursery setting, nursery-raised transplants and native (non-transplanted) plants in the same
mangrove habitat. In addition to this, we also assessed bacterial composition in the bulk sediment in order to ascertain if
the roots of mangrove plants affect sediment bacterial composition. We found that mangrove roots appear to influence
bacterial abundance and composition in the rhizosphere. Due to the sheer abundance of roots in mangrove habitat, such an
effect can have an important impact on the maintenance of bacterial guilds involved in nutrient cycling and other key
ecosystem functions. Surprisingly, we also noted a marked impact of initial nursery conditions on the rhizosphere bacterial
composition of replanted mangrove trees. This result is intriguing because mangroves are periodically inundated with
seawater and represent a highly dynamic environment compared to the more controlled nursery environment. Conclusions/Significance: In as far as microbial diversity and composition influences plant growth and health, this study
indicates that nursery conditions and early microbial colonization patterns of the replants are key factors that should be
considered during reforestation projects. In addition to this, our results provide information on the role of the mangrove
rhizosphere as a habitat for bacteria from estuarine sediments. Citation: Gomes NCM, Cleary DFR, Pinto FN, Egas C, Almeida A, et al. (2010) Taking Root: Enduring Effect of Rhizosphere Bacterial Colonization in
Mangroves. PLoS ONE 5(11): e14065. doi:10.1371/journal.pone.0014065 Received July 16, 2010; Accepted October 26, 2010; Published November 22, 2010 Received July 16, 2010; Accepted October 26, 2010; Published November 22, 2010 Copyright: 2010 Gomes et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was funded by the Deutsche Forschungsgemeinschaft SM59/4-1 and 4-2 (http://www.dfg.de/en/index.jsp), FAPERJ-Brazil (http://www.faperj. br/) and Centre for Environmental and Marine Studies (CESAM, Portugal) (http://www.cesam.ua.pt/). Abstract The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: gomesncm@ua.pt . These authors contributed equally to this work. . These authors contributed equally to this work. Taking Root: Enduring Effect of Rhizosphere Bacterial
Colonization in Mangroves Newton C. M. Gomes1*., Daniel F. R. Cleary1., Fernando N. Pinto2, Conceic¸a˜o Egas3,4, Adelaide
Almeida1, Angela Cunha1, Leda C. S. Mendonc¸a-Hagler5, Kornelia Smalla6 1 CESAM and Department of Biology, University of Aveiro, Aveiro, Portugal, 2 Institute of Biology, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil, 3 Biocant-
Biotechnology Innovation Center, Cantanhede, Portugal, 4 Center for Neurosciences and Cellular Biology, University of Coimbra, Coimbra, Portugal, 5 Institute of
Microbiology, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil, 6 Julius Ku¨hn-Institute for Cultivated Crops, Braunschweig, Germany November 2010 | Volume 5 | Issue 11 | e14065 Introduction In return, the microorganisms
contribute to plant growth and health by nutrient solubilisation, N2
fixation, the production of plant hormones and the degradation of
phytotoxic compounds [9,10]. However, intertidal zones of the
mangrove forests are periodically inundated and it is unknown
whether roots from mangrove plants located in these extreme
environments can impose a similar selective pressure on microbial
communities as has been demonstrated for purely terrestrial plants. There was significant variation in OTU composition among
treatments (Adonis analysis: F3,15 = 3.518, R2 = 0.468, P,0.001). A principal coordinates analysis (PCO), using the Hellinger
distance, of OTU composition (Fig. 2) showed that the primary
axis of variation was between samples obtained from the nursery
and samples from the rhizosphere and bulk sediment in the
mangrove sampling site. The transplanted samples, however, had
the greatest similarity (of the mangrove samples) to the nursery
samples, with several dominant OTU’s in common. Along Axis 2,
the greatest difference was between the transplanted and the bulk
sediment samples; native plant samples were intermediate. In line with the PCO, the RDP classification of the OTU’s
showed that the nursery samples contained the most distinct
composition of the major taxonomic groups, e.g., significantly
higher relative abundances of Acidobacteria, Actinobacteria, Verrucro-
microbia, Burkholderiales, Caulobacterales and Rhizobiales and signifi-
cantly lower relative abundances of Chloroflexi, Firmicutes and
Desulfobacterales (Fig. 3). Nursery samples also contained fewer
phyla than the mangrove samples. Interestingly, the Bacteroidetes
were markedly more abundant in the rhizosphere of native plants
than in either the nursery or bulk sediment samples. Proteobacteria
was the most abundant phylum in all samples and comprised from
36% to 40% of total reads. The relative abundance of the most
dominant orders within the Proteobacteria are also shown in Fig. 3. Desulfobacterales was the most abundant proteobacterial order
detected in Trn, Nat and Bul samples, with 17, 23 and 30% of
the total reads assigned to this order, respectively. Chromatiales was
the second most abundant order, almost equally distributed
among mangrove samples, with only a few representatives
detected in nursery samples. Three key questions need to be addressed in order to ascertain
whether
manipulation
of
the
microbial
community
in
the
rhizosphere can be exploited in the restoration of mangrove
habitats. First of all, it is essential to investigate if mangrove plants
can influence the composition of microorganisms colonizing the
sediment surrounding their roots as has been observed for terrestrial
plants [12–14]. Introduction Next, it is important to ascertain whether the initial
growth conditions of nursery raised trees have a long-term effect on
the microbial community of replant rhizospheres. Finally, it is
necessary to evaluate if microbial rhizoengineering during initial
growth conditions in the nursery can enhance plant growth and
survival. Various studies have already demonstrated that plant
diversity can influence such ecosystem processes as stability,
productivity, nutrient dynamics and vulnerability to invasive species
[15] although this remains to be shown for microbial diversity. A
more diverse microbial community may, however, buffer a plant
from potentially dangerous pathogens and include a diverse array of
functional groups of species that facilitate plant growth. In this study, we address the first two questions, namely if
mangrove plants influence the composition of bacterial commu-
nities colonizing the sediment surrounding their roots (rhizosphere
effect) and if the initial growth conditions have a significant and
long-term
effect
on
the
bacterial
community
of
replanted
mangrove trees. We also compare the microbial communities of
bulk sediment and the rhizosphere of native mangrove plants. In
addition to comparing microbial diversity and composition among
treatments, we also make an in depth analysis of the dominant
bacterial populations in order to see if known beneficial microbes
are enhanced in transplants and native mangrove plants compared
to the bulk sediment. Of the orders shown in Fig. 3, Rhizobiales, Campylobacterales,
Methylococcales and Vibrionales tend to be more abundant in the
rhizosphere samples than in the bulk sediment. Rhizobiales
populations were interestingly also significantly more abundant in
nursery and transplant samples than in native and bulk sediment
samples. Transplant rhizospheres showed about three times more
abundant OTU’s assigned to Rhizobiales than native plants. The
relative abundance of this order in nursery samples was in turn
about five times higher than in transplanted samples. The OTU’s
assigned
to
Methylococcales
were
all
assigned
to
the
family
Methylococcaceae (mainly Methylomonas) (using the RDP Classifier)
and were at least twice as abundant in mangrove rhizosphere
samples (Trn and Nat) than in bulk sediment. Curiously, in Fig. 4A
(the ternary plot of dominant OTU’s distributed in the rhizosphere
samples) specific OTU’s related to aerobic methanotrophs (41 and
47) assigned to members of the family Methylococcaceae (Table 1),
were specifically enhanced in the transplanted plants. The
abundance and diversity of Vibrionales was also much higher in
mangrove rhizosphere samples than either nursery or bulk sediment
samples. Introduction Commission funded a project for mangrove restoration in Sri
Lanka, which resulted in more than 60,000 replanted mangrove
saplings [5]. Mangrove forests are unique and diverse coastal ecosystems
located in tropical and subtropical regions. These forests are both
ecologically and economically important. In addition to protecting
coastal areas from erosion, mangroves also diminish the impact of
Tsunamis and serve as critical nurseries for juvenile fish [1,2]. Despite the well known benefits of maintaining healthy man-
groves, they are highly threatened ecosystems and at present are
disappearing at a rate of 1 to 2% per year across their range [3]. Due to the growing concern that mangroves may disappear in a
relatively short time frame (,100 years) [3] and the need to
reverse ongoing destruction, several international and community-
based rehabilitation programs have been established across the
globe [4,5]. International organizations that support mangrove
rehabilitation include the European Union, the World Bank and
the World Wide Fund for Nature. In 2005, for example, the EU Natural regeneration is often the first strategy to be adopted for
recovery of degraded mangroves. When this is hampered,
restoration projects may be established that involve growing
mangrove seedlings in nurseries and subsequently transplanting
these to degraded areas [6–8]. However, reforestation approaches
using nursery-raised plants often show highly variable survival
rates and knowledge is lacking about the biology of the whole
process. Surprisingly, despite the well-known mutual dependence
between plant roots and soil microbial communities [9,10], no
studies have hitherto made an in depth analysis of how initial
growth
conditions
and
transplantation
affect
the
microbial
communities of replants. The interaction between plants and
microorganisms has in fact only recently become a focal topic in
restoration ecology [11]. Previous studies have demonstrated that PLoS ONE | www.plosone.org PLoS ONE | www.ploso November 2010 | Volume 5 | Issue 11 | e14065 1 November 2010 | Volume 5 | Issue 11 | e14065 PLoS ONE | www.plosone.org Mangrove Rhizosphere Bacteria samples also contained a very large proportion of ‘rare’ OTU’s
compared to samples from other treatments. soil microorganisms are essential for nutrient cycling, soil structure
generation and decomposition and are thus key players in the
regulation of plant productivity and plant community dynamics
[11]. Several plant species have been, furthermore, shown to
influence the microorganisms colonizing their root environment
(the ‘rhizosphere effect’) [12–14]. Introduction The great majority of these OTU’s were assigned to the
genus Vibrio (using the RDP Classifier). PLoS ONE | www.plosone.org November 2010 | Volume 5 | Issue 11 | e14065 Results The data retrieved from the cluster analyses of the Ribosomal
Database Project (RDP) pyrosequencing pipeline was used to
estimate operational taxonomic units (OTU’s) richness and
compare composition among treatments. The dominance-diversi-
ty plots and species rarefaction curve of each sample revealed
marked differences among treatments (Fig. 1A,B). Samples taken
from transplanted (Trn) plants had the highest number of OTU’s. Nursery
(Nur)
samples,
in
contrast,
exhibited
pronounced
dominance of a few OTU’s but contained much fewer OTU’s
compared to samples from other treatments. In the Bulk sediment
(Bul) samples, the dominance of the most abundant OTU’s was
much less pronounced but there were more ‘rare’ OTU’s than in
the nursery samples. Rhizosphere samples from the native (Nat)
saplings exhibited somewhat more dominance and fewer ‘rare’
OTU’s compared to the sediment samples. The rhizosphere effect
on bacterial diversity is, however, much more pronounced for
transplanted samples that were raised in a ‘terrestrial’ soil matrix. In addition to the pronounced dominance, the transplanted The ternary plots of dominant OTU’s (Fig. 4A,B) show a
dominant bacterial population (OTU 64), which was almost
equally distributed in all rhizosphere samples, but was not detected
in the bulk sediment. This OTU was assigned to Bacteria with high
confidence (94%) but could not be assigned to any known Phylum
(Table 1). Three dominant OTU’s (1, 49 and 431) found in
nursery and transplanted rhizospheres, were classified as diazo-
trophic bacteria belonging to the order Rhizobiales (Table 1). Several other dominant OTU’s were much more abundant or
only detected in the nursery samples and seem not to be able to
persist in the mangrove environment after transplantation; for November 2010 | Volume 5 | Issue 11 | e14065 2 Mangrove Rhizosphere Bacteria Figure 1. Diversity analyses of rhizosphere samples from nursery (Nur), transplanted
plants and from the bulk sediment (Bul). A) Dominance-diversity plots. Each panel plots logarit
of species for each sample. The blue horizontal lines represent observed (raw) data. The red and yello
Zipf-Mandelbrot models respectively. The best fits were obtained with the ‘radfit()’ function in the v
each sample data set using; error bars represent a single standard deviation. d i 10 1371/j
l
0014065 001 Figure 1. Diversity analyses of rhizosphere samples from nursery (Nur), transplanted (Trn) and native (Nat) Rhizophora mangle
plants and from the bulk sediment (Bul). A) Dominance-diversity plots. Mangrove Rhizosphere Bacteria The rhizosphere effect is typically characterized by
a reduced diversity in the rhizosphere compared to the bulk
sediment and increased abundance of root specialized bacterial
guilds [16]. This effect in the mangrove plants is, however, much
more pronounced for transplants that were raised in a ‘terrestrial’
soil matrix. In addition to the pronounced dominance, the
transplants showed a larger proportion of ‘rare’ OTU’s compared
to bulk sediment. This is, therefore, an atypical rhizosphere effect
and suggests that the unique initial growth conditions (for mangrove
plants) and transplantation, as it was performed in this study, has
had a marked impact on the diversity and composition of the
bacterial communities of replants. The RDP classifier analysis of all bacterial OTU’s revealed
several bacterial guilds colonizing mangrove samples which are
known for their importance in the marine biogeochemical cycling of
carbon, nitrogen and sulphur. Marine members of the Bacteroidetes
were more abundant in the rhizosphere of native plants and are
known degraders of particulate organic matter in the ocean [17]. However, their potential ecological role in mangrove rhizospheres is
unknown. The Proteobacteria was the most abundant phylum in all
samples. This group is metabolically highly diverse, widely
distributed in marine environments, and is an important player in
nutrient cycling [18]. The potential effect of mangrove roots on
sediment proteobacterial populations may influence several envi-
ronmentally relevant processes in mangrove ecosystems. Root
production in a tropical mangrove dominated by R. mangle can also
be much higher than in inland forests; mangrove roots form a
complex below-ground net with a growth of about 28 tons of dry
biomass per hectare per year [19,20]. The ability of such root
systems to facilitate the growth of specific microbial guilds, may be
essential for nutrient cycling and ecological resilience. Figure 2. Principal coordinates (PCO) analysis of operational
taxonomic unit (OTU) composition. The first two axes of a PCO
ordination are shown based on a matrix of OTU composition of
rhizosphere samples from nursery (Nur), transplanted (Trn) and native
(Nat) Rhizophora mangle plants and from the bulk sediment (Bul). Grey
symbols represent individual OTU’s where the size of the symbol
corresponds to its total abundance (see legend in plot). Coloured symbols
represent sample sites where the size corresponds to OTU richness. doi:10.1371/journal.pone.0014065.g002 example, members of the order Caulobacterales, which are known
chemoorganotrophic aerobic organisms. The relative abundance of dominant OTU’s present in the
mangrove samples (Fig. Mangrove Rhizosphere Bacteria 4B) revealed several OTU’s with strong
associations to the mangrove rhizospheres (Trn and Nat). While
the OTU’s 24, 93 and 231 were more abundant in Nat
rhizospheres, OTU’s 27, 31, 41, 47, 49, 65, 72 and 239 were
more prevalent in Trn rhizospheres. The taxonomic assignment of
these OTU’s and their known putative ecophysiological traits are
presented in Table 1. In general, in agreement with the relative
abundance analyses of the order Campylobacterales (Fig. 3), OTU’s
assigned to the genus Sulfurovum (65 and 239) were more abundant
in
the
rhizosphere
samples
(with
stronger
associations
to
transplants) but were again rare in the bulk sediment (Fig. 4B
and see Table 1). The rhizosphere of transplants also appeared to
have enhanced the colonization of bacterial populations related to
Methylomonas (OTU’s 41 and 47). In contrast, OTU’s assigned to
the diazotrophic bacteria Listonella (231) and Vibrio (93) were
mainly detected in the rhizosphere of native plants. Our results showed that specific proteobacterial groups involved
in the biogeochemical sulphur cycle were the most abundant
bacterial guilds in the mangrove samples. The Desulfobacterales was
the most abundant order detected in Trn, Nat and Bul samples. This order encompasses primarily sulfate-reducing bacteria (SRB)
which are important players in the process of anoxic mineralization
of organic matter and pollutants, such as anthropogenic hydrocar-
bons [21,22]. Chromatiales was the second most abundant proteo-
bacterial order and was detected in all mangrove samples (but not in
nursery samples). This order is represented by anaerobic or
microaerophilic microorganisms specialized in sulfur-anoxygenic
photosynthesis and are able to oxidize hydrogen sulfide (H2S) to
elemental sulphur [23]. Campylobacterales were also abundant and
mainly detected in the mangrove samples (Trn, Nat and Bul) with a
marked increased abundance in rhizosphere samples. Members of
this order are sulfide-oxidizing denitrifying bacteria [24]. In this study, some dominant bacterial OTU’s associated to
known plant-beneficial organisms were only detected in nursery or
transplanted samples. Rhizobacterial populations acquired during
nursery growth were, therefore, presumably able to survive in the
mangrove environment and remained abundant in the rhizo-
sphere up to 202 days after planting (dap). Several dominant
OTU’s,
furthermore,
showed
specific
associations with the
rhizosphere of native and transplanted plants and were assigned
to microbial guilds known to influence nutrient cycling (Fig. 3,
Fig. 4A,B; also see Table 1). Mangrove Rhizosphere Bacteria The ternary plots of dominant OTU’s also showed increased
abundance of the Campylobacterales belonging to the genus Sulfurovum
in mangrove rhizosphere samples. This genus is known to be an
important player in the process of sulfide-oxidation and denitrifi-
cation in marine environments [24–26]. A previous study [27] also
showed that R. mangle can oxidize the sediment rhizosphere and
thereby contribute to the reduction of hydrogen sulfide in the
sediment. However, no study has investigated the potential role of Mangrove Rhizosphere Bacteria Mangrove Rhizosphere Bacteria Figure 2. Principal coordinates (PCO) analysis of operational
taxonomic unit (OTU) composition. The first two axes of a PCO
ordination are shown based on a matrix of OTU composition of
rhizosphere samples from nursery (Nur), transplanted (Trn) and native
(Nat) Rhizophora mangle plants and from the bulk sediment (Bul). Grey
symbols represent individual OTU’s where the size of the symbol
corresponds to its total abundance (see legend in plot). Coloured symbols
represent sample sites where the size corresponds to OTU richness. doi:10.1371/journal.pone.0014065.g002 mangrove ecology and virtually all mangrove reforestation projects. Mangroves are unique coastal marine intertidal environments and
as such are periodically inundated with seawater. Therefore the
mangrove root bacterial communities have to adapt to living in a
habitat which is exposed to extreme changes on a daily basis due to
tidal cycles. In this study, diversity plots and PCO analysis revealed
marked differences between rhizosphere (Nur, Trn and Nat) and
bulk sediment communities. These results agree with the concept of
the so-called ‘rhizosphere effect’, a phenomenon well described for
terrestrial plants. The rhizosphere effect is typically characterized by
a reduced diversity in the rhizosphere compared to the bulk
sediment and increased abundance of root specialized bacterial
guilds [16]. This effect in the mangrove plants is, however, much
more pronounced for transplants that were raised in a ‘terrestrial’
soil matrix. In addition to the pronounced dominance, the
transplants showed a larger proportion of ‘rare’ OTU’s compared
to bulk sediment. This is, therefore, an atypical rhizosphere effect
and suggests that the unique initial growth conditions (for mangrove
plants) and transplantation, as it was performed in this study, has
had a marked impact on the diversity and composition of the
bacterial communities of replants. mangrove ecology and virtually all mangrove reforestation projects. Mangroves are unique coastal marine intertidal environments and
as such are periodically inundated with seawater. Therefore the
mangrove root bacterial communities have to adapt to living in a
habitat which is exposed to extreme changes on a daily basis due to
tidal cycles. In this study, diversity plots and PCO analysis revealed
marked differences between rhizosphere (Nur, Trn and Nat) and
bulk sediment communities. These results agree with the concept of
the so-called ‘rhizosphere effect’, a phenomenon well described for
terrestrial plants. November 2010 | Volume 5 | Issue 11 | e14065 Results Each panel plots logarithmic species abundance against the rank order
of species for each sample. The blue horizontal lines represent observed (raw) data. The red and yellow lines represent the best fits, namely Zipf and
Zipf-Mandelbrot models respectively. The best fits were obtained with the ‘radfit()’ function in the vegan library in R. B) Species rarefaction curve of
each sample data set using; error bars represent a single standard deviation. doi:10.1371/journal.pone.0014065.g001 Figure 1. Diversity analyses of rhizosphere samples from nursery (Nur), transplanted (Trn) and native (Nat) Rhizophora mangle
plants and from the bulk sediment (Bul). A) Dominance-diversity plots. Each panel plots logarithmic species abundance against the rank order
of species for each sample. The blue horizontal lines represent observed (raw) data. The red and yellow lines represent the best fits, namely Zipf and
Zipf-Mandelbrot models respectively. The best fits were obtained with the ‘radfit()’ function in the vegan library in R. B) Species rarefaction curve of
each sample data set using; error bars represent a single standard deviation. doi:10.1371/journal.pone.0014065.g001 November 2010 | Volume 5 | Issue 11 | e14065 PLoS ONE | www.plosone.org 3 Discussion The mutual dependence between plants and microbes is a
fundamental biological interaction that has been largely ignored in PLoS ONE | www.plosone.org November 2010 | Volume 5 | Issue 11 | e14065 4 Mangrove Rhizosphere Bacteria Figure 3. Relative abundance of the most dominant bacterial groups. Each panel plots the relative abundance of different bacterial taxa
inhabiting rhizosphere samples from nursery (Nur), transplanted (Trn) and native (Nat) Rhizophora mangle plants and bulk sediments (Bul). All classes
are shown where the relative abundance in at least one treatment exceeds 1% (first three rows). The eight most abundant classified orders of
Proteobacteria are also shown (last two rows). Symbols above the bars represent significant deviations (*** P,0.001, ** 0.001,P,0.01,
* 0.01,P,0.05) from the relative abundance in the bulk sediment using an analysis of deviance (glm with ‘quasibinomial’ family). Note that we did
not control for multiple tests. doi:10.1371/journal.pone.0014065.g003
Mangrove Rhizosphere Bacteria Figure 3. Relative abundance of the most dominant bacterial groups. Each panel plots the relative abundance of different bacterial taxa
inhabiting rhizosphere samples from nursery (Nur), transplanted (Trn) and native (Nat) Rhizophora mangle plants and bulk sediments (Bul). All classes
are shown where the relative abundance in at least one treatment exceeds 1% (first three rows). The eight most abundant classified orders of
Proteobacteria are also shown (last two rows). Symbols above the bars represent significant deviations (*** P,0.001, ** 0.001,P,0.01,
* 0.01,P,0.05) from the relative abundance in the bulk sediment using an analysis of deviance (glm with ‘quasibinomial’ family). Note that we did
not control for multiple tests. doi:10 1371/journal pone 0014065 g003 rhizosphere can have a strong influence on microbial activity and
thus affect several processes of environmental relevance [29]. The
effect of nursery conditions and roots on the diversity and
abundance of methane consuming bacteria in the sediment
surrounding the roots of mangrove plants has not been previously
demonstrated. Such an effect can be important when considering
the global destruction of mangrove habitat and large scale
replanting approaches and merits further study. plant microbe interactions in the process of sulfide sediment
detoxification in mangrove ecosystems. Our results reveal for the
first time that R. mangle roots appear to be able to enhance the
abundance of bacterial sulfide oxidizers which in turn may have
further ecological implications for the process of sediment sulfide
detoxification. Discussion Curiously, the RDP and ternary plot analyses showed that
Rhizobiales populations were more abundant in nursery and
transplant samples than in native and bulk sediment samples. These results indicate that the nursery period was important for
recruitment of nitrogen-fixing rhizobia. Such phenomena can
favour the growth of mangrove replants in nitrogen-poor mangrove
sediment. Mangrove rhizospheres (Trn and Nat) also showed a
preferential enhancement of OTU’s assigned to the Methylococcaceae
family in comparison to bulk sediment samples, but in contrast to
the Rhizobiales, members of this family were absent from nursery
samples. Previous studies have shown that the Methylococcaceae family
encompasses aerobic methanotrophs, which are key players in the
methane flux from sediment (marine and fresh water) to the
atmosphere [28]. Our results suggest an important ecological role of
R. mangle roots in the selective enhancement of methanotrophic
populations in mangroves. The chemical properties of the p
g pp
y
Our analyses also suggest that mangrove roots are a preferred
habitat for Vibrio populations. The Vibrio genus includes mainly
aquatic bacteria, several of which are free-living and obligate
endosymbionts. Previous studies on nitrogen-fixation in mangrove
ecosystems have already identified a number of Vibrio species in the
rhizosphere of mangroves [30]. However, none of these studies
made comparative analyses of their relative abundance in mangrove
rhizosphere (transplanted and native) versus bulk sediment samples. Several dominant OTU’s were only detected in nursery or
transplanted samples, strengthening our observation that rhizobac-
terial populations acquired during nursery growth were introduced
into the mangrove environment and remained abundant in the
rhizosphere up to 202 dap. The ability of rhizo-competent bacteria
to survive during the first months of transplantation is an important Our analyses also suggest that mangrove roots are a preferred
habitat for Vibrio populations. The Vibrio genus includes mainly
aquatic bacteria, several of which are free-living and obligate
endosymbionts. Previous studies on nitrogen-fixation in mangrove
ecosystems have already identified a number of Vibrio species in the
rhizosphere of mangroves [30]. However, none of these studies
made comparative analyses of their relative abundance in mangrove
rhizosphere (transplanted and native) versus bulk sediment samples. Several dominant OTU’s were only detected in nursery or
transplanted samples, strengthening our observation that rhizobac-
terial populations acquired during nursery growth were introduced
into the mangrove environment and remained abundant in the
rhizosphere up to 202 dap. November 2010 | Volume 5 | Issue 11 | e14065 PLoS ONE | www.plosone.org Discussion The ability of rhizo-competent bacteria
to survive during the first months of transplantation is an important PLoS ONE | www.plosone.org November 2010 | Volume 5 | Issue 11 | e14065 November 2010 | Volume 5 | Issue 11 | e14065 5 Mangrove Rhizosphere Bacteria finding because this phase is the most critical for sapling survival
[31]. Anything that can significantly augment the transplantation
success of mangrove saplings will be of major importance to the
conservation and restoration of this important ecosystem. We also
hope that o r st d
ill f nction as a catal st to stim late long term
studies to understand
time in mangrove en
tation on community
In conclusion, our
marked heterogeneit
Figure 4. Ternary plots showing the ratio’s of the most abundant operational ta
[rhizosphere samples of nursery (Nur), transplanted (Trn), native Rhizophora mangle
rhizosphere (Nur, Trn and Nat) treatments (A) and mangrove (Trn, Nat and Bul) treatments (B) are
Table 1). In ternary plots each corner of the triangle represents a proportion of 100% for a given trea
that treatment. As the proportion of a given treatment increases in a sample then it moves towa
doi:10.1371/journal.pone.0014065.g004 Figure 4. Ternary plots showing the ratio’s of the most abundant operational taxonomic units (OTU’s) across treatments
[rhizosphere samples of nursery (Nur), transplanted (Trn), native Rhizophora mangle (Nat) and bulk sediment (Bul)]. Ratio’s in
rhizosphere (Nur, Trn and Nat) treatments (A) and mangrove (Trn, Nat and Bul) treatments (B) are shown. Numbers identify individual OTU’s (see
Table 1). In ternary plots each corner of the triangle represents a proportion of 100% for a given treatment with the other corners representing 0% of
that treatment. As the proportion of a given treatment increases in a sample then it moves towards the corner representing that treatment. doi:10.1371/journal.pone.0014065.g004 Figure 4. Ternary plots showing the ratio’s of the most abundant operational taxonomic units (OTU’s) across treatments
[rhizosphere samples of nursery (Nur), transplanted (Trn), native Rhizophora mangle (Nat) and bulk sediment (Bul)]. Ratio’s in
rhizosphere (Nur, Trn and Nat) treatments (A) and mangrove (Trn, Nat and Bul) treatments (B) are shown. Numbers identify individual OTU’s (see
Table 1). In ternary plots each corner of the triangle represents a proportion of 100% for a given treatment with the other corners representing 0% of
that treatment. Discussion As the proportion of a given treatment increases in a sample then it moves towards the corner representing that treatment. doi:10 1371/journal pone 0014065 g004 studies to understand how microbial communities change through
time in mangrove environments including the impact of transplan-
tation on community dynamics. finding because this phase is the most critical for sapling survival
[31]. Anything that can significantly augment the transplantation
success of mangrove saplings will be of major importance to the
conservation and restoration of this important ecosystem. We also
hope that our study will function as a catalyst to stimulate long-term In conclusion, our results reveal a strong treatment effect and
marked heterogeneity in OTU composition. An important finding November 2010 | Volume 5 | Issue 11 | e14065 PLoS ONE | www.plosone.org 6 Mangrove Rhizosphere Bacteria Table 1. Taxonomic assignment of partial 16S rRNA gene sequences of dominant bacterial populations (operational taxono
units $50 reads) and their known putative ecophysiological traits. OTU code/number of reads
Sequence classification
Known traits
839/78, 545/131, 249/50, 7/73,
91/66, 64/160, 257/127, 45/148,
165/121
Bacteria
Unknown Phylum. 923/65
Proteobacteria
The Phylum Proteobacteria is highly diverse, widely distributed in marine environments, and is kn
as an important player in the process of nutrient cycling. 171/62, 274/72, 1097/104,
75/135, 72/94
619/76
231/60
437/199
41/108, 47/89
1192/53
211/103, 319/77, 880/50
93/363
Gammaproteobacteria
Haliea
Listonella
Marinomonas
Methylomonas
Thiohalocapsa
Thiohalophilus
Vibrio
The Gammaproteobacteria includes populations that are able to decompose marine organic ma
Many OTU’s classified into this Class are known for their importance in nutrient cycling in mar
ecosystems. However, Haliea strains were recently isolated from marine environments and the
putative ecological functions are still unknown. The genus Listonella includes diazotrophic populations with some representatives previously
detected in mangrove rhizospheres. Marinomonas species have been previously isolated from salt marsh roots and evidence sugge
that members of this genus are involved in dimethylsulphoniopropionate (DMSP) catabolism in
rhizosphere of estuarine plants. The degradation of DMSP into dimethylsulfide is a key process in
transfer of sulphur from marine ecosystems to the atmosphere. The family Methylococcaceae includes aerobic methanotrophs such as Methylomonas spp. This fa
belongs to a group of methanotrophic bacteria, which are important in reducing the methane
from sediment (marine and fresh water) to the atmosphere. Members of the Thiohalocapsa genus are purple sulfur photolithoautotrophic bacteria with
halophilic growth response. The Thiohalophilus genus comprises sulphur-oxidizing
chemolithoautotrophic bacteria which are also capable of halophilic growth. Discussion The Vibrio genus includes mainly aquatic bacteria of which several are free-living and obligate
endosymbionts. 419/53
431/90
1/86, 49/186
567/98
Alphaproteobacteria
Rhizobiales
Bradyrhizobium
Asticcacaulis
The Alphaproteobacteria comprise several plant symbionts. In this study several OTU’s assigne
this class were related to rhizobia (Rhizobiales); these are well known for their ability to fix
atmospheric nitrogen in association with plants. Bradyrhizobium spp. are well known as root-no
bacteria, which are used as plant inoculants worldwide. The cluster 567 was assigned to the genus Asticcacaulis, which consists of chemoorganotroph
aerobic organisms. 84/50
Betaproteobacteria
Herbaspirillum
The genus Herbaspirillum also comprises endophytic diazotrophs. 70/100
34/65, 861/68
273/71
89/209
126/68
24/92, 581/58, 1077/59
938/52
Deltaproteobacteria
Desulfobacteraceae
Desulfatibacillum
Desulfosarcina
Desulfobacterium
Desulfobulbaceae
Desulfobulbus
Syntrophobacterales
Deltaproteobacteria, have been described as a key group of sulphate-reducing bacteria (SRB) i
marine sediments. The Desulfobacteraceae (Desulfatibacillum, Desulfosarcina and Desulfobacteri
and Desulfobulbaceae (Desulfobulbus) families are often detected in marine environments and
important players in the process of anoxic mineralization of organic matter. 357/160
65/59, 239/219
Epsilonproteobacteria
Helicobacteraceae
Sulfurovum
Members of the Helicobacteraceae family are involved in autotrophic nitrate reduction and sul
oxidation. The genus Sulfurovum (Campylobacterales) is an important player in the process of sul
oxidation and denitrification in marine environments. 57/102
215/75
Acidobacteria
Gp1
Gp23
Only a few Acidobacteria strains have been cultivated up to now. Therefore, the putative ecolo
function of this group still remains largely unknown. 749/56
Actinobacteria
Actinomycetales
Members of this order are best known from soils and plant rhizospheres. They are well known
efficient degraders of complex biopolymers (e.g. lignocellulose, keratin, and chitin). Only recently
actinomycetes were recognized as autochthonous marine microbiota
55/132
Bacteroidetes
The Chitinophagaceae family includes isolates that have been frequently detected in several
environmental samples and are well known as efficient degraders of biopolymers. Chitinophagaceae
10/61
Chloroflexi
Members of this phylum consist of facultatively aerobic, filamentous bacteria and are presuma Table 1. Taxonomic assignment of partial 16S rRNA gene sequences of dominant bacterial populations (operational taxonomic
units $50 reads) and their known putative ecophysiological traits. The Phylum Proteobacteria is highly diverse, widely distributed in marine environments, and is known
as an important player in the process of nutrient cycling. The Gammaproteobacteria includes populations that are able to decompose marine organic matter. Many OTU’s classified into this Class are known for their importance in nutrient cycling in marine
ecosystems. However, Haliea strains were recently isolated from marine environments and their
putative ecological functions are still unknown. Discussion The genus Listonella includes diazotrophic populations with some representatives previously
detected in mangrove rhizospheres. Marinomonas species have been previously isolated from salt marsh roots and evidence suggests
that members of this genus are involved in dimethylsulphoniopropionate (DMSP) catabolism in the
rhizosphere of estuarine plants. The degradation of DMSP into dimethylsulfide is a key process in the
transfer of sulphur from marine ecosystems to the atmosphere. The family Methylococcaceae includes aerobic methanotrophs such as Methylomonas spp. This family
belongs to a group of methanotrophic bacteria, which are important in reducing the methane flux
from sediment (marine and fresh water) to the atmosphere. Members of the Thiohalocapsa genus are purple sulfur photolithoautotrophic bacteria
halophilic growth response. The Thiohalophilus genus comprises sulphur-oxidizing g
g
g
chemolithoautotrophic bacteria which are also capable of halophilic growth. The Vibrio genus includes mainly aquatic bacteria of which several are free-living and obligate
endosymbionts. The Alphaproteobacteria comprise several plant symbionts. In this study several OTU’s assigned to
this class were related to rhizobia (Rhizobiales); these are well known for their ability to fix
atmospheric nitrogen in association with plants. Bradyrhizobium spp. are well known as root-nodule
bacteria, which are used as plant inoculants worldwide. The cluster 567 was assigned to the genus Asticcacaulis, which consists of chemoorganotrophic,
aerobic organisms. The genus Herbaspirillum also comprises endophytic diazotrophs. Deltaproteobacteria, have been described as a key group of sulphate-reducing bacteria (SRB) in
marine sediments. The Desulfobacteraceae (Desulfatibacillum, Desulfosarcina and Desulfobacterium)
and Desulfobulbaceae (Desulfobulbus) families are often detected in marine environments and are
important players in the process of anoxic mineralization of organic matter. Members of the Helicobacteraceae family are involved in autotrophic nitrate reduction and sulfide
oxidation. The genus Sulfurovum (Campylobacterales) is an important player in the process of sulfide-
oxidation and denitrification in marine environments. Only a few Acidobacteria strains have been cultivated up to now. Therefore, the putative ecological
function of this group still remains largely unknown. Members of this order are best known from soils and plant rhizospheres. They are well known as
efficient degraders of complex biopolymers (e.g. lignocellulose, keratin, and chitin). Only recently the
actinomycetes were recognized as autochthonous marine microbiota The Chitinophagaceae family includes isolates that have been frequently detected in several
environmental samples and are well known as efficient degraders of biopolymers. Members of this phylum consist of facultatively aerobic, filamentous bacteria and are presumably
involved in the degradation of carbohydrates and amino acids. Discussion The Deferribacteres class includes species involved in dissimilatory Fe reduction. The genus Caldithrix
includes a few isolates retrieved from extreme environments which were nitrate-reducing, anaerobe
chemo-organoheterotrophs capable of fermenting proteinaceous substrates. November 2010 | Volume 5 | Issue 11 | e14065 November 2010 | Volume 5 | Issue 11 | e14065 PLoS ONE | www.plosone.org 7 Mangrove Rhizosphere Bacteria OTU code/number of reads
Sequence classification
Known traits
488/63
27/350, 209/126
31/183, 430/99
Verrucomicrobia
Opitutus
Spartobacteria_genera_
incertae_sedis
Subdivision3_genera_
incertae_sedis
Verrucomicrobia OTU’s are frequently found in culture-independent surveys of a broad range of
environmental and non-environmental samples. However, there are only a few species belonging to
this group which have been successfully isolated and cultivated. In general they are mesophilic
carbohydrate degraders. Recently a few aerobic methanotrophs of Verrucomicrobia have been
found. The codes in bold refer to the OTUs’ code followed by the number of sequences reads assigned to each OTU. Sequences were assigned up to the lowest taxonomic rank with at least 50% bootstrap support. doi:10.1371/journal.pone.0014065.t001
Table 1. cont. OTU code/number of reads
Sequence classification
Known traits Verrucomicrobia OTU’s are frequently found in culture-independent surveys of a broad range of
environmental and non-environmental samples. However, there are only a few species belonging to
this group which have been successfully isolated and cultivated. In general they are mesophilic
carbohydrate degraders. Recently a few aerobic methanotrophs of Verrucomicrobia have been
found. The codes in bold refer to the OTUs’ code followed by the number of sequences reads assigned to each OTU. Sequences were assigned up to the lowest taxonomic rank with at least 50% bootstrap support. doi:10.1371/journal.pone.0014065.t001 The codes in bold refer to the OTUs’ code followed by the number of sequences reads assigned to each OTU. Sequences were assigned up to the lowest taxonomic rank with at least 50% bootstrap support. doi:10.1371/journal.pone.0014065.t001 from a given treatment were not clustered together so as to avoid
pseudo-replication. Each rhizosphere sample consisted of the total
root system with tightly adhering sediment of each individual plant
[16]. A spatula was used to remove the sediment that could be
easily detached from the roots. Only sediment adhering to the
plant root system was considered as the rhizosphere fraction. Each
rhizosphere sample consisted of the total root system. Each sample
was thoroughly mixed and microbial cells were detached from
rhizosphere and bulk sediment samples (5 grams) as previously
described [33]. Materials and Methods Initially a replanting approach was simulated in an urban
mangrove located in Guanabara Bay (Rio de Janeiro, Brazil)
(22u469530S/43u049160W). The sampling site characteristics have
been described previously [32]. Mature propagules of the
mangrove tree species Rhizophora mangle were collected from
mangrove forests located in Guanabara Bay and planted in
polyethylene bags containing a mixture of clay mineral and red
yellow podzolic soil (1:1). This mixture has been used successfully
for almost a decade in mangrove replanting projects in Rio de
Janeiro (Brazil), with plants supplied by Jose´ Luiz de Castro
Ferreira (Association ‘Amigos do Manguezal de Jequia´’, Rio de
Janeiro, Brazil). The plants were watered every day with fresh
water and marine water (3 times each) during 75 days. The use of
a soil mixture as substrate instead of mangrove sediment allows us
to evaluate whether distinct initial growing conditions would have
a long-term effect on the microbial communities of transplants. Before replanting, the saplings were carefully removed from the
plastic bags to avoid damage to the root system; loose soil, i.e., not
adhering to the roots, was discarded. Four replicate samples were
made of (1) the roots of nursery plants before planting (Nur), (2)
roots of transplanted saplings 202 dap (Trn), (3) roots of native
(non-transplanted) saplings (Nat) and (4) bulk sediment in the
replant area (each consisting of four cores ,20 cm of top sediment
with 4 cm diameter) (Bul). The transplanted plants appeared
healthy and were approximately 50 cm in height. An effort was
made to retrieve native saplings in a similar condition and growth
stage to the transplanted saplings. Replicate samples of bulk
sediment, native and transplanted saplings were made haphaz-
ardly over an area of 10 m2 and care was taken that replicates The selected pyrosequencing reads were aligned online using
the INFERNAL aligner algorithm [34]. Aligned sequences were
assigned (97% identity) to OTU’s (phylotype clusters) using the
Complete Linkage Clustering application of the RDP pyrose-
quencing pipeline [35]. The complete linkage cluster file was then
converted into a square matrix containing the presence and
abundance of OTU’s per sample using a self-written function in R
(Supplementary Data S1). All 454 sequences generated in this
study can be downloaded from the NCBI Short Read Archive,
accession number: SRA023845. The OTU richness rarefaction curve of each sample was
computed using a self-written function in R (Supplementary Data
S2). Discussion The microbial pellet was obtained and total
community DNA extraction was performed using a BIO-101
DNA extraction kit (Q Biogene) and mechanical lysis [33]. from this study is the observation that rhizo-competent bacteria
are able to colonize mangrove roots while the plants are still in the
nursery and are able to survive in the mangrove rhizosphere for an
extended period of time after transplantation. This is the first study
to demonstrate such an effect and suggests that the initial
conditions in which saplings are raised can have a pronounced
and long-term effect on the root microbial community. This effect
may help to explain the often highly variable success rate of
reforestation projects since both plant growth promoters and plant
pathogens may be introduced into the mangrove rehabilitation
area. A more thorough understanding of how nursery conditions
affect the microbial communities of transplants may yield new
insights into the potential of this phenomenon for the restoration
of degraded mangrove forests. The recent development of
molecular techniques such as massive parallel pyrosequencing will
greatly contribute to this task. Our results also contribute to
elucidate the role of mangrove roots as a habitat for estuarine
sediment bacteria. A barcoded pyrosequencing approach was used for character-
ization of bacterial communities. The V4 hyper-variable region of
the bacterial 16S rRNA gene was PCR amplified for each sample
(,248 bp) using primers and tags described in the pyrosequencing
pipeline of the Ribosomal Database Project (RDP) (Release 10,
Update 20) (http://rdp.cme.msu.edu/). Pyrosequencing libraries
were obtained using the 454 Genome Sequencer FLX platform
(Roche Diagnostics Ltd, West Sussex, UK). Only sequences
containing exact matches to primer sequences and barcode tags
were used for further analyses. The primers were trimmed and
sequences with reads below 150 bp or with ambiguous bases were
discarded. The relative abundance of the most dominant bacterial
groups in each treatment and the representative sequences of the
most dominant OTU’s ($50 reads) were determined according to
the Naive Bayesian rRNA Classifier (Version 1.0) of the RDP
(Release 10, Update 20) with 50% as bootstrap cut-off. The results
of this bootstrap value are close to the ones with 80% cut-off [33]. Sequences classified as plant organelles or not classified into the
Bacteria
domain
were
removed. After
quality
control,
the
sequencing effort yielded 5940, 10443, 6828 and 7428 reads for
the treatments Nur, Trn, Nat and Bul, respectively. November 2010 | Volume 5 | Issue 11 | e14065 References 16. Sørensen J (1997) The rhizosphere as a habitat for soil microorganisms. In: van
Elsas JD, Trevors JT, Wellington EMH, eds. Modern soil microbiology. New
York: Marcel Dekker Inc. pp 21–45. 1. Aburto-Oropeza O, Ezcurra E, Danemann G, Valdez V, Murray J, et al. (2008)
Mangroves in the Gulf of California increase fishery yields. Proc Natl Acad Sci
USA 105: 10456–10459. 17. Pinhassi J, Sala MM, Havskum H, Peters F, Guadayol O, et al. (2004) Changes
in bacterioplankton composition under different phytoplankton regimens. Appl
Environ Microbiol 70: 6753–6766. 2. Kathiresan K, Rajendran N (2005) Coastal mangrove forests mitigated tsunami
Estuar. Coast Shelf Sci 65: 601–606. 3. Duke NC, Meynecke J-O, Dittmann S, Ellison AM, Anger A, et al. (2007) World
Without Mangroves? Science 317: 41–42. 18. Kersters K, De Vos P, Gillis M, Swings J, Vandamme P, et al. (2006)
Introduction to the Proteobacteria. In: Dworkin M, Falkow S, Rosenberg E,
Schleifer K-H, Stackebrandt E, eds. The Prokaryotes. Berlin: Springer. pp 3–37. 4. Primavera JH, Esteban JMA (2008) A review of mangrove rehabilitation in the
Philippines: successes, failures and future prospects. Wetl Ecol Manag 16: 173–
253. 19. Vogt KA, Grier CC, Vogt DJ (1986) Production, turnover and nutrient
dynamics of above- and belowground detritus of world forests. Adv Ecol Res 15:
303–377. 5. Gattenlo¨hner U, Lampert S, Wunderlich K Mangrove Rehabilitation
Guidebook Published in the framework of the EU-ASIA PRO ECO II B Post
Tsunami Project in Sri Lanka (Global Nature Fund). Available: http://www. mangroverestoration.com/html/downloads.html. 20. Robertson AI, Phillips MJ (1995) Mangroves as filters of shrimp pond effluent:
predictions and biogeochemical research needs. Hydrobiologia 295: 311–321. 6. Elster C (2000) Reasons for reforestation success and failure with three
mangrove species in Colombia. Forest Ecol Manage 131: 201–214. 21. Lyimo TJ, Pol A, Harhangi HR, Jetten MSM, Op den Camp HJM (2009)
Anaerobic oxidation of dimethylsulfide and methanethiol in mangrove
sediments is dominated by sulfate-reducing bacteria. FEMS Microbiol Ecol
70: 151–160. 7. Toledo G, Rojas A, Bashan Y (2001) Monitoring of black mangroves restoration
with nursery-reared seedlings on arid coastal lagoon. Hydrobiologia 444: 101–109. 22. Chang BV, Yuan SY (2007) Anaerobic degradation of five polycyclic aromatic
hydrocarbons from river sediment in Taiwan. J Environ Sci Health Part B Pestic
Food Contam Agric Wastes 42: 63–69. 8. Acknowledgments We thank Jose´ Luiz de Castro Ferreira for his valuable support for raising
the mangrove saplings and his continuous efforts to help preserving
mangroves in Rio de Janeiro (Brazil). We would like to thank Shell Brazil
for infrastructure support to raise the mangrove saplings. Materials and Methods For an
explanation of the ecological mechanisms behind the models see
Wilson [37] although it should be noted that a good model fit does
not necessarily imply a given mechanism. Variation in composition
among treatments was assessed with Principal coordinates analysis
(PCO), using the cmdscale() function in the R base package and
wascores() function in vegan. Prior to the PCO, the raw data was
log10 (x+1) transformed and used to produce a distance matrix based
on the Hellinger distance with the decostand() function in vegan and
dist() base R function. Variation in OTU composition among
treatments was tested for significance using the adonis() function in
vegan. The adonis() function is an analysis of variance with distance
matrices using permutations that partitions distance matrices among
sources of variation; in this case treatments. In the adonis() analysis,
the Hellinger distance matrix of OTU composition was the response
variable with treatment as independent variable. The number of
permutations was set at 999; all other arguments used the default
values set in the function. Variation in the relative abundance of
dominant higher taxa was tested for significance with an analysis of
deviance using the glm() function in R. Because the data was
proportional, we first applied a glm with the family= argument set
as binomial. The ratio, however, of residual deviance to residual d.f. in the models substantially exceeded 1 so we set family = to
‘quasibinomial’. In the ‘quasibinomial’ family the dispersion
parameter is not fixed at one so that it can model over-dispersion. Author Contributions Conceived and designed the experiments: NCG LCMH KS. Performed
the experiments: NCG. Analyzed the data: NCG DFC CE. Contributed
reagents/materials/analysis tools: FNP CE AA AC LCMH KS. Wrote the
paper: NCG DFC FNP AA AC LCMH KS. Wrote the R functions:
DFRC. Variation in the distribution of the most dominant taxa ($50
reads) among treatments was assessed using ternary diagrams
representing the percent abundance of dominant bacterial OTU’s Variation in the distribution of the most dominant taxa ($50
reads) among treatments was assessed using ternary diagrams
representing the percent abundance of dominant bacterial OTU’s Supporting Information Data S1
R self-written function for conversion of complete linkage
cluster files (RDP pyrosequencing pipeline) into a square matrix
containing the presence and abundance of OTU’s per sample. g
p
p
p
Found
at:
doi:10.1371/journal.pone.0014065.s001
(0.02
MB
PDF) g
p
p
p
Found
at:
doi:10.1371/journal.pone.0014065.s001
(0.02
MB
PDF) Data S2
R self-written function for construction of OTU
richness rarefaction curves. Found
at:
doi:10.1371/journal.pone.0014065.s002
(0.02
MB
PDF) Materials and Methods Dominance-diversity plots were generated based on the
logarithmic species abundance against the rank order of species for
each sample using the radfit() function in the vegan package [36]. Best fit lines representing the Zipf and Zipf-Madelbrot models were
automatically generated. The Zipf model is a generalized linear November 2010 | Volume 5 | Issue 11 | e14065 PLoS ONE | www.plosone.org 8 Mangrove Rhizosphere Bacteria model (‘glm’) with logarithmic link function whereas the Zipf-
Mandelbrot adds one nonlinear parameter to the Zipf model. For an
explanation of the ecological mechanisms behind the models see
Wilson [37] although it should be noted that a good model fit does
not necessarily imply a given mechanism. Variation in composition
among treatments was assessed with Principal coordinates analysis
(PCO), using the cmdscale() function in the R base package and
wascores() function in vegan. Prior to the PCO, the raw data was
log10 (x+1) transformed and used to produce a distance matrix based
on the Hellinger distance with the decostand() function in vegan and
dist() base R function. Variation in OTU composition among
treatments was tested for significance using the adonis() function in
vegan. The adonis() function is an analysis of variance with distance
matrices using permutations that partitions distance matrices among
sources of variation; in this case treatments. In the adonis() analysis,
the Hellinger distance matrix of OTU composition was the response
variable with treatment as independent variable. The number of
permutations was set at 999; all other arguments used the default
values set in the function. Variation in the relative abundance of
dominant higher taxa was tested for significance with an analysis of
deviance using the glm() function in R. Because the data was
proportional, we first applied a glm with the family= argument set
as binomial. The ratio, however, of residual deviance to residual d.f. in the models substantially exceeded 1 so we set family = to
‘quasibinomial’. In the ‘quasibinomial’ family the dispersion
parameter is not fixed at one so that it can model over-dispersion. as determined by complete linkage cluster analysis of 16S rRNA
gene sequences. The ternary diagrams were obtained using the
ternaryplot() function of the vcd package in R. model (‘glm’) with logarithmic link function whereas the Zipf-
Mandelbrot adds one nonlinear parameter to the Zipf model. References Kirui BYK, Huxham M, Kairo J, Skov M (2008) Influence of species richness
and environmental context on early survival of replanted mangroves at Gazi
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of the epsilonproteobacterial chemolithoautotroph Sulfurimonas denitrificans. Appl Environ Microb 74: 1145–1156. 11. Harris J (2009) Soil microbial communities and restoration ecology: facilitators
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like bacteria. Chemosphere 76: 677–682. 12. Grayston SJ, Wang S, Campbell CD, Edwards AC (1998) Selective influence of
plant species on microbial diversity in the rhizosphere. Soil Biol Biochem 30:
369–378. p
27. Mckee KL (1993) Soil physicochemical patterns and mangrove species
distribution - reciprocal effects? J Ecol 81: 477–487. 13. Gomes NCM, Heuer H, Scho¨nfeld J, Costa R, Hagler-Mendonc¸a L, et al. (2001)
Bacterial diversity of the rhizosphere of maize (Zea mays) grown in tropical soil
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ROS-mediated plasmodesmal
regulation requires a network
of an Arabidopsis receptor-like
kinase, calmodulin-like proteins,
and callose synthases OPEN ACCESS
EDITED BY
Vinay Kumar,
Pune University, India
REVIEWED BY
Ezekiel Ahn,
United States Department of Agriculture
(USDA), United States
Siddhesh B. Ghag,
UM-DAE Centre for Excellence in Basic
Sciences, India
*CORRESPONDENCE
Rahul Mahadev Shelake
rahultnau@gmail.com
Jae-Yean Kim
kimjy@gnu.ac.kr
†PRESENT ADDRESS
Ritesh Kumar,
Department of Agronomy and Plant
Genetics, University of Minnesota,
St. Paul, MN, United States
Dhineshkumar Thiruppathi,
Donald Danforth Plant Science Center,
St Louis, MO, United States
SPECIALTY SECTION
This article was submitted to
Plant Abiotic Stress,
a section of the journal
Frontiers in Plant Science
RECEIVED 24 November 2022
ACCEPTED 29 December 2022 OPEN ACCESS
EDITED BY
Vinay Kumar,
Pune University, India
REVIEWED BY
Ezekiel Ahn,
United States Department of Agriculture
(USDA), United States
Siddhesh B. Ghag,
UM-DAE Centre for Excellence in Basic
Sciences, India
*CORRESPONDENCE
Rahul Mahadev Shelake
rahultnau@gmail.com
Jae-Yean Kim
kimjy@gnu.ac.kr
†PRESENT ADDRESS
Ritesh Kumar,
Department of Agronomy and Plant
Genetics, University of Minnesota,
St. Paul, MN, United States
Dhineshkumar Thiruppathi,
Donald Danforth Plant Science Center,
St Louis, MO, United States
SPECIALTY SECTION
This article was submitted to
Plant Abiotic Stress,
a section of the journal
Frontiers in Plant Science
RECEIVED 24 November 2022
ACCEPTED 29 December 2022
PUBLISHED 19 January 2023
CITATION
Vu MH, Hyun TK, Bahk S, Jo Y, Kumar R,
Thiruppathi D, Iswanto ABB, Chung WS,
Shelake RM and Kim J-Y (2023)
ROS-mediated plasmodesmal regulation
requires a network of an Arabidopsis
receptor-like kinase, calmodulin-like
proteins, and callose synthases. Front. Plant Sci. 13:1107224. doi: 10.3389/fpls.2022.1107224
COPYRIGHT
© 2023 Vu, Hyun, Bahk, Jo, Kumar,
Thiruppathi, Iswanto, Chung, Shelake and
Kim. This is an open-access article
distributed under the terms of the Creative
Commons Attribution License (CC BY). TYPE Original Research
PUBLISHED 19 January 2023
DOI 10.3389/fpls.2022.1107224 © 2023 Vu, Hyun, Bahk, Jo, Kumar,
Thiruppathi, Iswanto, Chung, Shelake and
Kim. This is an open-access article
distributed under the terms of the Creative
Commons Attribution License (CC BY). The
use, distribution or reproduction in other
forums is permitted, provided the original
author(s) and the copyright owner(s) are
credited and that the original publication in
this journal is cited, in accordance with
accepted academic practice. No use,
distribution or reproduction is permitted
which does not comply with these terms. callose, plasmodesmata, ROS perception, abiotic and biotic stress, receptor-like
kinase (RLK) ROS-mediated plasmodesmal
regulation requires a network
of an Arabidopsis receptor-like
kinase, calmodulin-like proteins,
and callose synthases The
use, distribution or reproduction in other Minh Huy Vu 1, Tae Kyung Hyun 2, Sungwha Bahk 1, Yeonhwa Jo 1,3,
Ritesh Kumar 1†, Dhineshkumar Thiruppathi 1†,
Arya Bagus Boedi Iswanto 1, Woo Sik Chung 1,4,
Rahul Mahadev Shelake
1* and Jae-Yean Kim
1,4,5* Minh Huy Vu 1, Tae Kyung Hyun 2, Sungwha Bahk 1, Yeonhwa Jo 1,3,
Ritesh Kumar 1†, Dhineshkumar Thiruppathi 1†,
Arya Bagus Boedi Iswanto 1, Woo Sik Chung 1,4,
Rahul Mahadev Shelake
1* and Jae-Yean Kim
1,4,5* 1Division of Applied Life Science (BK21 Four Program), Plant Molecular Biology and Biotechnology
Research Center, Gyeongsang National University, Jinju, Republic of Korea, 2Department of Industrial
Plant Science and Technology, College of Agricultural, Life and Environmental Sciences, Chungbuk
National University, Cheongju, Republic of Korea, 3College of Biotechnology and Bioengineering,
Sungkyunkwan University, Suwon, Republic of Korea, 4Division of Life Science, Gyeongsang National
University, Jinju, Republic of Korea, 5Research and Development Center, Nulla Bio Inc 501 Jinju-daero,
Jinju, Republic of Korea Plasmodesmata (PD) play a critical role in symplasmic communication,
coordinating plant activities related to growth & development, and
environmental stress responses. Most developmental and environmental stress
signals induce reactive oxygen species (ROS)-mediated signaling in the apoplast
that causes PD closure by callose deposition. Although the apoplastic ROS signals
are primarily perceived at the plasma membrane (PM) by receptor-like kinases
(RLKs), such components involved in PD regulation are not yet known. Here, we
show that an Arabidopsis NOVEL CYS-RICH RECEPTOR KINASE (NCRK), a PD-
localized protein, is required for plasmodesmal callose deposition in response to
ROS stress. We identified the involvement of NCRK in callose accumulation at PD
channels in either basal level or ROS-dependent manner. Loss-of-function mutant
(ncrk) of NCRK induces impaired callose accumulation at the PD under the ROS
stress resembling a phenotype of the PD-regulating GLUCAN SYNTHASE-LIKE 4
(gsl4) knock-out plant. The overexpression of transgenic NCRK can complement
the callose and the PD permeability phenotypes of ncrk mutants but not kinase-
inactive NCRK variants or Cys-mutant NCRK, in which Cys residues were mutated
in Cys-rich repeat ectodomain. Interestingly, NCRK mediates plasmodesmal
permeability in mechanical injury-mediated signaling pathways regulated by
GSL4. Furthermore, we show that NCRK interacts with calmodulin-like protein
41 (CML41) and GSL4 in response to ROS stress. Altogether, our data indicate that
NCRK functions as an upstream regulator of PD callose accumulation in response
to ROS-mediated stress signaling pathways. ROS-mediated plasmodesmal
regulation requires a network
of an Arabidopsis receptor-like
kinase, calmodulin-like proteins,
and callose synthases CITATION
Vu MH, Hyun TK, Bahk S, Jo Y, Kumar R,
Thiruppathi D, Iswanto ABB, Chung WS,
Shelake RM and Kim J-Y (2023)
ROS-mediated plasmodesmal regulation
requires a network of an Arabidopsis
receptor-like kinase, calmodulin-like
proteins, and callose synthases. Front. Plant Sci. 13:1107224. doi: 10.3389/fpls.2022.1107224 KEYWORDS 1 Introduction Over a decade, many PD-related receptor proteins have been
shown to regulate cell-to-cell movement via controlling the PD
aperture during stress and development, such as PD-LOCALIZED
PROTEIN 1 and 5 (PDLP1, PDLP5), QIĀN SHŎU KINASE (QSK1),
CYS-RICH RECEPTOR-LIKE KINASE 2 (CRK2), and several
members of PM-located Leucine-rich-repeat receptor-like kinases
(RLKs) family (Lee et al., 2011; Grison et al., 2019; Hunter et al.,
2019; Kimura et al., 2020; Fichman et al., 2021). Nevertheless, whether
PD localized-receptor proteins are activated by eH2O2 that may
participate in PD regulation by inducing callose deposition during
wounding stress are currently unknown. ROS, Ca2+, phosphorylation,
and electric signaling are rendered at the local wounding site to trigger
rapid plant response (Sager and Lee, 2014; Toyota et al., 2018; Vega-
Muñoz et al., 2020; Fichman et al., 2021; Shin et al., 2022). Previous
studies have shown that changes in cytosolic Ca2+ signals regulate
plasmodesmal flux and callose deposition following biotic and abiotic
stresses (Leba et al., 2012; Wu et al., 2020). Calmodulin and
Calmodulin-like (CML) proteins, Ca2+-dependent protein kinases
(CDPKs), and calcineurin-B-like proteins are the major families of
Ca2+-binding proteins in plants (Zeng et al., 2015; Yip Delormel and
Boudsocq, 2019). CML9 negatively controls callose deposition after
Pseudomonas syringae pv. tomato (Pto) DC3000 strain treatment in
Arabidopsis (Leba et al., 2012). The PD-localized CML41 positively
regulates the PD callose upon the flagellin epitope flg22 pathogen
perception (Xu et al., 2017). Overall, several PD-located receptor and
CML protein family members have been recently identified; but none
have been characterized for their interacting partner proteins in the
plasmodesmal regulation during the stress response. Biotic and abiotic stresses, such as pathogens, insects, drought,
waterlogging, and heat or cold, augment and intensify the risks to food
security and affect agricultural production (Lesk et al., 2016; Bailey-
Serres et al., 2019). The increased frequency and intensity of various
biotic and abiotic stresses highlight the need to understand the
mechanisms that help plants to resist such stresses (Zandalinas et al.,
2021; Shelake et al., 2022). Mechanical wounding caused by different
stresses activates plant defense pathways similar to those triggered by
insects and herbivores (León et al., 2001; Gatehouse, 2002; War et al.,
2012; Savatin et al., 2014). Also, wounded plant parts are prone to
nutrient loss and provide entry points to phytopathogens (Savatin et al.,
2014). 1 Introduction Reactive oxygen species (ROS) and calcium (Ca2+) waves play a
crucial role in integrating different stress response signaling networks
and activating the plant defense mechanisms. In addition to activating
acclimation mechanisms in the plant tissues exposed to stress, various
abiotic stresses, as well as mechanical injury, can trigger rapid systemic
responses at the whole plant level (Kaya et al., 2014; Cui and Lee, 2016;
Mangano et al., 2017; Toyota et al., 2018; Wu et al., 2018; Cheval et al.,
2020), resulting in the development of stress memory that protects the
plants from subsequent exposures to the same stress. Controlling the cell-to-cell movement via callose-mediated
plasmodesmal regulation is one of the prevalent modes of action
adopted by the plant immune system during stress (Iswanto et al.,
2021). Callose, a b-1,3-glucan, is synthesized and degraded
dynamically at the plasmodesmata (PD) neck, which modulates PD
pore size and regulates the diffusion of molecules and signals (Kumar
et al., 2015; Amsbury et al., 2018; Wu et al., 2018). ROS and callose were
proposed to be linked through the function of the GLUCAN
SYNTHASE-LIKE 4 (GSL4)/callose synthase 8 (CalS8) during wound
stress in Arabidopsis (Arabidopsis thaliana) (Cui and Lee, 2016). Hydrogen peroxide (H2O2) is a major ROS species involved in plant
signaling pathways due to its unique aspects, such as rapid movability
across the plasma membrane (PM), the relatively long half-life, oxidizes
proteins, and possess properties similar to water (Castro et al., 2021). H2O2 activates the signaling through a cascade of proteins and is
implicated in plant adaptation to developmental and stress responses
(Mittleretal.,2022).ExtracellularH2O2(eH2O2)attheapoplastissensed
by receptors and transduced to the cytosol. It then enables alterations in
different protein structures, kinases/phosphatase molecular switches,
localization, and protein-protein interactions (Castro et al., 2021). A
primary mechanism for H2O2 sensing is the oxidative modification of
Cysteine (Cys) residues, in which the availability of different oxidation
states results in a diverse range of post-translational modifications
(Waszczak et al., 2015). Recently, the apoplast ROS sensor named
HYDROGEN-PEROXIDE-INDUCED CALCIUM INCREASES 1
(HPCA1) perceives the H2O2 molecule by modifying the
conformation of the extracellular domain (ED), also called hydrogen
peroxide (HP) domain (Wu et al., 2020). The HP domain, which
contains four Cys residues, is activated by eH2O2, increasing the kinase
activity and resulting in induced Ca2+ influx into the cytosol (Wu et al.,
2020). COPYRIGHT © 2023 Vu, Hyun, Bahk, Jo, Kumar,
Thiruppathi, Iswanto, Chung, Shelake and
Kim. This is an open-access article
distributed under the terms of the Creative
Commons Attribution License (CC BY). The
use, distribution or reproduction in other
forums is permitted, provided the original
author(s) and the copyright owner(s) are
credited and that the original publication in
this journal is cited, in accordance with
accepted academic practice. No use,
distribution or reproduction is permitted
which does not comply with these terms. 01 Frontiers in Plant Science frontiersin.org frontiersin.org Vu et al. 10.3389/fpls.2022.1107224 10.3389/fpls.2022.1107224 1 Introduction However, HPCA1 is essential for local and systemic cell-to-cell
ROS signaling in the local response to bacterial and salinity stress
treatments, but not wounding, suggesting that other extracellular ROS
receptors exist at PD that mediate the ROS-induced wounding stress
response (Fichman et al., 2022). Here, we characterize an Arabidopsis NOVEL CYS-RICH
RECEPTOR KINASE (NCRK; AT2G28250), which acts as a key
ROS receptor and is involved in callose-mediated PD regulation in
response to external ROS treatment and wounding stress. Moreover,
we characterize that NCRK is necessary for basal callose deposition
and ROS-dependent PD closure, and these processes are mediated by
its interactions with CML41 and GSL4. In addition, our study reveals
that NCRK phosphorylation is essential for the ROS-mediated
downstream signaling pathways during this process. We further
characterize the function of the Cys-rich motif positioned on ED of
NCRK (NCRK-ED) in ROS perception in response to eH2O2
treatment. Our findings indicate that NCRK plays a crucial role in
the apoplast ROS signaling, linking the callose accumulation that
requires the plant to acclimate to biotic and abiotic stresses. Frontiers in Plant Science frontiersin.org 2.1 NCRK is a PD-localized RLK and controls
basal callose deposition at the PD 2.1 NCRK is a PD-localized RLK and controls
basal callose deposition at the PD Our previous study on rice PD-proteome analysis identified
several members of the RLK family resided in the PD region,
including an unpublished Os02g20400 (Jo, 2011; Jo et al., 2011). By
using bioinformatic analysis, we identified an Arabidopsis RLK
protein family member (AT2G28250, named NCRK) with the
highest identity compared with Os02g20400 (named OsNCRK)
(Figure 1A). This protein was formerly reported as an NCRK, a
member of the RLK family with an unknown function (Figure 1B) Frontiers in Plant Science 02 10.3389/fpls.2022.1107224 Vu et al. Vu et al. A
B
D
E
F
C
FIGURE 1
NCRK is a plasmodesmata-localized RLK that regulates basal PD permeability. (A) Phylogenetic tree of NCRK across species. (B) Conserved cysteine-rich
repeat domain homolog sequence across species. (C) OsNCRK (upper panels) and NCRK (middle and bottom panels) co-localize with PD markers during
transient expression, and NCRK (bottom panels) co-localizes with callose in the transgenic plant. (D) PD callose staining and its quantification in the leaf
epidermal cells of wild-type Col-0 and ncrk-1 (n≥12). Scale bar: 20 mm. (E) DANS assays on Col-0 and ncrk-1. The diameter of the fluorescent circle
shows the level of cell-to-cell permeability of symplasmic dye (n≥12). Scale bar: 200 mm. (F) Plant images and quantification of rosette leaf diameter,
Col-0 (n=31), ncrk-1 (n=31), gsl4 (n=34). Scale bar: 1 cm. The data are summarized in box plots in which the line within the box marks the median, the
box signifies the upper and lower quartiles. The whiskers go down to the smallest value and up to the largest. Data were analyzed by a Student’s t-test. ns, not significant. **P <0.01, ***P < 0.001. A B B C C D E E F NCRK is a plasmodesmata-localized RLK that regulates basal PD permeability. (A) Phylogenetic tree of NCRK across species. (B) Conserved cysteine-rich
repeat domain homolog sequence across species. (C) OsNCRK (upper panels) and NCRK (middle and bottom panels) co-localize with PD markers during
transient expression, and NCRK (bottom panels) co-localizes with callose in the transgenic plant. (D) PD callose staining and its quantification in the leaf
epidermal cells of wild-type Col-0 and ncrk-1 (n≥12). Scale bar: 20 mm. (E) DANS assays on Col-0 and ncrk-1. The diameter of the fluorescent circle
shows the level of cell-to-cell permeability of symplasmic dye (n≥12). Scale bar: 200 mm. Frontiers in Plant Science frontiersin.org 2.1 NCRK is a PD-localized RLK and controls
basal callose deposition at the PD Interestingly, the ncrk-1 mutant plant
displayed enhanced developmental growth compared with the wild-
type, similar to the gsl4 mutant plant, showing a reduced PD callose
level and increased PD permeability (Figure 1F). These data indicate
that NCRK is a plasmodesmal RLK protein family member, which
controls basal callose accumulation and PD permeability. accumulation, but the Cys-rich motif seems specifically involved in
ROS-induced callose regulation. The HPCA1 contains the Cys
residues, which could sense the eH2O2 in Arabidopsis (Wu et al.,
2020). To investigate whether the Cys residues of ED in NCRK could
respond to eH2O2, we pursued a domain-swapping approach
replacing the ED and transmembrane domain (TMD) of NCRK
with the ED and TMD of HPCA1 that yielded the chimeric H/N
(HPCA1/NCRK) protein. The predicted structure of the H/N
receptor was comparable with the native NCRK protein except for
the ED region (Figure 3A). However, the chimeric H/N protein was
localized at PM but not in the PD, as observed in the transient N. benthamiana assay (Figure 3B). Further, Arabidopsis transgenic plants in the ncrk-1 mutant
background with a chimeric H/N protein driven by the endogenous
NCRK promoter were generated, and eH2O2 treatment was applied
(Figure 3A). We found that the chimeric H/N protein could not
complement the callose in normal growth conditions but partially
rescued the impaired callose deposition phenotype in the case of
eH2O2 treatment (Figures 3C, D). These data suggest that NCRK-ED
may serve a similar function as the HPCA1-HP domain regarding the
H2O2-induced callose response. Overall, these results demonstrate the
role of NCRK in eH2O2 perception at PD, and the Cys-rich motif of
NCRK-ED might be involved in eH2O2-induced callose deposition. 2.3 NCRK requires a CML41-GSL4 complex
for ROS-induced callose regulation Changes in eH2O2 and intracellular H2O2 were found to be related
to an increase in Ca2+ and consequently activate several CML protein
family members (Magnan et al., 2008; Leba et al., 2012; Wu et al., 2017;
Xu et al., 2017). Also, the CML members are localized at the nucleus,
cytoplasm, and PM, and some are co-localized with NCRK
(Supplementary Figure 4). This subcellular co-localization might
suggest the interaction of NCRK and the other partners. We
hypothesized that NCRK might control ROS-induced callose
deposition by directly interacting with CMLs members or GSL
members and regulating its activity. To understand how NCRK
controls the callose deposition during ROS stress and to identify the
interaction partners of NCRK, we performed bimolecular fluorescent
complementation (BiFC), co-immunoprecipitation (Co-IP) assay, and
genetic experiments. As a positive control, RDK1-YFPn and ABI4-YFPc
resulted in strong reconstituted YFP signals at PM (Kumar et al., 2017). However, a fluorescence signal was not observed in the leaves co-
expressing NCRK-YFPn and ABI4-YFPc (Figure 4A). We detected a
YFP signal on the PM when the NCRK-YFPn was co-expressed with the
YFPc fused to CML19, CML20, CML41, GSL4, and GSL8. This data
indicates that NCRK showed strong or weak interactions with several
CML (CML19, CML20, CML41) and GSL (GSL4, GSL8) family
members. We performed Co-IP assays to confirm the in planta
interaction of NCRK with the CML and GSL family members. The
result showed that NCRK coimmunoprecipitates with CML41, GSL4,
and GSL8 but not with negative control (GFP) (Figure 4B). Consequently, BiFC and Co-IP observations revealed that NCRK
strongly interacts with CML41, GSL4, and GSL8 in vivo. To test if
the ROS can induce the interaction of NCRK and CML41, we
performed the BiFC assay under the eH2O2 treatment. After 2 hours 2.1 NCRK is a PD-localized RLK and controls
basal callose deposition at the PD (F) Plant images and quantification of rosette leaf diameter,
Col-0 (n=31), ncrk-1 (n=31), gsl4 (n=34). Scale bar: 1 cm. The data are summarized in box plots in which the line within the box marks the median, the
box signifies the upper and lower quartiles. The whiskers go down to the smallest value and up to the largest. Data were analyzed by a Student’s t-test. ns, not significant. **P <0.01, ***P < 0.001. (Molendijk et al., 2008). In Arabidopsis, NCRK could be
hyperphosphorylated and partially co-localized with the small
GTPase ARABIDOPSIS RAB HOMOLOG F2A (RapF2a) in
endosomes (Molendijk et al., 2008). The amino acid alignment
between RLK orthologs reveals that the extracellular receptor
domain encoded by NCRK protein contains two repeats of about
40 amino acids sharing a unique and highly conserved Cys-rich motif,
WXCXCX13–18CX3CXC across the species, suggesting their specific
function (Figure 1B). both proteins, followed by fluorescence imaging using confocal
microscopy. OsNCRK and Arabidopsis NCRK showed punctate
spots at the cell periphery; a typical pattern was observed resembling
PD localization. To confirm if the spots are co-localized with PD, we
co-expressed NCRK-GFP with PD markers such as Turnip vein-
clearing virus (TVCV) movement protein, callose staining with
Aniline Blue dye, PDLP1 and PDLP5 (Thomas et al., 2008; Jo et al.,
2011; Lee et al., 2011). NCRK-GFP fluorescence spots co-localized well
with the four PD markers (Figure 1C and Supplementary Figure 1A). To quantify the percentage of NCRK co-localization at the PD area, we
measured the PD index by calculating the ratio of mean fluorescent
intensity of the PD enrichment area and its PM neighbor area First, we examined the subcellular localization of OsNCRK and
NCRK in the transient Nicotiana benthamiana plant by transiently
expressing green fluorescent protein (GFP) fusion to the C-terminal of 03 frontiersin.org Vu et al. Vu et al. 10.3389/fpls.2022.1107224 10.3389/fpls.2022.1107224 (Supplementary Figure 1C). NCRK was highly localized in the PD area,
similarly with PDLP1 or PDLP5, but not with RECEPTOR DEAD
KINASE1 (RDK1) used as PM markers (Kumar et al., 2017)
(Supplementary Figure 1B). Analyses of transgenic seedlings
expressing a GUS gene driven by Arabidopsis NCRK promoter (a)
showed that the underlying gene (gus) is broadly expressed throughout
the whole seedling and more prominently in the root, hypocotyl, and
cotyledon. In cotyledon, relatively strong expression was observed in
vascular tissues but to less extent in mesophyll cells (Supplementary
Figure 2B). 2.1 NCRK is a PD-localized RLK and controls
basal callose deposition at the PD Analyses of pNCRK::NCRK-mCherry transgenic lines
showed that the fluorescence was co-localized or localized to the
vicinity of the aniline blue-stained callose (Figure 1D). To investigate
the potential function of NCRK in PD regulation, we obtained T-DNA
insertion lines (ncrk-1, ncrk-2) (Supplementary Figure 2A) and
performed the PD callose staining experiment. ncrk-1 and ncrk-2
mutants showed less basal callose accumulation (Figure 1D and
Supplementary Figure 3C). ncrk-1 mutant plants exhibited increased
PD permeability and decreased PD callose compared with wild-type
plants in the leave and hypocotyl system (Figure 1E and Supplementary
Figures 3A, B), suggesting the role of NCRK in callose deposition and
PD permeability regulation. The callose turnover of the plant showed
defective growth and development or was even lethal except for some
mutant genes such as gsl4. Interestingly, the ncrk-1 mutant plant
displayed enhanced developmental growth compared with the wild-
type, similar to the gsl4 mutant plant, showing a reduced PD callose
level and increased PD permeability (Figure 1F). These data indicate
that NCRK is a plasmodesmal RLK protein family member, which
controls basal callose accumulation and PD permeability. (Supplementary Figure 1C). NCRK was highly localized in the PD area,
similarly with PDLP1 or PDLP5, but not with RECEPTOR DEAD
KINASE1 (RDK1) used as PM markers (Kumar et al., 2017)
(Supplementary Figure 1B). Analyses of transgenic seedlings
expressing a GUS gene driven by Arabidopsis NCRK promoter (a)
showed that the underlying gene (gus) is broadly expressed throughout
the whole seedling and more prominently in the root, hypocotyl, and
cotyledon. In cotyledon, relatively strong expression was observed in
vascular tissues but to less extent in mesophyll cells (Supplementary
Figure 2B). Analyses of pNCRK::NCRK-mCherry transgenic lines
showed that the fluorescence was co-localized or localized to the
vicinity of the aniline blue-stained callose (Figure 1D). To investigate
the potential function of NCRK in PD regulation, we obtained T-DNA
insertion lines (ncrk-1, ncrk-2) (Supplementary Figure 2A) and
performed the PD callose staining experiment. ncrk-1 and ncrk-2
mutants showed less basal callose accumulation (Figure 1D and
Supplementary Figure 3C). ncrk-1 mutant plants exhibited increased
PD permeability and decreased PD callose compared with wild-type
plants in the leave and hypocotyl system (Figure 1E and Supplementary
Figures 3A, B), suggesting the role of NCRK in callose deposition and
PD permeability regulation. The callose turnover of the plant showed
defective growth and development or was even lethal except for some
mutant genes such as gsl4. Frontiers in Plant Science 2.2 NCRK is involved in ROS-induced
callose deposition It has been shown that the Cys residue in the ED of RLK could
perceive the eH2O2 and respond to the ROS stress (Hunter et al.,
2019; Wu et al., 2020). Therefore, we investigated whether NCRK
could be involved in ROS-dependent callose accumulation. As
described earlier, the spraying of 10 mM H2O2 on the leaf surface
of wild-type plants was reported to stimulate ROS-mediated callose
accumulation after 2 hours of treatment (Cui and Lee, 2016). Therefore, we focused on the role of NCRK in ROS-induced callose
accumulation by eH2O2 application. We first observed that callose
deposition and PD permeability of the ncrk-1 mutant plant was
impaired in 10 mM H2O2 treatment (Figures 2A, B). To confirm
that the basal callose and ROS-induced callose impaired phenotype of
ncrk-1 was indeed caused by the NCRK mutation, we showed that
NCRK could rescue the ncrk-1 phenotype and callose accumulation
was restored in the complementation lines upon eH2O2 treatment
(Figures 2E, 3A). NCRK contains a highly conserved and unique Cys-
rich repeat sequence in the ED (Figure 1B). To explore whether these
extracellular Cys residues are essential for eH2O2 sensing, we
performed ncrk-1 complementation using NCRK mutant, which
included Cys mutations (Figures 2C, D). NCRK mutants with two
(Cys30 and Cys32 to Ala; 2CA), three (Cys46, Cys50, and Cys52 to
Ala; 3CA), or all five Cys residues to Ala (5CA) could complement the
basal callose level of ncrk-1 but not ROS-dependent callose phenotype
(Figure 2F), suggesting that NCRK-ED might not affect basal callose 04 Frontiers in Plant Science frontiersin.org Vu et al. Vu et al. 10.3389/fpls.2022.1107224 A
B
D
E
F
C
FIGURE 2
NCRK is required for H2O2-induced callose deposition. (A) Images of basal and ROS-dependent callose deposition in Col-0 and ncrk-1, gsl4 and gsl8+/-
mutant treated with H2O2 and their quantification. Scale bar: 20 mm. (n≥12). (B) Images of basal and ROS-dependent PD permeability of Col-0 and ncrk-
1 mutant treated with H2O2 and their quantification. Scale bar: 200 mm, (n≥12). (C, D) Diagram of NCRK extracellular domain and its mutants. The Cys
residues are marked as magenta, the Ala residues are marked as red. The disulfide bonds of the Cys-rich motif are shown. Quantification of basal and
ROS-dependent callose deposition of ncrk-1 lines complemented with NCRK and NCRK mutants for Cys residues overexpressed using native NCRK
promoter summarized in panel (E, F), respectively. Scale bar: 20 mm. (n≥12). 2.2 NCRK is involved in ROS-induced
callose deposition Data were analyzed by Student’s t-test. ns, not significant. ***P < 0.001. A
B D
C C D D E F E F FIGURE 2
NCRK is required for H2O2-induced callose deposition. (A) Images of basal and ROS-dependent callose deposition in Col-0 and ncrk-1, gsl4 and gsl8+/-
mutant treated with H2O2 and their quantification. Scale bar: 20 mm. (n≥12). (B) Images of basal and ROS-dependent PD permeability of Col-0 and ncrk-
1 mutant treated with H2O2 and their quantification. Scale bar: 200 mm, (n≥12). (C, D) Diagram of NCRK extracellular domain and its mutants. The Cys
residues are marked as magenta, the Ala residues are marked as red. The disulfide bonds of the Cys-rich motif are shown. Quantification of basal and
ROS-dependent callose deposition of ncrk-1 lines complemented with NCRK and NCRK mutants for Cys residues overexpressed using native NCRK
promoter summarized in panel (E, F), respectively. Scale bar: 20 mm. (n≥12). Data were analyzed by Student’s t-test. ns, not significant. ***P < 0.001. pathway. Our data demonstrated that NCRK, CML41, and GSL4 are
responsible for ROS-induced callose deposition pathways. of post-spraying, the reconstituted YFP fluorescence resulting from
NCRK-YFPn and CML41-YFPc was detected in the membrane and
punctate spots. When treated with eH2O2, the accumulation of NCRK-
CML41 interaction signal intensity was increased in leaves but not in
RDK1-ABI4 (positive control) and NCRK-CML20 signal intensity
(Figure 4C). These results suggest that eH2O2 may activate NCRK,
stimulating the interaction between NCRK and CML41. Frontiers in Plant Science 2.4 NCRK phosphorylation is required for
ROS-induced callose accumulation NCRK shares similar conserved catalytic subdomains with CRK2
and HPCA1, which require kinase activity for their functions (Stone
and Walker, 1995; Kornev et al., 2006) (Supplementary Figure 5). To
explore whether NCRK (NCRKCD) cytoplasmic domain has kinase
activity or functions as a scaffold, we prepared recombinant proteins
purified from E. coli. We could detect the autophosphorylation and
phosphorylation of myelin basic protein (MBP) only with wild-type
NCRKCD but not with the kinase-inactive mutants NCRK(K238E)CD Since we observed the direct interaction of NCRK and GSL
members, GSL4 and GSL8, we analyzed the role of GSL members
in the ROS-dependent pathway of NCRK. To determine whether
GSL4 is working in the NCRK-related pathway, we generated the ncrk
gsl4 double mutant plant by crossing the ncrk-1 and gsl4 mutant. The
callose intensity of the double mutant was similar to the ncrk-1 and
gsl4 single mutants in normal growth and ROS-treated conditions
(Figure 4D). It suggests that GSL4 acts in the NCRK-dependent-ROS 05 frontiersin.org Vu et al. 10.3389/fpls.2022.1107224 Vu et al. A
B
D
C
FIGURE 3
The extracellular domain of NCRK is required for basal and ROS-dependent callose deposition. (A) Diagram of NCRK and HPCA1/NCRK fusion protein and
3D protein structure of NCRK protein and HPCA1/NCRK fusion protein. SP, signal peptide; TMD, transmembrane domain; CRM-RD, Cys-rich motif-receptor
domain; KD, kinase domain; HPD, hydrogen peroxide domain; LRR-RD, Leu-rich repeat-receptor domain. (B) Localization of NCRK and HPCA1-NCRK fusion
protein in N. benthamiana. (C) Images of PD callose deposition of Col-0, ncrk-1, and HPCA1-NCRK complementation line treated with H2O2 and
quantification of callose levels depicted in (D) (n≥12). Scale bar: 20 mm. Data were analyzed by Student’s t-test. ns, not significant. ***P < 0.001. A B B D D C C FIGURE 3
The extracellular domain of NCRK is required for basal and ROS-dependent callose deposition. (A) Diagram of NCRK and HPCA1/NCRK fusion protein and
3D protein structure of NCRK protein and HPCA1/NCRK fusion protein. SP, signal peptide; TMD, transmembrane domain; CRM-RD, Cys-rich motif-receptor
domain; KD, kinase domain; HPD, hydrogen peroxide domain; LRR-RD, Leu-rich repeat-receptor domain. (B) Localization of NCRK and HPCA1-NCRK fusion
protein in N. benthamiana. (C) Images of PD callose deposition of Col-0, ncrk-1, and HPCA1-NCRK complementation line treated with H2O2 and
quantification of callose levels depicted in (D) (n≥12). Scale bar: 20 mm. Data were analyzed by Student’s t-test. ns, not significant. ***P < 0.001. (ATP binding) and NCRK(D339L)CD (catalytic) (Figure 5A). 2.4 NCRK phosphorylation is required for
ROS-induced callose accumulation Since
NCRK physically interacts with CML41, we tested whether NCRK
phosphorylates the CML41 in vitro. The ROP4 was used as a positive
control, and NCRK could phosphorylate ROP4 and CML41. NCRK
also could phosphorylate CML20, but it reduces the activity of NCRK
(Figure 5B). The ncrk-1 transgenic plants with constructs of these two
kinase inactive variants driven by the endogenous NCRK promoter
were generated. The complementation lines showed partially rescued
basal callose levels in mock condition but significantly reduced callose
levels compared to wild-type plants under the ROS stress condition
(Figure 5C). These data revealed that the NCRK phosphorylates
CML20 and CML41 in vitro, and NCRK phosphorylation status
influences ROS-induced callose accumulation at PD. 2002). To determine the effect of MV application on callose
deposition, wild-type plants were air sprayed with different MV
concentrations (0.1, 1, and 5 mM). Wild-type plants sprayed with 5
mM of MV showed considerably higher callose deposition at 2 hours
post-treatment (Supplementary Figure 6). Similarly, a higher level of
callose accumulation was observed in the ncrk-1 mutant plants
sprayed with 5 mM of MV (Figure 5D). This data revealed that
NCRK is independent of MV-induced callose deposition. Next, we
tested whether NCRK is required for apoplastic ROS-induced callose
deposition. Since GSL4 is specifically responsible for apoplasmic
ROS-mediated PD regulation during wounding stress and NCRK
acts as a mediator of GSL4 in eH2O2 response, we quantified callose
level after mechanical wounding to wild-type and ncrk-1 mutant
plants. This experiment showed the ncrk-1 mutant is impaired to gain
of callose intensity within 2 hours post-wounding (Figure 5E). These
data suggest that NCRK plays a role in wounding-dependent
callose deposition. Frontiers in Plant Science frontiersin.org 2.5 NCRK is required for wounding-induced
callose deposition The previous study has shown that CML41 is a responsive
component of callose deposition-pathogen defense, and an flg22 (P. syringae) also triggers apoplastic ROS in plant immunity (Xu et al.,
2017). To examine the function of NCRK mediating ROS-induced
plant signaling network during pathogenic stress, we infiltrated wild-
type and ncrk-1 mutant plants with flg22 to assess the impact of the
altered callose regulation. After 24 hours post-infiltration, PD callose
of ncrk-1 mutant plants was slightly reduced compared to wild-type
plants (Figures 5F, G). Additionally, we focused on assessing the Since the ncrk-1 mutant showed the deficiency in the callose
deposition under the eH2O2 treatment, we tested whether ROS
production triggered by another abiotic and biotic stress in the
plant could affect the callose deposition in NCRK mutant
background. Previously, herbicide methyl viologen (MV) treatment
was shown to induce ROS production in chloroplast and
mitochondria (Cui et al., 2019). The chloroplast and peroxisomes
are the primary sources of intracellular ROS in plants (Noctor et al., Frontiers in Plant Science 06 frontiersin.org Vu et al. Vu et al. 10.3389/fpls.2022.1107224 A
B
D
E
C
FIGURE 4
NCRK interacts with CML and GSL members. (A) Interaction of NCRK with its partners in vivo by BiFC assay. Scale bar: 20 mm. (B) Interaction of NCRK
with its partners by Co-IP. (C, D) ROS-induced NCRK interaction investigated using BiFC assay and visualized by confocal microscopy. (E) Callose
deposition of Col-0, ncrk-1, gsl4 single mutant, and ncrk gsl4 double mutant. Data were analyzed by Student’s t-test. ns, not significant. ***P < 0.001. A
B B B E D C E D FIGURE 4
NCRK interacts with CML and GSL members. (A) Interaction of NCRK with its partners in vivo by BiFC assay. Scale bar: 20 mm. (B) Interaction of NCRK
with its partners by Co-IP. (C, D) ROS-induced NCRK interaction investigated using BiFC assay and visualized by confocal microscopy. (E) Callose
deposition of Col-0, ncrk-1, gsl4 single mutant, and ncrk gsl4 double mutant. Data were analyzed by Student’s t-test. ns, not significant. ***P < 0.001. ANd-See (DANS) assay (Figures 1D, E). Previous reports have shown
that basal PD permeability was controlled by GSL4 and GSL8 in
different callose-related pathways (Cui and Lee, 2016). The plant
phenotype and callose level of the ncrk-1 mutant were like gsl4
mutants (Figures 1F, 2A), indicating that NCRK and GSL4 have
similar functions with respect to callose regulation. 2.5 NCRK is required for wounding-induced
callose deposition GSL4 contributes
to the maintenance of basal PD permeability, which relies on PDLP5
function (Cui and Lee, 2016), whereas GSL8 physically interacts with
PDLP5 (Saatian et al., 2018) and controls basal PD callose but does
not need it for functioning in ROS-dependent PD permeability
regulation (Cui and Lee, 2016). Therefore, GSL4 and GSL8 may
interact directly or indirectly with other proteins to control basal PD
permeability (Iswanto et al., 2022). Here, we found that the NCRK
interacts with GSL4 and GSL8 (Figure 4B), suggesting basal PD
permeability could be mediated by NCRK interactions with GSL4
or GSL8 and may be linked with PDLP5 functioning. Thus, NCRK
could interact with PDLP5 to regulate callose synthase activity, an
exciting aspect to explore in future work. significance of NCRK activity in bacterial resistance by surface
inoculation with P. syringae. After three days post-infection (3 dpi),
ncrk-1 mutant plants showed increased bacterial growth compared to
wild-type plants, indicating that NCRK might be involved in an flg22-
induced PD closure pathway. Frontiers in Plant Science 3 Discussion In this study, we have shown that NCRK accumulates at the
plasmodesmal region and regulates the callose deposition and, thereby,
PD permeability. NCRK also contributes to maintaining ROS-induced
callose deposition upon eH2O2 stress. The expression of NCRK mutated
at five Cys residues with Ala could not complement ncrk-1 callose level
after ROS treatment, suggesting Cys residues of NCRK-ED are essential
for ROS-dependent callose accumulation. As the H/N chimeric protein
could rescue the ncrk-1 phenotype, the NCRK-ED might serve as an
eH2O2 receptor. Furthermore, NCRK has kinase activity, which is
required for ROS-induced callose deposition. We also demonstrated
that NCRKcouldphysicallyinteractwithGSL4 andCML41andmediate
ROS-dependent callose response. Previously, the yeast two-hybrid assay showed NCRK interaction
with ROP11 (Molendijk et al., 2008). ROP11 also interacts with
RBOHF and regulates ROS production (Yan et al., 2016), indicating
the possible involvement of NCRK in ROS-induced callose response. To test this hypothesis, we performed a callose staining assay together NCRK is the plasmodesmal RLK responsible for basal callose
deposition, as demonstrated in the callose staining assay and Drop- 07 frontiersin.org 10.3389/fpls.2022.1107224 Vu et al. Vu et al. A
B
D
E
F
G
H
C
FIGURE 5
NCRK kinase activity is required for ROS-induced callose accumulation. (A) The kinase activity of NCRKCD wild-type, NCRK(K238E)CD and NCRK
(D339L)CD mutant proteins was analyzed by autoradiography. (B) NCRK phosphorylates ROP4 and CML41. (C) Partial complementation of the ncrk-1
phenotype by expressing kinase-inactive variants NCRK. (D) Fluorescence intensity quantification of callose deposition of Col-0 and ncrk-1 mutant
following paraquat (MV) treatment. (E) Fluorescence intensity quantification of callose deposition in Col-0 and ncrk-1 mutant following wounding
treatment. (F) Callose deposition of Col-0 and ncrk-1 mutant following flg22 treatment and (G) their intensity quantification. Scale bar: 200 mm. (n≥12). (H) Evaluation of ncrk-1 mutant plant susceptibility to Pst DC3000 cor-; quantification of bacterial growth in Col-0 and ncrk-1 upon 0 and 3 d post‐
inoculation of Pst DC3000 cor- suspension. Data were analyzed by Student’s t-test. ns, not significant. *P < 0.05, **P < 0.01, ***P < 0.001. A
B C E
H D D F G G F H H FIGURE 5
NCRK kinase activity is required for ROS-induced callose accumulation. (A) The kinase activity of NCRKCD wild-type, NCRK(K238E)CD and NCRK
(D339L)CD mutant proteins was analyzed by autoradiography. (B) NCRK phosphorylates ROP4 and CML41. (C) Partial complementation of the ncrk-1
phenotype by expressing kinase-inactive variants NCRK. Frontiers in Plant Science 3 Discussion (D) Fluorescence intensity quantification of callose deposition of Col-0 and ncrk-1 mutant
following paraquat (MV) treatment. (E) Fluorescence intensity quantification of callose deposition in Col-0 and ncrk-1 mutant following wounding
treatment. (F) Callose deposition of Col-0 and ncrk-1 mutant following flg22 treatment and (G) their intensity quantification. Scale bar: 200 mm. (n≥12). (H) Evaluation of ncrk-1 mutant plant susceptibility to Pst DC3000 cor-; quantification of bacterial growth in Col-0 and ncrk-1 upon 0 and 3 d post‐
inoculation of Pst DC3000 cor- suspension. Data were analyzed by Student’s t-test. ns, not significant. *P < 0.05, **P < 0.01, ***P < 0.001. responses (Hunter et al., 2019; Kimura et al., 2020; Wu et al., 2020). It
is also suggested that the activity of a candidate H2O2 receptor is
based on their extracellular Cys-rich motifs located at PD to respond
rapidly and trigger the callose defense upon ROS stress (Cheval et al.,
2020; Castro et al., 2021). Our callose staining assay showed that the
5CA (but not 3CA or 2CA) mutant failed to retain the function of
callose accumulation in ROS response (Figure 2F). It is reported that
the conserved Cys motif of Cys-rich receptor-like kinases could form
the disulfide bridges and control the thiol redox regulation (Wrzaczek
et al., 2010; Idänheimo et al., 2014). The predicted NCRK protein
structure revealed the orientation of Cys residues of NCRK-ED could with eH2O2 spraying to examine the callose level in ncrk-1 mutant
leaves. Using this assay, we compared the plasmodesmal callose of ncrk-
1 with wild-type, gsl4, and gsl8 heterozygous mutants. The callose
deposition of ncrk-1 mutant plants was impaired in eH2O2 response
(Figure 2A). Moreover, the ncrk gls4 double mutant also showed an
impaired callose deposition pattern similar to the ncrk-1 or gls4 single
mutant (Figure 4E). In summary, NCRK and GSL4 function in the
basal and ROS-dependent callose deposition pathways. As discussed earlier, Cys-rich motifs have been proposed as a
candidate for ROS sensors responsible for most of the communication
between the apoplast and intracellular environment in various stress 08 frontiersin.org Vu et al. Vu et al. 10.3389/fpls.2022.1107224 these different GSL or CML family members may modify the protein
conformation and transduce cellular signals generated by various
stresses to mediate plant defense responses. form disulfide bonds (Figure 2C). 3 Discussion These disulfide bonds could not be
formed when the five Cys residues were mutated with Ala
(Figure 2D), indicating that Cys residues might be sites of
eH2O2 sensing. Furthermore, H/N chimeric complementation plant can respond
to eH2O2 and trigger callose deposition in the ncrk-1 mutant
background (Figure 3D). Altogether, our data suggests that the Cys-
rich motif of NCRK serves as an ROS sensor, likewise the HP domain
of HPCA1 but needs more investigations. Therefore, considering the
disulfide bonds are required for ROS-induced callose deposition, and
NCRK-ED function is similar to that of HPCA1-ED, it is tempting to
speculate that Cys residues of NCRK-ED might also be modified by
eH2O2. Thus, further characterization of reduced-oxidized states of
the Cys-rich motif will provide molecular insights into NCRK
functioning in the ROS sensing mechanism. 4.1 Plant material and growth condition Seedlings of Arabidopsis thaliana, ecotype Columbia-0 (Col-0)
were grown in soil or in petri dishes in half-strength Murashige and
Skoog (MS) salts (Duchefa Biochemie, P.O. Box 809 2003 RV
Haarlem, The Netherlands), 1.0% (w/v) sucrose (BioShop, Canada
Inc., 5480 Mainway, Burlington, Ontario L7L 6A4, Canada), and 0.6%
(w/v) agar (Duchefa Biochemie) in controlled environmental rooms
or plant growth chambers (20-22°C). The fluency rate of white light
was approximately 100 mmol m-2s-1. The photoperiods were 16 hours/
8 hours light/dark cycles. For seedling culture, seeds were sterilized
with 20% bleach for 5 min, washed with sterilized water, and then
sown on soil or half-strength MS media, placed at 4°C for 3 days in
the dark, and then transferred to growth rooms or chambers. The
hydroponic assay was performed in half-strength of MS media, and
plants were grown at the culture room condition (22°C under 16
hours/8 hours light/dark cycles). T-DNA insertion mutants were
obtained from the Arabidopsis Biological Resource Center (https://
abrc.osu.edu). The T-DNA insertion mutants of NCRK
(SALK_045757 and SALK_202953 designated as ncrk-1 and ncrk-2,
respectively) identified by T-DNA insertion-based PCR using T-DNA
left-border primer (LBb1.3) and two pairs of NCRK-specific primers
(LP1-RP1 and LP2-RP2), respectively. Previous studies showed that the CRK2 could translocate to PD,
phosphorylate GSL6 (Cals1), and subsequently promote callose
deposition (Hunter et al., 2019). No significant changes were
observed in callose accumulation between non-treated and NaCl-
treated CRK2 kinase-dead mutant lines. Therefore, CRK2 kinase
activity is essential for salt-induced callose response. Similarly, our
results about quantification of callose deposition upon eH2O2 stress
or mock conditions of kinase-inactive lines showed no significant
differences (Figure 5B). In addition, the basal callose level of kinase-
inactive plants was not fully recovered compared to wild-type under
normal growth conditions. These results suggest that NCRK kinase
activity is crucial in basal and ROS-dependent callose regulation. Notably, CRK2 directly interacts with the GSL6 and phosphorylates it
(Hunter et al., 2019). Hence, considering the interaction of NCRK
with GSL4 and GSL8, it will be interesting to test whether NCRK
might phosphorylate GSL4 or GSL8. Moreover, NCRK shares the
conserved Threonine (Thr) residues similar to the kinase domains of
CRK2 and HPCA1 that were previously confirmed as primary
phosphorylation sites (Supplement Figure 5). Thus, the role of Thr
residues of NCRK could be studied to know their functions in
phosphorylation-mediated regulation of GSL4 or GSL8 during the
H2O2 stress. Frontiers in Plant Science 4.5 NCRK-mCherry subcellular localization
analysis and BiFC assays For hypocotyl experiments, five-day-old, etiolated Arabidopsis
seedlings were first cut at the base of the hook using a razor blade
(Iswanto et al., 2020). A coverslip was placed between each hypocotyl
cut surface and the MS agar plate. For dye loading, individual agar
blocks containing 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium
salt (5 mg/ml) were placed on the cut hypocotyl surface. After a 5 min
loading period, seedlings were washed in water for 15 min, and then
fluorescent probe movements were evaluated by confocal microscopy. For leave experiments, intact 2.5 to 3.5-week-old Arabidopsis plants
were air sprayed once with H2O2, MV, or wounding. Following a
designated incubation time, the fifth and sixth entire leaves were
subjected to DANS assays. DANS dye-loading assay using 1 mM 5(6)-
carboxy-2’-7’-dichlorofluorescein diacetate for cell-to-cell movement
assay as described (Cui and Lee, 2016). Briefly, the dye was loaded as a
1 µl droplet on the central regions of the adaxial side of each half-leaf
blade, followed by confocal imaging of the abaxial side after 5 min
loading and rinsing with a distilled water. Plasmodesmal callose
staining was performed by staining the mature leaves or hypocotyl,
for 1-1.5 hours in the dark before confocal imaging, with 0.02%
aniline blue solution containing 0.6 M glycine-NaOH, pH 9.5, and
0.001% silwet L-77 (Han et al., 2014; Cui and Lee, 2016). Quantifying
aniline blue stained plasmodesmal callose levels was performed using
ImageJ as described before (Zavaliev and Epel, 2015) by automatically
extracting plasmodesmata-associated fluorescent spots along the cell For analysis of NCRK-mCherry, HPCA1/NCRK-mCherry in stable
transgenic Arabidopsis lines, seedlings grown in half-strength MS media
inPetridishesfor7daysweresubjectedtomCherryconfocalimagingwith
theFV1000orFV1000MPEconfocalmicroscope(Olympus,Japan).Data
represent more than 3 independent lines examined, which displayed
similarmCherrysubcellularlocalization.Toanalyzewild-typeNCRKand
HPCA1/NCRK protein subcellular location, the transient expression of
fluorescentfusionproteinsinN.benthamianaleaftissuewasperformedas
previously described (Iswanto et al., 2020). The full-length coding
sequences of NCRK, HPCA1/NCRK were amplified from Col-0 and
fused with p35S and mCherry into pAGM4723 vector by Golden Gate
cloning. For BiFC assays, LR recombination of appropriated coding
sequence regions (NCRK, CML9, CML19, CML20, CML41, GSL4, and
GSL8) in pDONR207 were performed with the split-YFP destination
vectors pDEST-GWVYNE/pDEST-VYNE®GW, which allow C-terminal
fusions with C-terminal YFP moieties (Gehl et al., 2009). Agrobacterium
tumefaciens strain GV3101 containing the binary plasmid was grown in
LB medium with the appropriate antibiotics at 28°C for 24 hours for the
primary culture and then subculture at 28°C for 12 hours. 4.4 Histochemical GUS activity analysis Histochemical GUS staining of the NCRK promoter-driven GUS
(pNCRK::GFP-GUS) transgenic lines was detected by infiltration with
5-bromo-4-chloro-3indolyl-b-D-glucuronic acid (X-Gluc), as
described in an earlier report (Kumar et al., 2017). Briefly, plant
materials were incubated at 37°C overnight in a GUS staining buffer
containing 2 mM X-Gluc, 2mM potassium ferricyanide, 2 mM
potassium ferrocyanide, 0.2% Triton X-100, and 50 mM potassium
phosphate buffer, pH 7.0 and followed by chlorophyll clearing in 75%
ethanol. GUS images were taken using a stereomicroscope. 4.2 Constructs and transgenic lines Golden Gate cloning system (Werner et al., 2012) was used to
generate pNCRK::NCRK-mCherry, pNCRK::NCRK(C30A/C32A)-
mCherry, pNCRK::NCRK(C46A/C50A/C52A)-mCherry, pNCRK::NCRK
(C30A/C32A/C46A/C50A/C52A)-mCherry, pNCRK::NCRKK238E-
mCherry, pNCRK::NCRKD339L-mCherry, pNCRK::HPCA1ED-NCRKCD-
mCherry. The full-length NCRK genomic region (2 kb upstream and 1
kb downstream of the NCRK coding region), HPCA1 ED region (2 kb),
NCRK promoter region (2 kb), and NCRK1 full-length genomic were
amplified by PCR from genomic DNA. These fragments were cloned into
the pAGM4723 expression vector (Addgene #48015). Transgenic
Arabidopsis lines were generated by Agrobacterium-mediated
transformation (Zhang et al., 2006), and homozygous transgenic T3
lines carrying a single insertion were used. The ncrk-1 mutant was
complemented by a genomic fragment and NCRK-mCherry driven by
the native NCRK promoter. The site-directed mutagenesis was used to
introduce Cys-to-Ala mutations into the pNCRK::NCRK-mCherry
plasmid. For the pNCRK::GFP-GUS, p35S::CML19-GFP, p35S::CML19-
GFP, p35S::CML20-GFP, was cloned by Gateway cloning. The NCRK
promoter fragment, CML9, CML19, CML20, and CML41 cDNA were
amplified from genomic DNA. These fragments were moved into
pDONR207 plasmid (Invitrogen, 5791 Van Allen Way PO Box 6482
Carlsbad, California 92008, USA) and then cloned into the Gateway
binary vector pKGWFS7, pMDC43, and pMDC83, respectively. CML41 is a PD-localized Ca2+-binding protein required for
plant defense response during pathogen attack (Xu et al., 2017). In
addition, the interaction strength between NCRK and CML41
intensified following ROS treatment (Figures 4C, D). Since
CML41 (Figures 4C, D) and GSL4 (Figure 4E) mediates ROS-
induced callose accumulation, which is dependent on NCRK
function, the combination of NCRK and CML41 might
incorporate with GSL4 to modulate the callose accumulation in
response to ROS stress. We found herbicide (MV)-induced callose
deposition was independent of NCRK functioning. On the other
hand, biotic stressor (flg22) treatment showed slightly reduced
callose deposition in NCRK mutant background compared with
wild-type Col-0 (Figures 5D, F–H). We also demonstrated that
GSL4 requires NCRK to respond to the mechanical injury
(wounding)-induced stress and subsequently callose-mediated PD
regulation (Figure 5E). Overall, comparative analysis of wild-type
and ncrk mutant plants indicated that NCRK may or may not be
directly involved in the stress response pathway, depending on the
stress type. Given that the Arabidopsis genome encodes 12 GSL and
50 CML family members, NCRK interaction with a combination of Frontiers in Plant Science 09 frontiersin.org Vu et al. Vu et al. 10.3389/fpls.2022.1107224 4.6 Protein extraction
and co-immunoprecipitation For Co-IP in N. benthamiana, Golden Gate was used to clone PDRLK1
full-length genomic to generate HA-tagged fusions. pDONR207-CML19,
CML20, CML41 and CML9, GSL8 and ROP4 were recombined into
pMDC43 and pMCD83, respectively, to generate GFP-tagged fusions by
Gateway cloning. To clone the p35S::GSL4-GFP construct, GFP with Nos
terminator was amplified with additionally flanked with restriction sites for
SmaI and SpeI. The amplicon was excised and cloned into the construct
pDEST-GWVYNE-GSL4viaSmaI/SpeI.Theseconstructswereelectroporated
into A. tumefaciens strain GV3101 (pMP90). Agrobacterium suspensions
carrying p35S::PDRLK1-HA, p35S::GFP-CML19, p35S::GFP-CML20, and
p35S::GFP-CML41, p35S::GSL8-GFP, pDEST::GSL4-GFP were infiltrated in
variouscombinationsintoN.benthamianaleaves(OD600 = 0.3).Onegramof
leaftissuewasharvestedat48-72hpi,andtotalproteinwasresuspendedin1 ml
IP buffer (100 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 10%
glycerol, 3 mM DTT, 1 mM Na2MoO4, 1.5 mM sodium orthovanadate
[Na3VO4], 2 mM sodium fluoride [NaF], 1 mM phenylmethylsulfonyl
fluoride [PMSF], 50 µM MG132, a complete protease inhibitor [Roche] and
0.5%IGEPAL).The sampleswereclarifiedbycentrifugation at13,000rpm,at
4°C for 10 min twice. The supernatant was incubated with 10 ml of protein A
agaroseincubatedwithanti-GFPantibodyfor2hoursat4°Cwith360°rotating
shaker. Beads were washed five times with IP buffer. 40 ml of 2× SDS sample
buffer was added to the beads, and the beads were heated at 70°C for 15 min
and subjected to further SDS-PAGE and immunoblotting analysis (Kadota
et al., 2016; Kumar et al., 2017). A phylogenetic tree was created by multiple sequence alignment
method using MEGA (v11.0.9) (Tamura et al., 2021), in which
bootstrap was performed with 1,000 replicates, and gaps were treated
as pairwise deletions. The neighbor-joining method performed a
bootstrap with 1,000 replicates for the phylogenetic trees. Multiple
sequence alignments of the full-length amino acid sequence of
Figure 1B (OsNCRK, GRMZM2G063897, Glyma.11G144800,
Glyma.12G077300, and AtNCRK) and Supplementary Figure S5
(CRK2, HPCA, and NCRK) were aligned by ClustalW (https://www. ebi.ac.uk/Tools/msa/clustalo) (Sievers et al., 2011). The protein
structure of NCRK, H/N fusion protein, and NCRK-ED were
predicted by AlphaFold2 using MMseq2 (Mirdita et al., 2022). The
3D structure was visualized by the PyMOL program. Conflict of interest J-YK is a founder and CEO of Nulla Bio Inc. The remaining authors declare that the research was conducted in
the absence of any commercial or financial relationships that could be
construed as a potential conflict of interest. 4.5 NCRK-mCherry subcellular localization
analysis and BiFC assays The cells were
centrifuged for 15 min at 3500 rpm, resuspended to a final OD600 of 0.8,
and infiltrated into tobacco leaves by a needle-less syringe. Leaf pieces
were viewed under a confocal microscope after 2-3 days. Frontiers in Plant Science 10 frontiersin.org Vu et al. 10.3389/fpls.2022.1107224 reaction was stopped by adding 6 mL of 4 × SDS sample buffer and
boiling for 5 min. Samples containing 1 mg of protein from each
autophosphorylation reaction were separated by SDS-PAGE, and the
gel was stained, dried, and autoradiographed (Kim et al., 2017). boundaries of epidermal leaf pavement cells and plotting fluorescence
intensity per unit area. Author contributions MHV andJ-YK conceived the idea and designedthe experiments.YJ
andMHVdesignedexperiments,performedexperiments,analyzeddata,
and wrote the manuscript. TKH, SB, RK, DT, and ABBI performed the
experiments. RMS contributed to the interpretation and draft of the
output. RMS and J-YK revised the manuscript. All authors read and
approved the final manuscript. Data availability statement The original contributions presented in the study are included in
the article/Supplementary Material, further inquiries can be directed
to the corresponding author/s. 4.11 Statistical analysis All chemical treatments were performed by spraying them on
intact and mature plants at various concentrations before DANS
assays or plasmodesmal callose quantification. Mechanical wounding
was done by snipping the tip of the fifth or sixth leaf with a pair of
sharp forceps (Cui and Lee, 2016). Infected plants were grown in the
plant chamber and subjected to DANS assays or plasmodesmal callose
quantification 48 hours after infection. For flg22 treatment, either
distilled water or distilled water containing 1 µM of flg22 was
infiltrated into rosette leaves. After 24 hours, the infiltrated leaves
were incubated in callose staining buffer (aniline blue) for 1-1.5 hours
in the dark (Xu et al., 2017). For bacterial growth curve assay, we
performed infections by surface inoculation with the less virulent,
coronatine-deficient strain DC3000 (cor-). Briefly, Pst cultured at 28°C
and resuspended in MgCl2 to final A600 nm between 0.02 and 0.04
were generously infiltrated onto leaf abaxial surfaces of 5-6-week‐old
plants. Plants were covered, and leaf discs were taken 3 dpi from three
leaves per plant, with three plants per genotype per independent trial. Bacterial growth was assessed by colony counting (Kadota et al., 2014). Column plots were created using GraphPad Prism version 9.0.0
for windows, GraphPad Software, San Diego, California, USA (www. graphpad.com). Student’s t-test was performed to test the statistical
significance of differences. Funding This work was supported by the National Research Foundation of
Korea (grants NRF 2022R1A2C3010331, 2021R1A5A8029490,
2020M3A9I4038352, and 2020R1A6A1A03044344). Publisher’s note Recombinant protein purification and kinase assays were conducted
as previously described (Harper and Speicher, 2011). NCRKCD (wild
type, (K238E)CD and (D339L)CD) and other substrates (CML20, CML41,
and ROP4) were amplified and ligated into the pGEX4T-1 vector
containing GST tag. Overexpression of those proteins in E. coli strain
BL21(DE3) and purification on glutathione sepharose 4B R10 (GE
Healthcare, USA) were conducted as described. For the kinase assay,
these GST-fusion cytoplasmic domain kinase proteins (3 mg), GST (1 mg;
negative control) MBP (0.5 mg; positive control) were incubated in kinase
reaction buffer (25 mM Tris-HCl, pH 7.5, 1 mM DTT, 20 mM MgCl2, 2
mM MnCl2, and 50 mM [g-32P] ATP) and initiated using 1 mCi [g-32P]
ATP. The final reaction volume was 20 ml. After 30 min at 30°C, the All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated organizations,
or those of the publisher, the editors and the reviewers. Any product
that may be evaluated in this article, or claim that may be made by its
manufacturer, is not guaranteed or endorsed by the publisher. 4.9 Confocal microscopy and image processing For detection of aniline blue-stained plasmodesmal callose,
confocal images were taken under a 100X UplanXApo objective
with oil, using 405-nm laser excitation with a 420 to 480 nm
emission filter. Free or tagged mCherry was imaged with 543 nm
laser excitation and a 587 to 683 nm bandpass emission filter. GFP
was imaged with 488 nm laser excitation and a 505 to 550 nm bass
pass emission filter. DANS and HPTS assay images were taken under
a 10X UplanXApo objective using 488 nm Argon laser excitation,
with a 505 to 550 nm band pass emission filter. All confocal images
were acquired and processed using Olympus FV1000MPE or FV3000
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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
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view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a
credit line to the data. SHORT REPORT Open Access Abstract Background This study aimed to compare the complication rates of epithelium-off corneal cross-linking (epi-off
CXL) performed in an office-based setting with those of epi-off CXL performed in an operating room. Methods A retrospective cohort study, comprising 501 consecutive epi-off CXL procedures, performed in a non-ster‑
ile procedure room without laminar flow ventilation at the ELZA Institute in Zurich, Switzerland, between November
2015 and October 2021, was conducted. Results No cases of postoperative infectious keratitis were observed, while sterile infiltrates occurred in 10 out of 501
(2.00%) patients, all of whom responded well to topical steroid therapy. Delayed epithelialization (> 7 days) occurred
in 14 out of 501 (2.79%) patients. No other adverse events were noted. Conclusions Office-based epi-off CXL does not appear to be associated with an increased risk of complications
when compared to operating room settings. Keywords Corneal cross-linking, Office-based, Keratoconus, Cornea, Slit lamp, Epithelium-off, Infectious keratitis,
Sterile infiltrates Background Corneal cross-linking (CXL) is a surgical procedure com-
monly performed to halt the progression of corneal ecta-
sias like keratoconus or postoperative ectasia [1]. CXL
requires the corneal stroma to be saturated with ribo-
flavin, which is then irradiated with ultraviolet (UV)-A
light. This reaction results in the photochemical activa-
tion of riboflavin and the generation of reactive oxy-
gen species (ROS), which covalently cross-link stromal
molecules (predominantly collagen, but also proteogly-
cans), which renders a stiffer, biomechanically stronger
cornea more resistant to ectasia progression. The CXL 4 Faculty of Medicine, University of Geneva, Geneva, Switzerland
5 Dept. of Ophthalmology, University of Wenzhou, Wenzhou, China
6 Departmental Ophthalmology Unit, Alta Val d’Elsa Hospital, AUSL
Tuscany South-East, Siena, Italy 7 Postgraduate Ophthalmology School, University of Siena, Siena, Italy
8 Siena Crosslinking Center, Monteriggioni, Siena, Italy Rates of infectious keratitis and other ocular
surface adverse events in corneal cross‑linking
for keratoconus and corneal ectasias performed
in an office‑based setting: a retrospective
cohort study Rates of infectious keratitis and other ocular
surface adverse events in corneal cross‑linking
for keratoconus and corneal ectasias performed
in an office‑based setting: a retrospective
cohort study Farhad Hafezi1,2,3,4,5* , Emilio A. Torres‑Netto2, Leonard Kollros2, Nan‑Ji Lu2, Nikki Hafezi2, Cosimo Mazzotta6,7,8,
M. Enes Aydemir1,2 and Mark Hillen2 © The Author(s) 2023, corrected publication 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0
International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you
give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To
view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a
credit line to the data. Hafezi et al. Eye and Vision (2023) 10:36 Page 2 of 6 registered with the local ethics committee, the Zurich
Kantonale Ethikkommission (ZKE), under the reference
REG-2021-01121. As the study was anonymized and
retrospectively examined outcomes data only, the ethics
committee waived the need for written informed consent
and ethical approval for the study. To clarify, all patients
had submitted written informed consent to undergo the
original surgical procedure. procedure typically involves epithelial cell debridement
of the central 7–9 mm of the cornea (known as “epi-off”
CXL), since riboflavin is a large molecule (376.36 g/mol)
and does not pass through the epithelium easily [1, 2]. Once UV irradiation is complete, epithelial cells repop-
ulate the corneal surface over the next few days; during
this period, patients require topical antimicrobial and
pain management. Despite the associated costs and administrative burden,
CXL is typically performed in an operating room, as the
sterile environment is considered to be a safe setting for
performing epithelial debridement, riboflavin instilla-
tion, and the irradiation of a cornea with a large epithelial
defect for a period of up to 30 min [3]. However, the ROS
generated by the UV-riboflavin photochemical reaction
also reduce the microbial load on the cornea to such an
extent that riboflavin/UV CXL can be used to treat cases
of bacterial, fungal, or mixed bacterial/fungal infections
of the cornea [4] in a procedure called photoactivated
chromophore for keratitis-CXL (PACK-CXL) [5]. CXL procedure
All
ti
t
i All patients in this study underwent epi-off CXL. Ten
minutes prior to the procedure, patients received topical
ocular anesthesia drops: oxybuprocaine (oxybuprocaine
hydrochloride, 4 mg/mL, Théa Pharma SA, Clermont-
Ferrand, France) followed by one drop of Tetracain (tet-
racaine 1%, Théa Pharma SA, Clermont-Ferrand, France)
every 3 min for a 9-min period. After being brought into
the procedure room and the surgical area covered with
sterile drapes, the periorbital region was thoroughly dis-
infected with sterile cotton wool buds soaked in octeni-
dine hydrochloride (Octenisept; Schülke & Mayr GmbH,
Norderstedt, Germany). An eyelid speculum was placed,
and sterile surgical gauze was secured with surgical tape
laterally to the temporal canthus to absorb riboflavin
solution run-off. All persons involved wore masks and
sterile gloves, and all surfaces encountering the patient
were sterile. Epithelial debridement was performed mechanically
either with a hockey blade, an Amoils brush, or with
40% ethanol applied with a sterile cotton swab in a cir-
cular tapping manner for around 30–35 s, replaced with
a freshly soaked swab and tapped for a further 30–35 s
to loosen the epithelium, then wiped away in a circular
motion, then rinsed with balanced salt solution (Table 1). This step was performed either with the patient lying
supine in a reclining chair (n = 488) or seated upright at
a slit lamp (n = 13). Riboflavin instillation was performed
every 2 min for 10 min on all patients lying supine in a
reclining chair. Over this nearly seven-year period, only
two riboflavin solutions were used (Table 1): Ricro-
lin + (Sooft, Montegiorgio, Italy) was used on the first 331
eyes, then Ribo-Ker (EMAGine AG, Zug, Switzerland;
n = 170). Both solutions share hypo-osmolarity and the
absence of carriers like dextran or hydroxypropyl methyl-
cellulose (HPMC). The riboflavin used was changed
because of availability issues during the COVID-19 pan-
demic. The UV irradiation protocol used (in terms of irra-
diation duration and intensity) was dependent on the age
of the patient, severity of the ectasia, corneal thickness,
and the establishment of newer cross-linking protocols. CXL procedure
All
ti
t
i Irradiation was performed by the cross-linking devices
described in Table 1, and the duration of irradiation The focus of this paper is narrow: it does not report
on the clinical results of CXL for keratoconus, but solely
retrospectively examines the adverse event rates of CXL
performed in a procedure room over a five-year period
in a single office-based non-sterile setting in order to
compare these rates with published adverse event rates of
CXL performed in an operating room setting. PACK-
CXL has been successfully used as an infectious keratitis
monotherapy, as well as in combination with antimicro-
bial pharmacotherapy [5–7]. Considering the cost and
administrative burdens of utilizing an operating room [3,
8], its main advantage, sterility, appears to be negated by
the antimicrobial effect of CXL/PACK-CXL. The fact that
comprehensive antimicrobial prophylaxis is applied after
UV irradiation is complete raises the possibility that CXL
could be performed in a non-sterile procedure room
without an additional infection risk compared to CXL
performed in an operating room [3, 6]. This mirrors the
transition of intravitreal anti-vascular endothelial growth
factor (VEGF) therapies [9–12] and even cataract surgery
[13], from the operating theater to doctors’ offices or pro-
cedure rooms. Surgical technique
Patientsh This retrospective cohort study involved the analysis of
individuals who underwent epi-off CXL procedures for
the treatment of corneal ectasia in an office-based, non-
sterile setting (a 16 m2 procedure room without laminar
flow ventilation or humidity control) at the ELZA Insti-
tute in Zurich, Switzerland, between November 2015 and
October 2021, as previously described [3]. The study was Page 3 of 6 Hafezi et al. Eye and Vision (2023) 10:36 Table 1 Baseline demographics and cross-linking procedure
parameters used
SD = standard deviation; LASIK = laser in situ keratomileusis; PRK =
photorefractive keratectomy; UV = ultraviolet; CXL = corneal collagen cross-
Parameter
Value
Gender
Female
152
Male
349
Age (years)
Mean (SD)
30.7 (12.4)
Minimum, maximum
5.1, 71.7
Operated eye (OD, OS)
255, 246
Preoperative pachymetry (µm)
Mean (SD)
460.9 (60.3)
Minimum, maximum
152, 596
Ectasia type (patients, n)
Keratoconus
440
Post-LASIK ectasia
28
Pellucid marginal degeneration
25
Post-radial keratotomy
4
Terrien marginal degeneration
3
Post-PRK ectasia
1
Epithelium removal method (eyes, n)
Amoils brush
18
Ethanol/cotton swab
150
Hockey knife
333
Epithelial removal location (eyes, n)
Reclining chair (supine)
488
Slit lamp (sat upright)
13
Riboflavin applied (eyes, n)
Ricrolin +
298
Ribo-Ker
203
Riboflavin saturation duration (eyes, n)
10 min
72
20 min
429
UV irradiation duration (mm:ss)
Mean (SD)
14:56 (07:25)
Minimum, maximum
4:38, 30:00
Mode
10:00
UV irradiation intensity (eyes, n)
3 mW/cm2
173
9 mW/cm2
319
18 mW/cm2
9
UV irradiation location (eyes, n)
Operating theater-microscope
453
Slit lamp
48
UV irradiation device (eyes, n)
C-Eye
88
CXL-365 Vario
413 ranged from 5 to 30 min, intensity ranged from 3 to 18
mW/cm2 and fluence ranged from 5.4 to 10 J/cm2. l
g
J
Corneal pachymetry was measured using the SP-1000
(Tomey, Nagoya, Japan) at the thinnest points imme-
diately after riboflavin application and at the end of UV
irradiation. After the procedure, the eye was thoroughly
irrigated with balanced salt solution (BSS), and topical
antibiotics Tobradex (0.1% tobramycin–0.3% dexametha-
sone, Novartis Pharma, Basel, Switzerland) and Vigamox
(moxifloxacin 0.5%; Alcon, Geneva, Switzerland) were
administered immediately afterward, and a bandage con-
tact lens (Air Optix Night&Day; Ciba Vision AG) was
used to cover the eye. Finally, the speculum was removed. The post-procedural antimicrobial and pain prophylaxis
regimen was as previously described [14]. Analysis of postoperative infections After the procedure, we assessed the following param-
eters: signs of postoperative microbial infection (within
the first 14 days), sterile infiltrates (within the first
14 days), and delayed epithelialization (> 7 days), which
were observed via slit lamp biomicroscopy. Results A total of 501 patients with corneal ectasia received CXL
in an office-based, non-sterile setting between Novem-
ber 2014 and October 2021, with the majority (440/501,
87.82%) having keratoconus as the indication for the pro-
cedure. The other indications were post-LASIK ectasia
(28/501, 5.59%), pellucid marginal degeneration (25/501,
4.99%), radial keratotomy (4/501, 0.80%), Terrien mar-
ginal degeneration (3/501, 0.60%), and post-PRK ecta-
sia (1/501, 0.20%). No cases of infectious keratitis were
observed. Peripheral sterile infiltrates occurred in ten
cases (10/501, 2.00%), all of which reacted well to topi-
cal steroids. Delayed epithelialization of more than seven
days occurred in 14/501 (2.79%) patients, with all corneas
showing full epithelialization after 12 days (Additional
file 1: Table S1). SD = standard deviation; LASIK = laser in situ keratomileusis; PRK =
photorefractive keratectomy; UV = ultraviolet; CXL = corneal collagen cross-
linking Discussion described four cases of infectious keratitis in 117
eyes (3.42%) undergoing epi-off Dresden protocol [23,
24]. basal stroma. This measure aims to protect the corneal
endothelium from damage, as established by the Dresden
protocol. Most patients received 9 mW/cm2 UV intensity for
10 min. However, for certain groups of patients (predom-
inantly pediatric) with particularly aggressive disease,
the classic Dresden protocol (3 mW/cm2 for 30 min) [26]
was applied for maximal corneal strengthening effect. The study being performed by a single surgeon, has the
benefit of consistency and removing any variables that
may be introduced by multiple surgeons, but may also
limit the generalizability of the results. Finally, some pro-
cedures were performed with the patient sitting upright
at the slit lamp to receive the UV irradiation, whereas
other patients were irradiated lying supine. However, it
has been shown that the position in which the patient
receives UV irradiation does not materially influence the
riboflavin distribution or depth of cross-linking effect [3,
27, 28]. It is worth comparing the adverse event rates of CXL
with other ophthalmological procedures that were pre-
viously always performed in an operating room setting
and are now increasingly being performed as office-based
procedures. These include intravitreal injections (IVIs)
of anti-VEGF drugs for the treatment of neovascular dis-
eases of the retina [9–12] or cataract surgery [13], with
the intention of making cost and resource savings [9–13]. Undoubtedly, injecting a substance into the vitreous cav-
ity or performing intraocular surgery has the potential for
serious infectious consequences. Nevertheless, published
data show that IVI or cataract surgery performed in an
office-based or examination room setting does not result
in increased endophthalmitis rates [9–13]. For example,
one meta-analysis of 1,275,815 IVIs found no differ-
ence in endophthalmitis rates between those performed
in an office or an operating room setting [9]. Ianchulev
et al. reported the results of a large single-center retro-
spective study of office-based cataract surgery (13,507
patients; 21,501 eyes), finding that “office-based efficacy
outcomes were consistently excellent, with a safety pro-
file expected of minimally invasive cataract procedures
performed in ambulatory surgical centers and hospital
outpatient departments” [13]. The safety of intraocular
procedures and surgeries conducted in an office-based
setting has been shown to be comparable to that of pro-
cedures performed in an operating room. Discussion In this study of epi-off CXL performed in an office-
based, non-sterile procedure room setting, adverse
events were rare, with observed rates ranging from 0%
to 2.79%. These rates are comparable to epi-off CXL
complication rates reported in the literature. For exam-
ple, in 2009, Koller et al. described a case series of 117
eyes with corneal ectasia that underwent Dresden pro-
tocol CXL (30 min of 3 mW/cm2 UV irradiation at 3
mW/cm2 intensity) [15]. Sterile infiltrates occurred in C-Eye CXL-365 Vario Hafezi et al. Eye and Vision (2023) 10:36 Page 4 of 6 9/117 (7.69%) of eyes and stromal scarring in three eyes
(2.56%); no cases of infectious keratitis were observed. There have been several individual case reports describ-
ing infectious keratitis following epi-off CXL [16–20],
but there are few data on larger patient groups, with the
exception of Shetty et al. [21], who observed four cases
of infectious keratitis amongst 2350 patients (0.17%). Serraro et al. reviewed the adverse event rates of epi-
on and epi-off CXL procedures of 27 publications that
comprised a total of 9397 eyes, 9006 of which were
epi-off procedures [22]. In terms of epi-off procedures,
infectious, bacteria, viral and herpetic keratitis rates
were 2.26% (45/1990), 0.12% (2/1659), 0.62% (1/161)
and 0.18% (4/2182), respectively. Corneal infiltrate
rates were 2.0% (55/2776), and scarring occurred in
1.59% (49/3089). Reports by Dhawan et al. and Koppen
et al. described four cases of infectious keratitis in 117
eyes (3.42%) undergoing epi-off Dresden protocol [23,
24]. 9/117 (7.69%) of eyes and stromal scarring in three eyes
(2.56%); no cases of infectious keratitis were observed. There have been several individual case reports describ-
ing infectious keratitis following epi-off CXL [16–20],
but there are few data on larger patient groups, with the
exception of Shetty et al. [21], who observed four cases
of infectious keratitis amongst 2350 patients (0.17%). Serraro et al. reviewed the adverse event rates of epi-
on and epi-off CXL procedures of 27 publications that
comprised a total of 9397 eyes, 9006 of which were
epi-off procedures [22]. In terms of epi-off procedures,
infectious, bacteria, viral and herpetic keratitis rates
were 2.26% (45/1990), 0.12% (2/1659), 0.62% (1/161)
and 0.18% (4/2182), respectively. Corneal infiltrate
rates were 2.0% (55/2776), and scarring occurred in
1.59% (49/3089). Reports by Dhawan et al. and Koppen
et al. Ethics approval and consent to participate
ll
d
f
d
d All procedures were performed in accordance with relevant guidelines. The
ethics committee of Canton Zurich (Zurich Kantonale Ethikkommission,
BASEC-Nr. Req-2021-01121) waived the need for written informed consent
and ethical approval for the study. Availability of data and materials
l bl f
h Availability of data and materials Data are available from the corresponding author upon reasonable request. Supplementary Information these established facts, the findings from our study lend
further support to the concept of performing epi-off
CXL safely in a procedure room. For the purposes of this
discussion, a procedure room is defined as a room spe-
cifically designed and equipped for performing medical
procedures. It is characterized by a ventilation system
that ensures adequate airflow and minimizes the risk of
infection, and thus makes it an acceptable alternative to
an operating room. these established facts, the findings from our study lend
further support to the concept of performing epi-off
CXL safely in a procedure room. For the purposes of this
discussion, a procedure room is defined as a room spe-
cifically designed and equipped for performing medical
procedures. It is characterized by a ventilation system
that ensures adequate airflow and minimizes the risk of
infection, and thus makes it an acceptable alternative to
an operating room. Supplementary Information
The online version contains supplementary material available at https://doi.
org/10.1186/s40662-023-00354-1. Additional file 1: Table S1. Adverse events narrative. Received: 6 March 2023 Accepted: 16 July 2023
Published: 1 September 2023 Received: 6 March 2023 Accepted: 16 July 2023
Published: 1 September 2023 Received: 6 March 2023 Accepted: 16 July 2023
Published: 1 September 2023 Furthermore, as transepithelial procedures have
improved in their efficacy and are becoming more com-
monly performed [30], the more widespread adoption
of CXL that spares the corneal epithelium should fur-
ther reduce the risk of corneal infections during or after
CXL, providing further reassurance that office-based
CXL approaches can be performed as safely as CXL in an
operating room. Competing interests NH is CEO of EMAGine AG, a company producing a CXL device. FH holds a
patent on a UV light source (PCT/CH 2012/000090) and is CSO of EMAGine
AG. FH is an editorial board member of Eye and Vision. The other authors have
no proprietary or commercial interest in any of the materials discussed in this
article. Author contributions FH, ET, LK, and NL had full access to all the data in the study and take respon‑
sibility for the integrity of the data and the accuracy of the data analysis. Conception and design: FH, ET, LK. Data collection: all authors. Analysis and
interpretation: all authors. Drafting the manuscript: FH, MH, ET. Critical revi‑
sion of the manuscript: all authors. Supervision: FH. All authors reviewed and
approved the final version of the manuscript. Transitioning CXL from operating rooms to proce-
dural rooms should significantly reduce costs, enhancing
accessibility in low-to-middle income countries (LMICs)
where financial barriers limit care. This shift has broad
economic implications. Given the prevalence of vision
loss due to corneal ectasias, early CXL intervention is
crucial for vision preservation and prolonged economic
productivity. This cost reduction and increased access
could yield wider societal economic benefits, particularly
in LMICs that have higher levels of currently unmet clini-
cal need for CXL to treat corneal ectasias. Funding Funding
No funding was received for the study. Funding
No funding was received for the study. g
No funding was received for the study. Discussion In addition,
the UV-riboflavin photochemical reaction inherent in
CXL procedures is known to produce sufficient ROS to
reduce the microbial load significantly [4]. This reduc-
tion in microbial load is so substantial that CXL can be
successfully employed as a treatment for infectious kera-
titis [6], even as a stand-alone procedure [6, 29]. Given While it is recognized that environmental heat and
humidity can contribute to pathogen growth, and par-
tially explain regional differences in rates and types of
infectious keratitis, it is also reasonable to presume that
these environmental factors could also influence post-
procedural infection rates. However, given the strong
pathogen-killing effects of CXL, rendering the cornea
effectively “sterile” [5–7], the main drivers of post-proce-
dural infection risk are not the method, setting, or envi-
ronmental conditions that exist during the procedure. Rather, the drivers are in how carefully the cornea is han-
dled after CXL is complete, highlighting the importance
of patients carefully adhering to their post-procedural
topical antimicrobial drug regimen and not rubbing their
eyes [14].h This study has certain limitations. It is retrospective in
nature and compares adverse event rates with those pub-
lished in the literature, rather than having an operating
room control group. During the period under considera-
tion, the UV irradiation device and the riboflavin solu-
tion were changed. Even though the beam profiles were
similar and UV output intensities were matched, the
riboflavin solutions were similar in composition, both
being hypo-osmolar, HPMC, and dextran-free. Moreover,
different UV irradiation intensities and durations were
applied, reflecting the evolution of clinical practice in
CXL in Europe during this period. The study included both thin (330 to < 400 µm) and
ultra-thin (200 to < 330 µm) corneas treated with the
sub400 protocol [25]. This protocol adapts the UV flu-
ence delivered to patients’ individual thinnest-point
pachymetries to cross-link the cornea while maintaining
an approximately 70 µm uncross-linked safety margin of Hafezi et al. Eye and Vision (2023) 10:36 Page 5 of 6 Conclusionhi The findings from this retrospective analysis of 501 epi-
off CXL procedures indicate that there is no increase in
the risk of postoperative infectious keratitis when per-
forming epi-off CXL in a procedure room compared with
operating room-based procedures previously published
in the literature. This suggests that surgeons can be con-
fident that epi-off CXL can be safely performed outside
of the operating room setting. The fact that every CXL
procedure reduces the microbial load on the cornea due
to the UV-riboflavin photochemical reaction [5–7], and
has been shown to be effective enough to be used as a
monotherapy for the treatment of bacterial and fungal
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lagen crosslinking for the treatment of keratoconus. Am J Ophthalmol. 2003;135(5):620–7. •
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? Choose BMC and benefit from: 27. Salmon B, Richoz O, Tabibian D, Kling S, Wuarin R, Hafezi F. CXL at the slit
lamp: no clinically relevant changes in corneal riboflavin distribution dur‑
ing upright UV irradiation. J Refract Surg. 2017;33(4):281. 28. Hafezi F, Lu NJ, Assaf JF, Hafezi NL, Koppen C, Vinciguerra R, et al. Demar‑
cation line depth in epithelium-off corneal cross-linking performed at the
slit lamp. J Clin Med. 2022;11(19):5873. 29. Makdoumi K, Mortensen J, Sorkhabi O, Malmvall BE, Crafoord S. UVA-
riboflavin photochemical therapy of bacterial keratitis: a pilot study. Graefes Arch Clin Exp Ophthalmol. 2012;250(1):95–102. 30. Vinciguerra P, Rosetta P, Legrottaglie EF, Morenghi E, Mazzotta C, Kaye
SB, et al. Iontophoresis CXL with and without epithelial debridement
versus standard CXL: 2-year clinical results of a prospective clinical study. J Refract Surg. 2019;35(3):184–90.
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https://openalex.org/W4384827020
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https://www.frontiersin.org/articles/10.3389/fphar.2023.1206893/pdf
|
English
| null |
Multidisciplinary management in chronic myeloid leukemia improves cardiovascular risk measured by SCORE
|
Frontiers in pharmacology
| 2,023
|
cc-by
| 6,662
|
OPEN ACCESS OPEN ACCESS
EDITED BY
Olivier Feron,
Université catholique de Louvain,
Belgium
REVIEWED BY
Ibrahim C. Haznedaroglu,
Hacettepe University Hospital, Türkiye
Elisabetta Abruzzese,
University of Rome Tor Vergata, Italy
*CORRESPONDENCE
Carmen Díaz Pedroche,
cdiazp@salud.madrid.org
Rosa Ayala,
rayala@ucm.es
†These authors have contributed equally
to this work and share first authorship
‡These authors have contributed equally
to this work and share senior authorship
RECEIVED 16 April 2023
ACCEPTED 10 July 2023
PUBLISHED 19 July 2023
CITATION
Blanco Sánchez A, Gil Manso R,
Carreño-Tarragona G, Paredes Ruiz D,
González Olmedo J, Martínez-López J,
Díaz Pedroche C and Ayala R (2023),
Multidisciplinary management in chronic
myeloid leukemia improves
cardiovascular risk measured by SCORE. Front. Pharmacol. 14:1206893. doi: 10.3389/fphar.2023.1206893 OPEN ACCESS
EDITED BY
Olivier Feron,
Université catholique de Louvain,
Belgium
REVIEWED BY
Ibrahim C. Haznedaroglu,
Hacettepe University Hospital, Türkiye
Elisabetta Abruzzese,
University of Rome Tor Vergata, Italy
*CORRESPONDENCE
Carmen Díaz Pedroche,
cdiazp@salud.madrid.org
Rosa Ayala,
rayala@ucm.es
†These authors have contributed equally
to this work and share first authorship
‡These authors have contributed equally
to this work and share senior authorship
RECEIVED 16 April 2023
ACCEPTED 10 July 2023
PUBLISHED 19 July 2023
CITATION
Blanco Sánchez A, Gil Manso R,
Carreño-Tarragona G, Paredes Ruiz D,
González Olmedo J, Martínez-López J,
Díaz Pedroche C and Ayala R (2023),
Multidisciplinary management in chronic
myeloid leukemia improves
cardiovascular risk measured by SCORE. Front. Pharmacol. 14:1206893. EDITED BY
Olivier Feron,
Université catholique de Louvain,
Belgium REVIEWED BY
Ibrahim C. Haznedaroglu,
Hacettepe University Hospital, Türkiye
Elisabetta Abruzzese,
University of Rome Tor Vergata, Italy
*CORRESPONDENCE
Carmen Díaz Pedroche,
cdiazp@salud.madrid.org
Rosa Ayala,
rayala@ucm.es REVIEWED BY
Ibrahim C. Haznedaroglu,
Hacettepe University Hospital, Türkiye
Elisabetta Abruzzese,
University of Rome Tor Vergata, Italy Alberto Blanco Sánchez1†, Rodrigo Gil Manso1†,
Gonzalo Carreño-Tarragona1, Diana Paredes Ruiz2,
Jesús González Olmedo2, Joaquín Martínez-López1,
Carmen Díaz Pedroche2*‡ and Rosa Ayala1*‡ Alberto Blanco Sánchez1†, Rodrigo Gil Manso1†,
Gonzalo Carreño-Tarragona1, Diana Paredes Ruiz2,
Jesús González Olmedo2, Joaquín Martínez-López1,
Carmen Díaz Pedroche2*‡ and Rosa Ayala1*‡ 1Department of Hematology, Hospital Universitario 12 de Octubre, Madrid, Spain, 2Department of
Medicine, Hospital Universitario 12 de Octubre, Madrid, Spain †These authors have contributed equally
to this work and share first authorship ‡These authors have contributed equally
to this work and share senior authorship Introduction: Cardiovascular events are one of the main long-term complications
in patients with chronic myeloid leukemia (CML) receiving treatment with tyrosine
kinase inhibitors (TKIs). TYPE Original Research
PUBLISHED 19 July 2023
DOI 10.3389/fphar.2023.1206893 TYPE Original Research
PUBLISHED 19 July 2023
DOI 10.3389/fphar.2023.1206893 KEYWORDS chronic myeloid leukemia (CML), cardiovascular risk, tyrosine kinase inhibitor (TKI),
SCORE, multidisciplinary management, coronary disease, thromboembolic disease COPYRIGHT Results: At the time of diagnosis, 60% had some type of risk factor, 20% had a high
or very high risk SCORE, 40% had an intermediate risk, and 40% belonged to the
low risk category. During follow-up, the main cardiovascular adverse event
observed was hypertension (diagnosed in 8 patients, 23%). 66% of patients quit
smoking, achieving control of blood pressure in 95%, diabetes in 50%, weight in
76%, and dyslipidemia in 92%. 5.7% of patients suffered a thrombotic event and a
significant percentage of patients showed a reduction in their SCORE. © 2023 Blanco Sánchez, Gil Manso,
Carreño-Tarragona, Paredes Ruiz,
González Olmedo, Martínez-López, Díaz
Pedroche and Ayala. This is an open-
access article distributed under the terms
of the Creative Commons Attribution
License (CC BY). The use, distribution or
reproduction in other forums is
permitted, provided the original author(s)
and the copyright owner(s) are credited
and that the original publication in this
journal is cited, in accordance with
accepted academic practice. No use,
distribution or reproduction is permitted
which does not comply with these terms. Conclusion: Our study shows the benefit of controlling cardiovascular risk factors
through follow-up in a specialized consultation for patients with CML treated
with TKI. CITATION Blanco Sánchez A, Gil Manso R,
Carreño-Tarragona G, Paredes Ruiz D,
González Olmedo J, Martínez-López J,
Díaz Pedroche C and Ayala R (2023),
Multidisciplinary management in chronic
myeloid leukemia improves
cardiovascular risk measured by SCORE. Front. Pharmacol. 14:1206893. doi: 10.3389/fphar.2023.1206893 Methods: This study evaluated the cardiovascular risk of a cohort of patients with
CML at diagnosis and after follow-up in a specialized cardiovascular risk
consultation. In order to do this, we performed data analysis from 35 patients
who received TKIs and were referred to the aforementioned consultation between
2015 and 2018 at our center. Cardiovascular risk factors were analyzed separately,
as well as integrated into the cardiovascular SCORE, both at diagnosis and at the
last visit to the specialized consultation. frontiersin.org OPEN ACCESS The proper choice of TKI and the adequate management
of risk factors may reduce cardiovascular comorbidity in this population. RECEIVED 16 April 2023 ACCEPTED 10 July 2023 PUBLISHED 19 July 2023 chronic myeloid leukemia (CML), cardiovascular risk, tyrosine kinase inhibitor (TKI),
SCORE, multidisciplinary management, coronary disease, thromboembolic disease 1 Introduction The introduction of tyrosine kinase inhibitors (TKIs) in the treatment of chronic
myeloid leukemia (CML) marked a significant change in the management and prognosis
of this disease (Berman, 2022). This family of drugs allowed higher survival rates of these
patients to a level like that of the general population (Hochhaus et al., 2020). Moreover, TKIs Frontiers in Pharmacology 01 frontiersin.org 10.3389/fphar.2023.1206893 Blanco Sánchez et al. cardiovascular risk factors. Only symptomatic patients are referred
to other specialized consultation (cardiology or angiology). helped in achieving symptom control, total clearance of the tumor
clone, and significantly reducing the rate of acute transformation
(Cortes et al., 2021). To analyze the impact of this intervention, we have used the
SCORE (Systematic Coronary Risk Evaluation) model, which
estimates
the risk
of
death
from
cardiovascular
causes
in
10 years. It has the advantage of being adjusted to different
European countries, and estimates mortality associated with all
atherothrombotic manifestations and not just coronary mortality,
unlike the Framingham score. Moreover, SCORE is straightforward
to calculate because it includes few parameters: age, sex, systolic
blood pressure, total cholesterol and smoking (Visseren et al., 2021). However, TKI treatment poses new challenges in the management of
CML, like those associated with the numerous interactions of these drugs
and the adverse effects derived from their use (Haouala et al., 2011). Among the latter, the most frequent and concerning are cardiovascular
side effects (Douxfils et al., 2016) (Dahlén et al., 2016), which raise the
need for strict control of cardiovascular risk factors at the time of
diagnosis or those emerging over the follow-up (Barber et al., 2017). Currently, five TKIs with similar efficacy rates and a different
toxicity profile are approved for the treatment of CML (García-
Gutiérrez
&
Hernández-Boluda,
2019). Generally,
patients
experience some type of (mostly mild) adverse effect, that may
sometimes prompt a change in TKI (Cortes & Kantarjian, 2016). This model has already been used by other researchers to
evaluate the risk of developing cardiovascular events in patients
with CML treated with different TKIs, demonstrating its predictive
value at diagnosis (Breccia et al., 2015; Caocci et al., 2019). The mechanism by which TKIs cause cardiovascular damage is not
fully characterized, although it appears to be related to endothelial
damage through non-specific inhibition of tyrosine kinases (“off-target”
effect), alteration of glycemic metabolism, direct hypertensive effect or
glomerular impairment (Chaar et al., 2018). 2.1 Study design There are no studies comparing directly second-generation TKIs
(dasatinib, nilotinib, bosutinib), but the results of studies comparing
these with imatinib show a higher rate of cardiovascular events with
this generation of TKIs, so imatinib may be a preferable option in
patients with a high risk of cardiovascular disease (Cortes, 2020). This is a retrospective, single-center observational study that
analyzed a total of 35 patients diagnosed with CML at the 12 de
Octubre University Hospital, referred to the cardiovascular
disease consultation between 2015 and 2018, who received
treatment with one of the approved TKIs for this indication
(imatinib, dasatinib, nilotinib, bosutinib and ponatinib). The
patients
received
outpatient
follow-up
in
hematology
consultation and by an internal medicine specialist in the
aforementioned cardiovascular control consultation. Furthermore, no clear consensus exists on when to refer a
patient with CML from the hematology consultation to another
specialist for the evaluation and management of cardiovascular risk. Guidelines on this matter recommend doing so in the case of a
history of cardiovascular disease (Seguro et al., 2021), high risk of
cardiovascular disease (Zamorano et al., 2016) or presence of risk
factors when starting high risk TKI such as nilotinib (NCCN Clinical
Practice Guidelines in Oncology, 2023). There are no specific
recommendations to this effect from the European Leukemia Net. The diagnosis of CML was made following criteria established
by the latest classification of hematological neoplasms published by
the WHO (Swerdlow et al., 2017). The following prognostic scores
for CML were applied to the diagnosis: Sokal, Hasford, EUTOS and
ELTS. Regarding the criteria used to define cardiovascular risk
factors, they are explained below. However, at the time of diagnosis, patients diagnosed with CML
presented a high prevalence of cardiovascular risk factors, which
seems to be higher than that of the general population (Seguro et al.,
2021). One study showed, at the time of CML diagnosis, a prevalence
of 30% of hypertension, 11% of diabetes and 18% of dyslipemia
(Coutinho et al., 2017). Frontiers in Pharmacology 2.2 Cardiovascular variables Imatinib, n (%)
33 (94.3%)
Suspension
20 (60.6%)
Reasons
Lack of response
8 (40%)
Toxicity
7 (35%)
Clinical Trial
5 (25%)
Dasatinib, n (%)
16 (45.7%)
Suspension
8 (50%)
Reason
AE: 8 (100%)
Nilotinib, n (%)
15 (42.86%)
Suspension
9 (60%)
Reasons
Lack of response
1 (11.11%)
Toxicity
8 (88.89%)
Bosutinib, n (%)
3 (8.6%)
Suspension
1 (33.34%)
Reason: Toxicity
1 (100%)
Ponatinib, n (%)
1 (2.9%)
Suspension
1 (100%)
Reason: Clinical Trial
1 (100%)
TKI, total number used during follow-up, n (%)
1
15 (42.8%)
2
10 (28.6%)
3
9 (25.7%)
4
1 (2.9%) TABLE 1 Baseline characteristics of patients (n = 35). Gender
Male, n (%)
19 (54.3%)
Female, n (%)
16 (45.7%)
Median age at diagnosis (SD)
50.51 years (13.57)
Sokal Index
Low, n (%)
18 (51.4%)
Intermediate, n (%)
9 (25.7%)
High, n (%)
8 (22.9%)
Hasford Score
Low, n (%)
21 (60%)
Intermediate, n (%)
10 (28.6%)
High, n (%)
2 (5.7%)
Unknown, n (%)
2 (5.7%)
EUTOS Score
Low, n (%)
6 (17.1%)
High, n (%)
27 (77%)
Unknown, n (%)
2 (5.7%)
ELTS Score
Low, n (%)
21 (60%)
High, n (%)
12 (34.3%)
Unknown, n (%)
2 (5.7%) TABLE 1 Baseline characteristics of patients (n = 35). Control of diabetes was defined according to the targets specified by
these guidelines. Alcohol abuse: defined following criteria from DSM-V (APA,
2013). SCORE
(Systematic
Coronary
Risk
Evaluation):
defined
following ESC criteria (Visseren et al., 2021). the Sokal index, 60% according to the Hasford score, and 60%
according to the ELTS score. However, most patients belonged to the
high risk group according to the EUTOS Score (77.1%). 2.2 Cardiovascular variables Arterial
hypertension:
defined
as
systolic
blood
pressure ≥140 mmHg and/or diastolic blood pressure ≥90 mmHg,
following the criteria used by the ESC/ESH Guidelines for the
management of arterial hypertension (Williams et al., 2018). Arterial hypertension was considered to be controlled according
to the target for general and specific subgroups of hypertensive
patients, following the mentioned guidelines. Most of our knowledge about the efficacy and adverse effects of
TKIs comes from clinical trials. Nevertheless, their results could
underestimate the development of cardiovascular comorbidity,
considering the exclusion of patients with insufficient control of
cardiovascular risk factors, or the younger average age of patients
included in the main first-line trials with dasatinib (Kantarjian et al.,
2010) or nilotinib (Saglio et al., 2010). Therefore, real world evidence studies are essential, as they
are able to show the prevalence of complications arising from the
use of TKIs in a routine clinical practice scenario. One of the
largest studies to date (Coutinho et al., 2017), showed a
prevalence of almost 80% of cardiovascular risk factors at
5 years after the diagnosis of CML. Dyslipidemia: defined as hypertriglyceridemia (triglycerides
level
>200 mg/dL)
and/or
hypercholesterolemia
(cholesterol
level >200 mg/dL), following the criteria from the ESC/EAS
Guidelines for the management of dyslipidaemias (Mach et al.,
2020). Dyslipidemia was considered to be controlled following
the criteria defined by these guidelines. Diabetes mellitus: defined as an A1C ≥6.5%; fasting blood
glucose ≥126 mg/dL; blood glucose ≥200 mg/dL 2 hours after a
75 mg intake of glucose; or a casual blood glucose ≥200 mg/dL,
according to the ESC Guidelines on diabetes (Cosentino et al., 2020). In this study, we report our experience in the management of
cardiovascular risk factors at our center, where patients are referred
to a specialized internal medicine consultation at diagnosis or during
follow-up. The purpose of this strategy is to optimize the control of 02 frontiersin.org 10.3389/fphar.2023.1206893 Blanco Sánchez et al. TABLE 2 TKI (tyrosine kinase inhibitor) received for CML. 2.3 Statistics g
g
p
g
Regarding the prescribed TKI (Table 2), all except 2 patients
received imatinib (median time of exposition, 20.3 months), 45.7%
received dasatinib (median time of exposition, 24 months), 42.9%
received nilotinib (median time of exposition, 15.5 months),
3
patients
received
bosutinib
(median
time
of
exposition
4.1 months), and 1 received ponatinib (7.3 months of exposition). 60.6% of patients treated with imatinib had to stop it (in 40% of these
cases due to lack of optimal response, 35% as a result of adverse
effects, and 25% because of clinical trial protocol). Patients who
stopped dasatinib (57.7% of those who received this drug) did so for
reasons related to adverse effects. The discontinuation rate with
nilotinib was 60% (in one because of lack of efficacy and in the
remaining 88.9% as a consequence of toxicity). Out of the three
patients treated with bosutinib, one stopped it due to toxicity, and
the only patient treated with ponatinib stopped it because of clinical
trial protocol. Frequencies were calculated as percentages for qualitative
variables and as means and standard deviations for quantitative
variables. Comparison of variables was carried out using the
McNemar-Broker test. A p < 0.05 was considered statistically
significant. Statistical analysis was conducted using the SPSS
computer program version 25.0 (IBM, Chicago, IL). Frontiers in Pharmacology frontiersin.org 3 Results Table 1 summarizes the main characteristics of the 35 patients
included in the study. The mean age at the time of referral to the
cardiovascular
control
consultation
was
50
years
(standard
deviation, 13.5). 45.7% of patients were women. Over half of the
patients (51.4%) were classified in the low risk category according to 03 frontiersin.org 10.3389/fphar.2023.1206893 Blanco Sánchez et al. TABLE 3 Cardiovascular Risk Factors at first visit and last follow-up in Specialized Consultation. Months from diagnosis to referral, mean (SD)
53.80 (75.1)
Smoking, n (%)
At first visit
At last follow-up
Active
6 (17.14%)
2 (5.71%)
Previous
10 (28.57%)
14 (40.00%)
No smoking
19 (54.29%)
19 (54.29%)
Alcohol abuse, n (%)
4 (11.4%)
2 (5.7%)
Arterial Hypertension, n (%)
12 (34.3%)
20 (57.14%)
Improvement in blood pressure levels
20 (100%)
Control of hypertension
19 (95%)
Diabetes mellitus type 2, n (%)
3 (8.6%)
6 (17.2%)
Improvement in glucose levels
6 (100%)
Control of diabetes
3 (50%)
Dyslipidemia, n (%)
14 (40%)
24 (68.57%)
Improvement in lipid levels
24 (100%)
Control of dyslipidemia
22 (91.66%)
Cardiovascular Disease, n (%)
7 (20.00%)
11 (31.4%)
Coronary Disease
2 (5.7%)
3 (8.57%)
Cerebrovascular Disease
2 (5.7%)
2 (5.7%)
Peripheral Arterial Disease
3 (8.6%)
6 (17.2%)
COPD
1 (2.90%)
1 (2.9%)
Chronic kidney disease
2 (5.70%)
2 (5.7%)
SD: standard deviation; COPD: chronic obstructive pulmonary disease FIGURE 1
SCORE at the time of the first visit and at last follow up. FIGURE 1
SCORE at the time of the first visit and at last follow up. FIGURE 1
SCORE at the time of the first visit and at last follow up. FIGURE 1
SCORE at the time of the first visit and at last follow up. 04 04 Frontiers in Pharmacology frontiersin.org Blanco Sánchez et al. 10.3389/fphar.2023.1206893 Blanco Sánchez et al. 10.3389/fphar.2023.1206893 Table 3 shows the proportion of patients who had some type of
cardiovascular risk factor either at the time of referral or at the last visit
to the cardiovascular control consultation. The time elapsed between
diagnosis and consultation was approximately 54 months on average. At the time of the consultation 17.1% had an active tobacco habit and
28.6% had stopped smoking. 11.4% had alcohol consumption in the
range of abuse according to the previously stated criteria. 34.3% had
hypertension, 8.6% had DM, and 40% had dyslipidemia. 4 Discussion In this paper we present the results of cardiovascular control in
patients with CML under treatment with TKI in a specific
consultation. A reduction in cardiovascular risk factors was
achieved with at least a 20% improvement in cardiovascular score. The baseline characteristics of our cohort are similar to those
reported previously in patients with CML: an average age of 57 years
and a slight predominance in males (Dahlén et al., 2016). As for the
cardiovascular risk factors in our series, the data are comparable to
those reported by other authors. The study by Coutinho et al. (Coutinho et al., 2017) showed a rate of hypertension of
approximately 30%, like that of our population, and 11% of
diabetes (in our study 8.6%). The high proportion of patients
with dyslipidemia (40% compared to 18% in the aforementioned
study) in our cohort is striking, a difference that may be due to
heterogeneity of criteria used to define this condition. The most frequent cardiovascular disease in our cohort was PAOD
(6 patients developed PAOD after CML diagnosis, 3 of them before
referral to Internal Medicine Department and 3 of them afterwards). The
median age at the time of PAOD diagnosis was 63.5 years, with a median
time of 13.15 years from the introduction of TKI treatment. Regarding
the former 3 patients, 2 were receiving nilotinib and 1 dasatinib. Two of
them belonged to the intermediate risk and 1 to the very high risk
SCORE category. An improvement of SCORE was reached in 2 of them. The latter 3 cases with PAOD were diagnosed with a median of
2.5 years after the first consultation. All of them were receiving
imatinib (with a median time of exposition of 13.7 years). One of
them belonged to the high risk group and 2 to the intermediate risk
group. All of them remained in the same SCORE category, despite
the adequate control of cardiovascular risk factors. The presence of cardiovascular risk factors or comorbidities is
important, on the one hand, for the choice of TKI, given the different
toxicity profile of each one, and on the other hand, for the
management of such comorbidity (Latagliata et al., 2021). 4 Discussion Thus,
given that most of our patients received treatment with imatinib and
we have a small proportion of patients who received new generation
TKIs, it is difficult to make inferences about the relative risk for the
development of cardiovascular comorbidity regarding the TKI. The Figure 1 shows the distribution of patients according to the
cardiovascular SCORE at the time of the first consultation and the
last one. We have observed an increased number of patients
belonging to the low risk group at the expense of a decrease in
those assigned to the intermediate, high, and very high risk groups,
with a difference that reaches statistical significance. However, according to previous studies, it seems that nilotinib is
more associated with the development or worsening of arterial
hypertension (Roa-Chamorro et al., 2021), as well as coronary
disease together with dasatinib (Barber et al., 2017). Nilotinib is
especially associated with stroke (Chen et al., 2021), as well as
peripheral arterial disease together with dasatinib (Chen et al.,
2021). However, treatment with ponatinib has been the most
associated
with
hypertension
(17%
vs. 10%)
for
all
new-
generation TKI in a pooled analysis of hypertension incidence
(Mulas et al., 2021) and thrombotic risk (10% patients developed
cerebrovascular or vaso-occlusive disease) (Jain et al., 2015). The distribution of the patients among the different groups
before and after follow-up is low risk (21 versus 14 patients),
intermediate risk (10 versus 14) and high risk (3 versus 2). Only
one out of three patients remained in the very high risk category. The distribution of the patients among the different groups
before and after follow-up is low risk (21 versus 14 patients),
intermediate risk (10 versus 14) and high risk (3 versus 2). Only
one out of three patients remained in the very high risk category. Regarding arterial thrombosis, one patient receiving treatment
with dasatinib presented an episode of acute coronary syndrome. He
had a history of hypertension, dyslipidemia and coronary disease
prior to TKI initiation, belonging to the high risk SCORE category
when starting follow-up. Regarding arterial thrombosis, one patient receiving treatment
with dasatinib presented an episode of acute coronary syndrome. He
had a history of hypertension, dyslipidemia and coronary disease
prior to TKI initiation, belonging to the high risk SCORE category
when starting follow-up. 3 Results Seven patients
had already developed cardiovascular disease at the time of the
consultation (2 in the form of coronary disease, 2 stroke, and
3 peripheral arterial obstructive disease). One patient had a
diagnosis of chronic obstructive pulmonary disease COPD and
2 had chronic kidney disease. to the diagnosis of CML received imatinib without new thrombotic
events after the start of this drug. No patient treated with second
generation TKI or ponatinib developed venous thrombotic events. to the diagnosis of CML received imatinib without new thrombotic
events after the start of this drug. No patient treated with second
generation TKI or ponatinib developed venous thrombotic events. At the end of follow-up, 8 patients (22.9%) had been referred to
the vascular surgery and angiology consultation. Out of the 8 patients,
7 were referred because of intermittent claudication and 1 for
multidisciplinary assessment due to very high cardiovascular risk. These patients were evaluated with lower limb and carotid
doppler. Half of them showed carotid atherosclerosis, but only
one presented with significative stenosis (more than 50% of
arterial diameter reduction). Ten patients underwent lower limb doppler in order to rule out
significant arterial obstruction. Three patients showed findings of
arterial
obstruction
(those
diagnosed
with
PAOD),
four
atherosclerotic plaques and three did not reveal pathologic findings. During a mean follow-up of 31.25 months, 3 patients were
diagnosed with diabetes, 8 developed hypertension (13.3% of
patients with nilotinib, 12.5% with dasatinib and 12.1% with
imatinib), 10 dyslipidemia and 3 peripheral arterial obstructive
disease (PAOD). Strict control of hypertension was achieved in
all but one patient, control of dyslipidemia in all but 2 and only
3 patients did not reach adequate diabetes control. However, there
was an improvement of blood pressure, glucose level and lipids in all
patients. In 12 patients, it was necessary to change either the type or
dosage of TKI because of interactions with concomitant medication,
with statins being the main reason in 75% of these cases. Frontiers in Pharmacology frontiersin.org 4 Discussion With respect to the data on thromboembolic disease, only one
patient (receiving imatinib as TKI) presented a venous thrombotic event
in the form of deep vein thrombosis in the lower limb, arising in the
postoperative context of a major abdominal surgery. A patient with a
history of antiphospholipid syndrome and deep vein thrombosis prior For this reason, patient-based therapy has become increasingly
important in the treatment of CML (Ciftciler & Haznedaroglu, 2021). The availability of several TKIs has made it possible to choose the most
appropriate drug for each patient based on individual factors such as
age, comorbidities and availability in each center (Rabian et al., 2019). It 05 frontiersin.org Blanco Sánchez et al. Blanco Sánchez et al. 10.3389/fphar.2023.1206893 10.3389/fphar.2023.1206893 is important to consider factors such as the patient’s overall health
status, potential side effects, and the risk of developing resistance to the
TKI when selecting the best option. In summary, a personalized
approach to CML treatment can improve outcomes by maximizing
the benefits of therapy while minimizing side effects and reducing risk
of treatment resistance (Ciftciler & Haznedaroglu, 2021). As results have shown, a significant percentage of patients
achieved a change in their risk stratification according to the
SCORE, in all cases achieving a better prognosis category than
before follow-up, which was achieved thanks to the control of blood
pressure, dyslipidemia, or smoking cessation. Two studies have shown a correlation between the SCORE and the
occurrence of cardiovascular events in patients with CML and TKI
treatment (although both only included patients with ponatinib) (Breccia
et al., 2015; Caocci et al., 2019). Both showed a higher incidence of
cardiovascular events in the high and very high risk groups, with a
significant difference. In the study by Breccia et al., none of the patients
with a low risk SCORE developed cardiovascular disease. Given that most of our patients received treatment with imatinib in
first line and we have a small proportion of patients who received new
generation TKIs, it isdifficult to make inferencesabout the relative riskfor
the development of cardiovascular comorbidity according to the TKI in
our CML cohort. Nevertheless, with a median follow-up of 27.8 months,
none of the patients who received second generation TKI and who had
previous arterial hypertension showed a worsening of this condition (only
a patient with imatinib had a deficient control of hypertension during
follow-up). 4 Discussion 23% of patients were diagnosed of hypertension at some point
after TKI initiation. This percentage is slightly lower than that showed by
the large cohort of Jain et al. (2019). The only patient who received
ponatinib was under antihypertensive treatment before diagnosis of CML
and showed an adequate control of hypertension during TKI therapy. The importance of preventing cardiovascular disease lies in the fact
that it is the second leading cause of mortality in cancer patients (Sulpher
et al., 2015). For this reason, the importance of a multidisciplinary
management of patients with malignant hematological disorders is
increasingly been recognized, although we do not find in the
literature studies on multidisciplinary management of cardiovascular
risk in patients with CML, even when various groups have called
attention to this need (García-Gutiérrez et al., 2016; Basile et al., 2022). Our study shows that this approach to CML patients, in
coordination
with
specialists
is
feasible
and
results
in
an
improved
control
of
cardiovascular
risk
factors. The
main
limitation of our study is its retrospective nature and the limited
number of patients analyzed. In addition, there has been no
prolonged
follow-up
of
patients
that
could
demonstrate
a
reduction in cardiovascular events in patients with a better
prognosis SCORE. Among the strengths of the study, it includes
patients treated with different TKIs, and the use of a standardized
and population-targeted cardiovascular risk model. The importance of preventing cardiovascular disease lies in the fact
that it is the second leading cause of mortality in cancer patients (Sulpher
et al., 2015). For this reason, the importance of a multidisciplinary
management of patients with malignant hematological disorders is
increasingly been recognized, although we do not find in the
literature studies on multidisciplinary management of cardiovascular
risk in patients with CML, even when various groups have called
attention to this need (García-Gutiérrez et al., 2016; Basile et al., 2022). Although the associated thrombotic risk is assessed as a class effect of
TKI, the difference in targets of each of the different TKI may explain the
differences observed. The Swedish registry showed that patients with
CML have an overall risk of venous thromboembolic events and arterial
thromboembolic events 1.5 and 2 times higher than general population,
respectively (Dahlén et al., 2016). Moreover, second generation TKI and
ponatinib seem to confer greater risk than imatinib (Douxfils et al.,
2016). Data availability statement The raw data supporting the conclusion of this article will be
made available by the authors, without undue reservation. An appropriate approach to estimating the risk of developing
cardiovascular events are prognostic scores, such as the Framingham
score, the Pooled Cohort Equations score or the SCORE. Among them,
the SCORE model shows many advantages: there are many country-
specific versions derived from local data, it is easy to calculate, and it is
capable of predicting mortality derived from myocardial infarction,
stroke or heart failure over the next 10 years (Caocci et al., 2019). 5 Conclusion The adverse effects of tyrosine kinase inhibitors are one of the
main concerns when treating patients with chronic myeloid leukemia. These are usually related to their off-target effects and each TKI has a
different toxicity profile. Cardiovascular events are among their most
frequent and life-threatening complications, and their occurrence can
influence the choice or switch of TKI. The development of these
events can be prevented by controlling risk factors, which often
requires an interdisciplinary management. Our study shows that
follow-up in a specialized consultation is an attainable feasible
approach that can reduce cardiovascular risk of these patients. Another
important
aspect
to
consider
when
controlling
cardiovascular risk factors through pharmacological measures is the
potential interactions of the TKIs. In our cohort, this had a fundamental
impact on the use of statins, as previously seen (Haouala et al., 2011),
and for which rosuvastatin or pravastatin are usually recommended, as
they are not substrates of CYP3A4 (Osorio et al., 2018). Frontiers in Pharmacology 4 Discussion In our cohort, the rate of thromboembolic events was low, and
these only occurred in patients with strong risk factors. The comparison
with other studies is difficult due to difference of median follow-up (Jain
et al., 2019). However, these data suggest that follow-up in the specialized
consultation may have been effective in preventing thrombotic events. Our study shows that this approach to CML patients, in
coordination
with
specialists
is
feasible
and
results
in
an
improved
control
of
cardiovascular
risk
factors. The
main
limitation of our study is its retrospective nature and the limited
number of patients analyzed. In addition, there has been no
prolonged
follow-up
of
patients
that
could
demonstrate
a
reduction in cardiovascular events in patients with a better
prognosis SCORE. Among the strengths of the study, it includes
patients treated with different TKIs, and the use of a standardized
and population-targeted cardiovascular risk model. PAOD rate was surprisingly high in comparison to other
cardiovascular events. Other studies show a greater percentage of
coronary or cerebrovascular events, with an incidence lower than 1%
of PAOD among patients treated with imatinib (Chen et al., 2021). Half
of our patients were receiving imatinib at the time of PAOD diagnosis,
although nilotinib seems to be more associated with PAOD than other
TKI (Douxfils et al., 2016). Yet our patients had a long history of
exposition and many cardiovascular risk factors. Our high rate of PAOD
could be a consequence of the high suspicion degree maintained in the
specific consultation. There are many comorbidities causing lower limbs
pain and, unlike cerebrovascular or coronary disease, PAOD is often
mildly symptomatic and thus misdiagnosed (Nordanstig et al., 2023). For this reason, nearly one-third of patients underwent a Doppler study
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approved by Comité de Ética de la Investigación (CEI) Hospital
universitario 12 de Octubre. The patients/participants provided
their written informed consent to participate in this study. 06 frontiersin.org Blanco Sánchez et al. Blanco Sánchez et al. Blanco Sánchez et al. 10.3389/fphar.2023.1206893 Funding All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated organizations,
or those of the publisher, the editors and the reviewers. Any product
that may be evaluated in this article, or claim that may be made by its
manufacturer, is not guaranteed or endorsed by the publisher. This work was funded by the Instituto de Salud Carlos III (PI19/
01518 and PI22/1088) and the CRIS Foundation. Co-funded by the
European Regional Development Fund (ERDF). Conflict of interest CD, DP, JG designed research and collected data, RG and GC-T
interpreted the data and performed statistical analysis, AB and RG
wrote the manuscript, CD, RA, GC-T, and JM-L designed and
supervised the overall study. All authors contributed to the article
and approved the submitted version. The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be
construed as a potential conflict of interest. References Available at: https://www.nccn.org/professionals/physician_gls/pdf/cml.pdf (Accessed
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assessment in a population with chronic myeloid leukemia. Sci. Rep. 11 (1), 14637. doi:10.1038/s41598-021-94127-2 Visseren, F. L. J., Mach, F., Smulders, Y. M., Carballo, D., Koskinas, K. C., Bäck, M.,
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V. A. M., et al. (2021). Blanco Sánchez et al. References Recommendations for the management of cardiovascular risk in
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stratification, treatment and monitoring. Hematol. Transfus. Cell Ther. 43 (2), 191–200. doi:10.1016/j.htct.2020.04.009 Zamorano, J. L., Lancellotti, P., Rodriguez Muñoz, D., Aboyans, V., Asteggiano, R.,
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practice guidelines: The task force for cancer treatments and cardiovascular toxicity
of the European society of cardiology (ESC). Eur. Heart J. 37 (36), 2768–2801. doi:10. 1093/eurheartj/ehw211 Sulpher, J., Mathur, S., Graham, N., Crawley, F., Turek, M., Johnson, C., et al. (2015). Clinical experience of patients referred to a multidisciplinary cardiac 08 08 Frontiers in Pharmacology frontiersin.org
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Hydrological control of dissolved organic carbon dynamics in a rehabilitated &lt;i&gt;Sphagnum&lt;/i&gt;–dominated peatland: a water-table based modelling approach
| null | 2,017
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Hydrological control of dissolved organic carbon
dynamics in a rehabilitated Sphagnum-dominated
peatland: a water-table based modelling approach
Léonard Bernard-Jannin, Stéphane Binet, Sébastien Gogo, Fabien Leroy,
Christian Défarge, Nevila Jozja, Renata Zocatelli, Laurent Perdereau, Fatima
Laggoun-Défarge Léonard Bernard-Jannin, Stéphane Binet, Sébastien Gogo, Fabien Leroy,
Christian Défarge, Nevila Jozja, Renata Zocatelli, Laurent Perdereau, Fatima
Laggoun-Défarge To cite this version: Léonard Bernard-Jannin, Stéphane Binet, Sébastien Gogo, Fabien Leroy, Christian Défarge, et al.. Hydrological control of dissolved organic carbon dynamics in a rehabilitated Sphagnum-dominated
peatland: a water-table based modelling approach. Hydrology and Earth System Sciences, 2018, 22,
pp.4907-4920. 10.5194/hess-2017-578. insu-01778100 Hydrological control of dissolved organic carbon
dynamics in a rehabilitated Sphagnum-dominated
peatland: a water-table based modelling approach
Léonard Bernard-Jannin, Stéphane Binet, Sébastien Gogo, Fabien Leroy,
Christian Défarge, Nevila Jozja, Renata Zocatelli, Laurent Perdereau, Fatima
Laggoun-Défarge Hydrological control of dissolved organic carbon dynamics in a
rehabilitated Sphagnum–dominated peatland: a water-table based
modelling approach
d
d
i 1 2 3
h
i
1 2 3 4
b
i
1 2 3
bi
1 2 3
h i i Hydrological control of dissolved organic carbon dynamics in a
rehabilitated Sphagnum–dominated peatland: a water-table based
modelling approach
Léonard Bernard-Jannin1,2,3, Stéphane Binet1,2,3,4, Sébastien Gogo1,2,3, Fabien Leroy1,2,3, Christian
Défarge1,2,3,5, Nevila Jozja5, Renata Zocatelli5, Laurent Perdereau1,2,3, Fatima Laggoun-Défarge1,2,3
5
1Université d’Orléans, ISTO, UMR 7327, 45071, Orléans, France
2CNRS, ISTO, UMR 7327, 45071, Orléans, France
3BRGM, ISTO, UMR 7327, 45071, Orléans, France
4ECOLAB, Université de Toulouse, CNRS, UPS, INPT – UMR 5245, Toulouse, France
5CETRAHE, Université d’Orléans, 45072, Orléans, France
10 Léonard Bernard-Jannin , , , Stéphane Binet , , , , Sébastien Gogo , , , Fabien Leroy , , , Christia
Défarge1,2,3,5, Nevila Jozja5, Renata Zocatelli5, Laurent Perdereau1,2,3, Fatima Laggoun-Défarge1,2,3
5
1Université d’Orléans, ISTO, UMR 7327, 45071, Orléans, France
2CNRS, ISTO, UMR 7327, 45071, Orléans, France
3BRGM, ISTO, UMR 7327, 45071, Orléans, France
4ECOLAB, Université de Toulouse, CNRS, UPS, INPT – UMR 5245, Toulouse, France
5CETRAHE, Université d’Orléans, 45072, Orléans, France
10 Léonard Bernard-Jannin
, Stéphane Binet
, Sébastien Gogo
,
Défarge1,2,3,5, Nevila Jozja5, Renata Zocatelli5, Laurent Perdereau1,2,3, Fatim
5
1Université d’Orléans, ISTO, UMR 7327, 45071, Orléans, France
2CNRS, ISTO, UMR 7327, 45071, Orléans, France
3BRGM, ISTO, UMR 7327, 45071, Orléans, France
4ECOLAB, Université de Toulouse, CNRS, UPS, INPT – UMR 5245, Toulouse, France
5CETRAHE, Université d’Orléans, 45072, Orléans, France
10 Abstract Hydrological disturbances could increase dissolved organic carbon (DOC) exports through runoff and leaching, reducing the
potential carbon sink function of peatlands. The objective of this study was to assess the impact of hydrological restoration
15
on hydrological processes and DOC dynamics in a rehabilitated Sphagnum–dominated peatland. A conceptual hydrological
model calibrated on the water table and coupled with a biogeochemical module was applied to La Guette peatland (France),
which experienced a rewetting action on February 2014. The model (ten calibrated parameters) reproduced water table and
pore water DOC concentration time series (01/04/2014 to 15/07/2017) in two contrasted locations (rewetted and control) of the peatland. Hydrological restoration was found to impact the water balance through a decrease in slow deep drainage and
20
an increase in fast superficial runoff. Observed DOC concentrations were higher in summer in the rewetted location
compared to the control and were linked with a difference in dissolved organic matter composition analyzed by fluorescence. Hydrological conditions, especially the severity of the water table drawdown, were identified as the major factors controlling
DOC concentration dynamics. The results of the simulation suggest that the hydrological restoration did not affect DOC loads, at least on a short-term period (3 years). However, it impacted the temporal dynamics of DOC exports, which were the
25
most episodic and mainly transported through fast surface runoff in the area affected by the restoration while slow deep
drainage dominated DOC exports in the control area. In relation with dominant hydrological processes, exported DOC is
expected to be derived from more recent organic matter of the top peat layer in the rewetted area than in the control area. Since it is calibrated on water table and DOC concentration, the model presented in this study proved to be a relevant tool to loads, at least on a short-term period (3 years). However, it impacted the temporal dynamics of DOC exports, which were the
25
most episodic and mainly transported through fast surface runoff in the area affected by the restoration while slow deep
drainage dominated DOC exports in the control area. In relation with dominant hydrological processes, exported DOC is
expected to be derived from more recent organic matter of the top peat layer in the rewetted area than in the control area. HAL Id: insu-01778100 L’archive ouverte pluridisciplinaire HAL, est
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abroad, or from public or private research centers. Hydrol. Earth Syst. Sci. Discuss., https://doi.org/10.5194/hess-2017-578
Manuscript under review for journal Hydrol. Earth Syst. Sci. Discussion started: 1 November 2017
c⃝Author(s) 2017. CC BY 4.0 License. Abstract Since it is calibrated on water table and DOC concentration, the model presented in this study proved to be a relevant tool to loads, at least on a short-term period (3 years). However, it impacted the temporal dynamics of DOC exports, which were the
25
most episodic and mainly transported through fast surface runoff in the area affected by the restoration while slow deep
drainage dominated DOC exports in the control area. In relation with dominant hydrological processes, exported DOC is
expected to be derived from more recent organic matter of the top peat layer in the rewetted area than in the control area. Since it is calibrated on water table and DOC concentration, the model presented in this study proved to be a relevant tool to identify the main hydrological processes and factors controlling DOC dynamics in different areas of the same peatland. It is
30
also a suitable alternative to a discharge calibrated catchment model when the outlet is not easily identifiable. identify the main hydrological processes and factors controlling DOC dynamics in different areas of the same peatland. It is
30
also a suitable alternative to a discharge calibrated catchment model when the outlet is not easily identifiable. 1 1 Hydrol. Earth Syst. Sci. Discuss., https://doi.org/10.5194/hess-2017-578
Manuscript under review for journal Hydrol. Earth Syst. Sci. Discussion started: 1 November 2017
c⃝Author(s) 2017. CC BY 4.0 License. Hydrol. Earth Syst. Sci. Discuss., https://doi.org/10.5194/hess-2017-578
Manuscript under review for journal Hydrol. Earth Syst. Sci. Discussion started: 1 November 2017
c⃝Author(s) 2017. CC BY 4.0 License. 1 Introduction Sphagnum–dominated peatlands represent a major stock of the global soil carbon (C) pool (Gorham, 1991). Dissolved
organic carbon (DOC) exports through runoff and leaching could account for up to 25% of the C fluxes (Yu, 2012), reducing
the potential C storage function of peatlands (Billett et al., 2004) and impacting downstream water quality (Ritson et al.,
2014). DOC dynamics in peatlands has been found to be strongly controlled by site hydrology, especially by the water table
5
depth (WTD) (e.g. Hribljan et al., 2014; Jager et al., 2009; Strack et al., 2008, 2015). Therefore, hydrological disturbances
such as drainage can lead to increased DOC exports in relation with WTD variations (Strack et al., 2008; Worrall et al.,
2007). Where disturbances have occurred, hydrological restoration can be undertaken to reestablish peatland functioning
(Menberu et al., 2016), with a potential impact on DOC dynamics and exports (Glatzel et al., 2003; Strack et al., 2015; Sphagnum–dominated peatlands represent a major stock of the global soil carbon (C) pool (Gorham, 1991). Dissolved
organic carbon (DOC) exports through runoff and leaching could account for up to 25% of the C fluxes (Yu, 2012), reducing
the potential C storage function of peatlands (Billett et al., 2004) and impacting downstream water quality (Ritson et al., 2014). DOC dynamics in peatlands has been found to be strongly controlled by site hydrology, especially by the water table
5
depth (WTD) (e.g. Hribljan et al., 2014; Jager et al., 2009; Strack et al., 2008, 2015). Therefore, hydrological disturbances
such as drainage can lead to increased DOC exports in relation with WTD variations (Strack et al., 2008; Worrall et al.,
2007). Where disturbances have occurred, hydrological restoration can be undertaken to reestablish peatland functioning
(Menberu et al., 2016), with a potential impact on DOC dynamics and exports (Glatzel et al., 2003; Strack et al., 2015; 2014). DOC dynamics in peatlands has been found to be strongly controlled by site hydrology, especially by the water table
5
depth (WTD) (e.g. Hribljan et al., 2014; Jager et al., 2009; Strack et al., 2008, 2015). Therefore, hydrological disturbances
such as drainage can lead to increased DOC exports in relation with WTD variations (Strack et al., 2008; Worrall et al.,
2007). 1 Introduction Where disturbances have occurred, hydrological restoration can be undertaken to reestablish peatland functioning
(Menberu et al., 2016), with a potential impact on DOC dynamics and exports (Glatzel et al., 2003; Strack et al., 2015; Worrall et al., 2007). 10
In peatlands, as in many terrestrial ecosystems, DOC dynamics is controlled on the one hand, by its production to
consumption ratio in pore water, and, on the other hand, by lateral water fluxes that drive its exports. DOC production
through organic matter decomposition is known to increase with temperature (Clark et al., 2009; Freeman et al., 2001) and
DOC consumption, mainly due to heterotrophic bacterial activity, is also positively correlated to temperature and can lead to Worrall et al., 2007). 10
In peatlands, as in many terrestrial ecosystems, DOC dynamics is controlled on the one hand, by its production to
consumption ratio in pore water, and, on the other hand, by lateral water fluxes that drive its exports. DOC production
through organic matter decomposition is known to increase with temperature (Clark et al., 2009; Freeman et al., 2001) and
DOC consumption, mainly due to heterotrophic bacterial activity, is also positively correlated to temperature and can lead to Worrall et al., 2007). 10
In peatlands, as in many terrestrial ecosystems, DOC dynamics is controlled on the one hand, by its production to
consumption ratio in pore water, and, on the other hand, by lateral water fluxes that drive its exports. DOC production
through organic matter decomposition is known to increase with temperature (Clark et al., 2009; Freeman et al., 2001) and
DOC consumption, mainly due to heterotrophic bacterial activity, is also positively correlated to temperature and can lead to decreased DOC concentrations during drought (Clark et al., 2009; Pastor et al., 2003). The export of the DOC produced in
15
pore water is mainly controlled by peatland hydrology (Pastor et al., 2003; Strack et al., 2008), especially by the partitioning
between quick near surface flow and groundwater flow (Birkel et al., 2014). While changes in DOC net production resulting from WTD drawdown can be assessed through field monitoring, the relative
contributions of DOC production and consumption cannot be evaluated (Strack et al., 2008). Process-based biogeochemical p
p
(
,
)
g
models can be relevant tools to understand DOC dynamics (Evans et al., 2005) and can help to identify factors controlling its
20
production and consumption in such environments. 1 Introduction In particular, conceptual models are appropriate because they are
parsimonious in terms of their number of calibrated parameters, avoiding overparametrization issues (Birkel et al., 2017;
Seibert et al., 2009). Another advantage of using conceptual models is that they usually require common measured data (e.g. precipitation and water discharge or water level) so they can be applied to numerous study sites where such data are
available, making them a suitable tool to compare sites with different settings. 25 models can be relevant tools to understand DOC dynamics (Evans et al., 2005) and can help to identify factors controlling its
20
production and consumption in such environments. In particular, conceptual models are appropriate because they are
parsimonious in terms of their number of calibrated parameters, avoiding overparametrization issues (Birkel et al., 2017;
Seibert et al., 2009). Another advantage of using conceptual models is that they usually require common measured data (e.g. precipitation and water discharge or water level) so they can be applied to numerous study sites where such data are available, making them a suitable tool to compare sites with different settings. 25
When studying DOC dynamics in peatlands, existing conceptual models are composed of a DOC module combined with a
hydrological model (Birkel et al., 2014; Futter et al., 2007; Lessels et al., 2015). In these studies, the hydrological model is
usually adapted to the catchment and calibrated on stream discharge. However, stream discharge in peatlands is difficult to
monitor because the diffuse runoff that occurs in these flat areas can result in multiple outlets. Furthermore, while WTD is a ,
g
p
g
When studying DOC dynamics in peatlands, existing conceptual models are composed of a DOC module combined with a
hydrological model (Birkel et al., 2014; Futter et al., 2007; Lessels et al., 2015). In these studies, the hydrological model is
usually adapted to the catchment and calibrated on stream discharge. However, stream discharge in peatlands is difficult to
monitor because the diffuse runoff that occurs in these flat areas can result in multiple outlets. Furthermore, while WTD is a key parameter to explain DOC dynamics (Strack et al., 2008), it is usually not considered for calibration, and water
30
discharge is preferred instead. 2.1.1 Site description
15 The La Guette peatland (150 m.a.s.l., 47°19N, 2°16’E, 20 ha), located in the Sologne forest (Neuvy-sur-Barangeon, France)
is an acidic fen mainly composed of moss patches (Sphagnum cuspidatum, S. rubellum and S. palustre) and of Calluna
vulgaris and Erica tetralix. The peatland has been invaded by Molinia caerulea and Betula spp for 70 years with an
acceleration of the invasion in recent decades (Gogo et al., 2011). This was partly caused by a road ditch located near the
outlet that accelerated the peatland drainage (Fig. 1). In February 2014, hydrological restoration was undertaken in the road
20
ditch to raise the WTD and reduce its fluctuations in order to promote soil rewetting. 1 Introduction The hydrological model was calibrated on WTD which is an important driver of the DOC dynamics in Lessels et al., 2015). In these cases, DOC production and consumption equations are modified using terms related to
5
temperature and soil moisture. In this study, we propose to couple an existing WTD dependent hydrological model specially developed for simulating
peatland hydrology (Binet et al., 2013) with a biogeochemical module simulating DOC production and consumption as first
order rate processes. The hydrological model was calibrated on WTD which is an important driver of the DOC dynamics in peatland hydrology (Binet et al., 2013) with a biogeochemical module simulating DOC production and consumption as first
order rate processes. The hydrological model was calibrated on WTD which is an important driver of the DOC dynamics in
peatland. The model was applied to two sites of a Sphagnum-dominated peatland, one of them having experienced a
10
rewetting action. The objectives were to identify the main hydrological processes and the factors controlling DOC dynamics
in the study sites and to assess the impact of the rewetting on DOC export in a Sphagnum-dominated peatland. peatland. The model was applied to two sites of a Sphagnum-dominated peatland, one of them having experienced a
10
rewetting action. The objectives were to identify the main hydrological processes and the factors controlling DOC dynamics
in the study sites and to assess the impact of the rewetting on DOC export in a Sphagnum-dominated peatland. peatland. The model was applied to two sites of a Sphagnum-dominated peatland, one of them having experienced a
10
rewetting action. The objectives were to identify the main hydrological processes and the factors controlling DOC dynamics
in the study sites and to assess the impact of the rewetting on DOC export in a Sphagnum-dominated peatland. 1 Introduction Therefore, while these models have proven to be well adapted when modelling a catchment
containing a peatland area (Birkel et al., 2014; Futter et al., 2007; Lessels et al., 2015), where the outlet is well defined, they
are more difficult to apply when considering the peatland alone. In this case, the model should focus on the simulation of the key parameter to explain DOC dynamics (Strack et al., 2008), it is usually not considered for calibration, and water
30
discharge is preferred instead. Therefore, while these models have proven to be well adapted when modelling a catchment
containing a peatland area (Birkel et al., 2014; Futter et al., 2007; Lessels et al., 2015), where the outlet is well defined, they
are more difficult to apply when considering the peatland alone. In this case, the model should focus on the simulation of the key parameter to explain DOC dynamics (Strack et al., 2008), it is usually not considered for calibration, and water
30
discharge is preferred instead. Therefore, while these models have proven to be well adapted when modelling a catchment
containing a peatland area (Birkel et al., 2014; Futter et al., 2007; Lessels et al., 2015), where the outlet is well defined, they
are more difficult to apply when considering the peatland alone. In this case, the model should focus on the simulation of the 2 Hydrol. Earth Syst. Sci. Discuss., https://doi.org/10.5194/hess-2017-578
Manuscript under review for journal Hydrol. Earth Syst. Sci. Discussion started: 1 November 2017
c⃝Author(s) 2017. CC BY 4.0 License. WTD, especially when studying DOC dynamics in peatland pore water. Furthermore, a model based on WTD can also
provide interesting information about the spatial variability of the dominant hydrological processes when applied to different
locations within the same peatland. Models simulating DOC dynamics are usually based on a simple mass balance and DOC
production and consumption rates, usually expressed as first order rate processes (Birkel et al., 2014; Futter et al., 2007; Lessels et al., 2015). In these cases, DOC production and consumption equations are modified using terms related to
5
temperature and soil moisture. In this study, we propose to couple an existing WTD dependent hydrological model specially developed for simulating
peatland hydrology (Binet et al., 2013) with a biogeochemical module simulating DOC production and consumption as first
order rate processes. 2.1.2 Data collection and analysis Rainfall was measured with a tipping bucket rain gauge and potential evapotranspiration (PET) computed with
10
the FAO Penman-Monteith equation at an hourly time step (Allen et al., 1998) using local solar radiation, wind speed,
relative humidity and temperature measurements. areas (Fig. 1). Rainfall was measured with a tipping bucket rain gauge and potential evapotranspiration (PET) computed with
10
the FAO Penman-Monteith equation at an hourly time step (Allen et al., 1998) using local solar radiation, wind speed,
relative humidity and temperature measurements. The effect of hydrological conditions (dry period from 1st of June to 30th of November and wet period from 1st of December The effect of hydrological conditions (dry period from 1st of June to 30th of November and wet period from 1st of December
to 31st of May) and location (rewetted or control) on [DOC] and DOM composition were tested using two-way ANOVA and
Tukey’s post hoc tests were used to identify the locations of the significant differences identified between the factors. 15 The effect of hydrological conditions (dry period from 1st of June to 30th of November and wet period from 1st of December
to 31st of May) and location (rewetted or control) on [DOC] and DOM composition were tested using two-way ANOVA and The effect of hydrological conditions (dry period from 1st of June to 30th of November and wet period from 1st of December
to 31st of May) and location (rewetted or control) on [DOC] and DOM composition were tested using two-way ANOVA and
Tukey’s post hoc tests were used to identify the locations of the significant differences identified between the factors. 15 Tukey’s post hoc tests were used to identify the locations of the significant differences iden
15 Tukey’s post hoc tests were used to identify the locations of the significant differences identified between the factors. 15 2.2 Model description The modeling approach used in this study combines a conceptual hydrological model with a biogeochemical model
simulating DOC dynamics. The hydrological model is based on a conceptual water table dependent hydrological model that
has already been successfully applied in the study area (Binet et al., 2013). This model is coupled with a module based on
functions describing DOC production and consumption in pore-water which was developed for this study. The model is
20
described in detail in the following sub sections. 2.2.1 Hydrological model The hydrological model is based on the model described by Binet et al. (2013). It is a daily time step, reservoir model
specifically developed for peatland hydrology integrating a WTD dependent runoff. Compared to the original model, a few
modifications were made in this study in order to improve the model. The overall structure of the new model is presented in
25
Fig. 2. The hydrological model is based on the model described by Binet et al. (2013). It is a daily time step, reservoir model
specifically developed for peatland hydrology integrating a WTD dependent runoff. Compared to the original model, a few
modifications were made in this study in order to improve the model. The overall structure of the new model is presented in
25
Fig. 2. The relation between soil water content and WTD was improved. In the original version the user had to know the relation
between WTD and soil water content. Now the model automatically computes the soil water content based on the porosity of
the percolation reservoir (ϴmin), the porosity at the surface (ϴmax), and peat depth (Hmax) (Fig. 2). The porosity of the
percolation reservoir is considered to be constant over the depth and equal to ϴmin. The porosity of the Sm reservoir is equal
30
to 0 at the maximum depth (Hmax) and increases linearly with the storage until the surface where it reaches ϴmax-ϴmin, The hydrological model is based on the model described by Binet et al. (2013). It is a daily time step, reservoir model
specifically developed for peatland hydrology integrating a WTD dependent runoff. Compared to the original model, a few p
y
p
p
y
gy
g
g
p
p
g
,
modifications were made in this study in order to improve the model. The overall structure of the new model is presented in
25
Fig. 2. The relation between soil water content and WTD was improved. In the original version the user had to know the relation
between WTD and soil water content. Now the model automatically computes the soil water content based on the porosity of
the percolation reservoir (ϴmin), the porosity at the surface (ϴmax), and peat depth (Hmax) (Fig. 2). The porosity of the percolation reservoir is considered to be constant over the depth and equal to ϴmin. 2.1.2 Data collection and analysis WTD and DOC concentrations ([DOC]) in pore-water were monitored in two locations of the peatland. One is affected by
the restoration work and is called ―rewetted‖ while the other is not and is called ―control‖ (Fig. 1). WTD were recorded in
piezometers since February 2014 at a 15 min time step using vented-pressure probes (Orpheus mini, OTT Hydromet). Pore-
25
water was sampled in 4 wells surrounding each piezometer (each of them less than 5m from the piezometer) during 12
campaigns that took place every 1 to 4 months between February 2014 and July 2017. The water samples were filtered using
0.45 µm PES filters on the field and transported in an ice box to the lab where DOC concentrations were determined with a
TOC analyzer (TOC-L, Shimadzu) within 2 days following sampling (samples stored at 4°C). 3 3 Hydrol. Earth Syst. Sci. Discuss., https://doi.org/10.5194/hess-2017-578
Manuscript under review for journal Hydrol. Earth Syst. Sci. Discussion started: 1 November 2017
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Manuscript under review for journal Hydrol. Earth Syst. Sci. Discussion started: 1 November 2017
c⃝Author(s) 2017. CC BY 4.0 License. Pore water dissolved organic matter (DOM) was characterized by its fluorescence properties through three-dimensional
excitation emission matrices (EEMs; Fellman et al., 2010) acquired with F-2500 and F-7000 spectrofluorometers (Hitachi). EEMs were recorded using a 10 x 10 mm quartz mirrored cell, at a photomultiplier voltage of 400 V, with a scan speed of
1500 nm/min, over ranges of excitation of 220–500 nm, in 10 nm steps, and emission of 230–550 nm, in 1 nm steps,
respectively; the slit widths of both monochromators were set at 5 nm. A parallel factor analysis (PARAFAC) was
5
performed using the drEEM toolbox according to the processing described in Murphy et al. (2013). The method was applied
to analyze the samples of two campaigns, those of March 2015 (wet conditions) and September 2015 (dry conditions) in
order to compare DOM composition for two contrasted hydrological settings. Meteorological data were recorded at an hourly time step from a station located within the peatland between the two studied Meteorological data were recorded at an hourly time step from a station located within the peatland between the two studied
areas (Fig. 1). 2.2.1 Hydrological model Water from precipitation
first fills the Sm reservoir, and the Sr reservoir starts to be filled only when Sm is full (Sm=Smmax). The order in which (4) evapotranspiration is removed from the 3 reservoirs is now Sr, Sm and Se. 20
Finally a discharge coefficient was added to compute the flow from the new Sr reservoir,
(5)
where O is the overland flow from the Sr reservoir (mm), αo is the discharge coefficient of the Sr reservoir (-) and Sr is the
volume of water in the Sr reservoir (mm). Finally a discharge coefficient was added to compute the flow from the new Sr reservoir,
(5)
where O is the overland flow from the Sr reservoir (mm), αo is the discharge coefficient of the Sr reservoir (-) and Sr is the
l
f
t
i th S
i (
) (5) (5) where O is the overland flow from the Sr reservoir (mm), αo is the discharge coefficient of the Sr reservoir (-) and Sr is the
volume of water in the Sr reservoir (mm). This flux is added to the total discharge which is now computed according to
25
(6)
where Q is the total discharge (mm), D is the percolation rate from the Se reservoir (mm) and R is the runoff rate from the
Sm reservoir (mm). Given the structure of the model, D represents the drainage of the retention reservoir and can be assimilated to slow deep (6) where Q is the total discharge (mm), D is the percolation rate from the Se reservoir (mm) and R is the runoff rate from the
Sm reservoir (mm). where Q is the total discharge (mm), D is the percolation rate from the Se reservoir (mm) and R is the runoff rate from the
Sm reservoir (mm). Given the structure of the model, D represents the drainage of the retention reservoir and can be assimilated to slow deep Given the structure of the model, D represents the drainage of the retention reservoir and ca Given the structure of the model, D represents the drainage of the retention reservoir and can be assimilated to slow deep
drainage. R and O represent the drainage of the macroporosity and the overland flow and can be assimilated to fast
30
superficial drainage. Given the structure of the model, D represents the drainage of the retention reservoir and can be assimilated to slow deep
drainage. 2.2.1 Hydrological model The porosity of the Sm reservoir is equal
30
to 0 at the maximum depth (Hmax) and increases linearly with the storage until the surface where it reaches ϴmax-ϴmin, 4 4 Hydrol. Earth Syst. Sci. Discuss., https://doi.org/10.5194/hess-2017-578
Manuscript under review for journal Hydrol. Earth Syst. Sci. Discussion started: 1 November 2017
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Manuscript under review for journal Hydrol. Earth Syst. Sci. Discussion started: 1 November 2017
c⃝Author(s) 2017. CC BY 4.0 License. Hydrol. Earth Syst. Sci. Discuss., https://doi.org/10.5194/hess-2017-578
Manuscript under review for journal Hydrol. Earth Syst. Sci. Discussion started: 1 November 2017
c⃝Author(s) 2017. CC BY 4.0 License. ϴmax being the total porosity of the Se and Sm reservoirs at the surface. The new relation between WTD and soil moisture
content is given by ϴmax being the total porosity of the Se and Sm reservoirs at the surface. The new relation between WTD and soil moisture
content is given by
( )
(
⁄
)
(
)
(1)
( )
( ⁄ )
(2) (1) (2) where H is the WTD (m) and ϴ is the sum of the porosities in Sm and Se at a given H. 5
With this modification, the maximum amount of water stored in the Se reservoir (Semax) that was a calibrated parameter in
the original version of the model is now automatically computed with
(3)
Overall, this definition improved the relation between WTD and the water content. In the original version of the model, the (3) porosity of the Sm reservoir was equal to 1, while it now depends on the WTD, to better represent reality (Bourgault et al.,
10
2017). A third reservoir was added, Sr (overland flow storage), in order to differentiate the overland flow water (Sr) from the water
in the peat macroporosity (Sm), that were not differentiated in the original model. While it might not significantly affect the
hydrological model, this was done to prepare for the addition of biogeochemical processes which are different for these two reservoirs. Following the addition of this Sr reservoir, a maximum amount of water contained in the Sm reservoir is defined
15
(Smmax) and is computed according to
(4)
The routing was also slightly modified to take into account the addition of the new reservoir (Sr). 2.2.1 Hydrological model The computation of the following processes remained unchanged: infiltration from Sm to Se (ISe), percolation (P) and runoff
(R). The reader is referred to Binet et al. (2013) for a more detailed description of the computation of these processes. The modified hydrological model is now controlled by 9 parameters (Tab. 1). Three input parameters describing the peat
5
structure (Hmax, ϴmin and ϴmax) and 6 calibrated parameters controlling water fluxes in the model: Kcd and Kcg for ET,
Imax for the ISe, and a discharge coefficient for each reservoir (αp, αr and αo). The forcing variables remained daily
precipitation and PET as in the original model. The computation of the following processes remained unchanged: infiltration from Sm to Se (ISe), percolation (P) and runoff
(R). The reader is referred to Binet et al. (2013) for a more detailed description of the computation of these processes. The modified hydrological model is now controlled by 9 parameters (Tab. 1). Three input parameters describing the peat
5
structure (Hmax, ϴmin and ϴmax) and 6 calibrated parameters controlling water fluxes in the model: Kcd and Kcg for ET,
Imax for the ISe, and a discharge coefficient for each reservoir (αp, αr and αo). The forcing variables remained daily
precipitation and PET as in the original model. 2.2.2 DOC model
10 To simulate DOC dynamics, a module was developed based on first order production and loss, and mass balance, similarly
to what can be found in the literature (Birkel et al., 2014; Lessels et al., 2015). Production and loss are computed in the Se
and Sm reservoirs only since the main biogeochemical processes linked to DOC dynamics occur in soil storage and no
reaction takes place in the Sr reservoir. DOC production was based on a production coefficient and two additional modifiers based on soil water content and air temperature as usually considered in DOC production models (Birkel et al., 2014; Futter
15
et al., 2007; Lessels et al., 2015). The effect of the temperature was based on a Q10 formulation (the factor by which the rate
of a reaction increases for every 10-degree rise in the temperature) with a value of 2 according to the value commonly used
in DOC production models (Lessels et al., 2015; Michalzik et al., 2003; Tjoelker et al., 2001). The rate modifier based on
water content was expressed with a quadratic function to represent the non-linear production of DOC with the variation in soil moisture. Therefore, the higher the soil moisture, the more DOC is produced (Birkel et al., 2014). DOC production is
20
computed as follows: (7) where PDOC is the DOC production rate (mg day-1 m-2), is the production constant (day-1), SOC is the amount of
organic carbon per mm of peat per square meter (mg mm-1 m-2), T is the air temperature (°C), S is the amount of water in the
considered reservoir (mm) and Smax is the maximum amount of water in the considered reservoir (mm). 25 considered reservoir (mm) and Smax is the maximum amount of water in the considered reservoir (mm). 25
DOC loss was based on a loss coefficient and is linked to air temperature in the same way as DOC production. DOC loss is
computed according to
⁄
(8)
where LDOC is the DOC loss rate (mg day-1 m-2), is the loss constant (day-1), [DOC] is the DOC concentration in pore co s de ed ese vo
(
) a d S a
s t e
a
u
a ou t o wate
t e co s de ed ese vo
(
). 5
DOC loss was based on a loss coefficient and is linked to air temperature in the same way as DOC production. 2.2.1 Hydrological model R and O represent the drainage of the macroporosity and the overland flow and can be assimilated to fast
30
superficial drainage. Concerning evapotranspiration, the crop coefficient used to compute evapotranspiration (ET) from ETP was separated into drainage. R and O represent the drainage of the macroporosity and the overland flow and can be assimilated to fast
30
superficial drainage. Concerning evapotranspiration, the crop coefficient used to compute evapotranspiration (ET) from ETP was separated into
the dormant (Kcd) and the growing (Kcg) season. The latter runs from May to September with a linear relation between the 5
superficial drainage. Concerning evapotranspiration, the crop coefficient used to compute evapotranspiration (ET) from ETP was separated into
the dormant (Kcd) and the growing (Kcg) season. The latter runs from May to September with a linear relation between the 5 Hydrol. Earth Syst. Sci. Discuss., https://doi.org/10.5194/hess-2017-578
Manuscript under review for journal Hydrol. Earth Syst. Sci. Discussion started: 1 November 2017
c⃝Author(s) 2017. CC BY 4.0 License. two coefficients during April and October. This was done to take into account the impact of vascular vegetation growth in
peatlands. Finally, a condition was added so that the water level in Sm cannot be lower than the water level in Se. two coefficients during April and October. This was done to take into account the impact of vascular vegetation growth in
peatlands. Finally, a condition was added so that the water level in Sm cannot be lower than the water level in Se. The computation of the following processes remained unchanged: infiltration from Sm to Se (ISe), percolation (P) and runoff
(R). The reader is referred to Binet et al. (2013) for a more detailed description of the computation of these processes. The modified hydrological model is now controlled by 9 parameters (Tab. 1). Three input parameters describing the peat
5
structure (Hmax, ϴmin and ϴmax) and 6 calibrated parameters controlling water fluxes in the model: Kcd and Kcg for ET,
Imax for the ISe, and a discharge coefficient for each reservoir (αp, αr and αo). The forcing variables remained daily
precipitation and PET as in the original model. two coefficients during April and October. This was done to take into account the impact of vascular vegetation growth in
peatlands. Finally, a condition was added so that the water level in Sm cannot be lower than the water level in Se. 2.2.3 Model setup The hydrological and biogeochemical model parameters were calibrated for each piezometer of the peatland for the period The hydrological and biogeochemical model parameters were calibrated for each piezometer of the peatland for the period
ranging from 01/04/2014 to 01/05/2016. This period was chosen because it includes a relatively wet (2014) and relatively
10
dry (2015) summer. The validation period started from 01/10/2016 to 15/07/2017. The period from 01/05/2016 to
30/09/2016 was not simulated because exceptionally heavy rainfall occurred on 31/05/2016, causing extensive flooding in
the whole region. The definition of the model is not suitable for these exceptional events because the water coming from
flooded rivers is not taken into account in the model. ϴmin and ϴmax were set at 0.2 and 1, respectively, and Hmax at 0.6
m, based on field data. was 2 mg L-1 according to measurements performed on rain water and SOC was set at 833
15
103 mg mm-1 m-2 following measurements performed on peat samples. ranging from 01/04/2014 to 01/05/2016. This period was chosen because it includes a relatively wet (2014) and relatively
10
dry (2015) summer. The validation period started from 01/10/2016 to 15/07/2017. The period from 01/05/2016 to
30/09/2016 was not simulated because exceptionally heavy rainfall occurred on 31/05/2016, causing extensive flooding in
the whole region. The definition of the model is not suitable for these exceptional events because the water coming from
flooded rivers is not taken into account in the model. ϴmin and ϴmax were set at 0.2 and 1, respectively, and Hmax at 0.6 m, based on field data. was 2 mg L-1 according to measurements performed on rain water and SOC was set at 833
15
103 mg mm-1 m-2 following measurements performed on peat samples. 2.2.2 DOC model
10 The additional forcing variable is air
temperature. The DOC model is controlled by 6 parameters (Tab. 1). Two input parameters (SOC and [DOC]rain) and four calibrated
5
parameters controlling DOC dynamics (production and loss constant in two reservoirs). The additional forcing variable is air
temperature. 2.2.4 Model evaluation The parameters were calibrated with a Nelder-Mead algorithm (Varadhan et al., 2016) implemented in the R software (R
Core Team, 2012) using the Nash-Sutcliffe coefficient (NS, Nash and Sutcliffe, 1970) as the objective function for the
hydrological module and the root-mean-square error (RMSE) for the DOC concentrations. The hydrological module was
20
calibrated first because substantially more water table data were available than DOC concentrations. The DOC module was
then calibrated over the calibrated hydrological model. Sensitivity analysis was performed using a latin-hypercube one-
factor-at-a-time (LHOAT) procedure (Zambrano-Bigiarini and Rojas, 2014) implemented in the R software. The sensitivity
analysis was based on NS for the hydrological model and on RMSE for the DOC model. 2.2.2 DOC model
10 DOC loss is
computed according to
⁄
(8) DOC loss was based on a loss coefficient and is linked to air temperature in the same way as DOC production. DOC loss is
computed according to (8) where LDOC is the DOC loss rate (mg day-1 m-2), is the loss constant (day-1), [DOC] is the DOC concentration in pore where LDOC is the DOC loss rate (mg day-1 m-2), is the loss constant (day-1), [DOC] is the DOC concentration in pore
water (mg L-1) and S is the amount of water in the considered reservoir (mm). 30 where LDOC is the DOC loss rate (mg day-1 m-2), is the loss constant (day-1), [DOC] is the DOC concentration in pore
water (mg L-1) and S is the amount of water in the considered reservoir (mm). 30
Finally, the mass balance of DOC is computed in the Sm and Se reservoirs
( )
(9) water (mg L-1) and S is the amount of water in the considered reservoir (mm). 30
Finally, the mass balance of DOC is computed in the Sm and Se reservoirs
( )
(9) water (mg L ) and S is the amount of water in the considered reservoir (mm). 30
Finally, the mass balance of DOC is computed in the Sm and Se reservoirs
( )
(9) ( )
(9) (9) 6 Hydrol. Earth Syst. Sci. Discuss., https://doi.org/10.5194/hess-2017-578
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c⃝Author(s) 2017. CC BY 4.0 License. Hydrol. Earth Syst. Sci. Discuss., https://doi.org/10.5194/hess-2017-578
Manuscript under review for journal Hydrol. Earth Syst. Sci. Discussion started: 1 November 2017
c⃝Author(s) 2017. CC BY 4.0 License. (10) (10)
where the exponent represents the time step, the subscript indicates the reservoir considered (Sm or Se), is the
DOC concentration in rain water (mg L-1), Ise is the infiltration from Sm to Se (mm) and Ism is the infiltration from Sr to Sm
(mm). (10) (10) where the exponent represents the time step, the subscript indicates the reservoir considered (Sm or Se), is the
DOC concentration in rain water (mg L-1), Ise is the infiltration from Sm to Se (mm) and Ism is the infiltration from Sr to Sm
(mm). The DOC model is controlled by 6 parameters (Tab. 1). Two input parameters (SOC and [DOC]rain) and four calibrated
5
parameters controlling DOC dynamics (production and loss constant in two reservoirs). 3.1 Observed hydrology and DOC A large increase in the contribution of C relative to the contribution of A was observed in dry conditions in the rewetted
area (p<0.001) while the ratio was similar for control and rewetted sites in wet conditions. Similarly a significant increase in
the contribution of M relative to the contribution of A was observed during dry conditions in the rewetted site compared to
wet conditions in control and rewetted areas (p<0.001). 3.1 Observed hydrology and DOC The ratio of the contribution of component C over the contribution of A and of the contribution of referred to here under its original name as C. The second component (ex 330, em 407) can be described as low-molecular-
15
weight and is referred to here as M. The third component (ex 250, em 446) can be described as high molecular weight and
humic and is referred to here as A. Component A is known to be more aromatic than C (Fellman et al., 2010), even if in our
case, the shorter emission wavelength for component A than for C may also indicate that C is more aromatic than A
(McKnight et al., 2001). The ratio of the contribution of component C over the contribution of A and of the contribution of referred to here under its original name as C. The second component (ex 330, em 407) can be described as low-molecular-
15
weight and is referred to here as M. The third component (ex 250, em 446) can be described as high molecular weight and
humic and is referred to here as A. Component A is known to be more aromatic than C (Fellman et al., 2010), even if in our
case, the shorter emission wavelength for component A than for C may also indicate that C is more aromatic than A
(McKnight et al., 2001). The ratio of the contribution of component C over the contribution of A and of the contribution of component M over the contribution of A in pore water samples of the wet and dry campaigns are presented in Fig. 4 (c and
20
d). A large increase in the contribution of C relative to the contribution of A was observed in dry conditions in the rewetted
area (p<0.001) while the ratio was similar for control and rewetted sites in wet conditions. Similarly a significant increase in
the contribution of M relative to the contribution of A was observed during dry conditions in the rewetted site compared to
wet conditions in control and rewetted areas (p<0.001). component M over the contribution of A in pore water samples of the wet and dry campaigns are presented in Fig. 4 (c and
20
d). 3.1 Observed hydrology and DOC The mean annual precipitation (P) of the area was 821 mm yr-1 and the mean annual PET 931 mm yr-1 for the period ranging
from 01/04/2014 to 01/04/2016 (Tab. 2). WTD and DOC exhibited different dynamics between rewetted and control areas
(Fig. 3b, c). The water table was close to the surface level in each piezometer during the wet season but the length of this 7 7 season depended on the severity of the water table drawdown that occurred during the previous drier season. In 2014, a
particularly wet year (P=906 mm and PET=904 mm from 01/04/2014 to 01/04/2015), the water table reached the surface in
December 2014 while for the following season, which was relatively dry (P=736 mm and PET=960 mm from 01/04/2015 to
01/06/2016), it reached the surface in May 2016. The WTD was lower on average and with a greater variability in the
control than in the rewetted area but the main difference between the sites was the severity of the maximum water table
5
drawdown which was 19 cm in the rewetted and 43 cm in the control site with the same climatic conditions for both season depended on the severity of the water table drawdown that occurred during the previous drier season. In 2014, a
particularly wet year (P=906 mm and PET=904 mm from 01/04/2014 to 01/04/2015), the water table reached the surface in
December 2014 while for the following season, which was relatively dry (P=736 mm and PET=960 mm from 01/04/2015 to
01/06/2016), it reached the surface in May 2016. The WTD was lower on average and with a greater variability in the control than in the rewetted area but the main difference between the sites was the severity of the maximum water table
5
drawdown which was 19 cm in the rewetted and 43 cm in the control site, with the same climatic conditions for both
locations. The average of [DOC] measurements was 13.3±4.6 mg L-1 in the control site and 21.6±7.2 mg L-1 in the rewetted one. [DOC] were globally higher in the rewetted than in the control site (p<0.001) but this was especially true in the dry period. 3.1 Observed hydrology and DOC 5 control than in the rewetted area but the main difference between the sites was the severity of the maximum water table
5
drawdown which was 19 cm in the rewetted and 43 cm in the control site, with the same climatic conditions for both
locations. The average of [DOC] measurements was 13.3±4.6 mg L-1 in the control site and 21.6±7.2 mg L-1 in the rewetted one. [DOC] were globally higher in the rewetted than in the control site (p<0.001) but this was especially true in the dry period. Overall, [DOC] were higher in dry periods than in wet periods for the rewetted site while this difference was not observed in
10
the control site (Fig. 4a). Finally, when considering the temporal evolution of [DOC], the main difference was observed
between April and October 2015 where [DOC] rose in the rewetted but decreased in the control site (Fig. 5). The PARAFAC analysis revealed three main components characterizing the DOM (Fig. 4b). According to the review by
Fellman et al. (2010), the first component (ex 360, em 466) can be described as high-molecular-weight and humic and is Overall, [DOC] were higher in dry periods than in wet periods for the rewetted site while this difference was not observed in
10
the control site (Fig. 4a). Finally, when considering the temporal evolution of [DOC], the main difference was observed
between April and October 2015 where [DOC] rose in the rewetted but decreased in the control site (Fig. 5). The PARAFAC analysis revealed three main components characterizing the DOM (Fig. 4b). According to the review by
Fellman et al. (2010), the first component (ex 360, em 466) can be described as high-molecular-weight and humic and is Fellman et al. (2010), the first component (ex 360, em 466) can be described as high-molecular-weight and humic and is
referred to here under its original name as C. The second component (ex 330, em 407) can be described as low-molecular-
15
weight and is referred to here as M. The third component (ex 250, em 446) can be described as high molecular weight and
humic and is referred to here as A. Component A is known to be more aromatic than C (Fellman et al., 2010), even if in our
case, the shorter emission wavelength for component A than for C may also indicate that C is more aromatic than A
(McKnight et al., 2001). 3.2 Hydrological modeling
25 Simulated and observed WTD dynamics are shown in Fig. 3. Overall the model performed well for both locations with NS
greater than 0.3 for all validation and calibration periods and reaching values greater than 0.8 for calibration periods (Tab. 3). The decrease in the model efficiency in validation compared to calibration periods can be explained by the exceptional event
that occurred prior to the validation period and that may have disturbed the hydrological balance. The most important point is that the model is able to reproduce two different WTD dynamics using the same input data. These differences are
30
explained by the difference in calibrated parameter values. The evapotranspiration coefficient, maximum infiltration rates
and Se discharge coefficient are higher in the control than in the rewetted site, and Sr and Sm discharge coefficients are
higher in the rewetted site than in the control one (Tab. 3). These differences are reflected in the water balance of each is that the model is able to reproduce two different WTD dynamics using the same input data. These differences are
30
explained by the difference in calibrated parameter values. The evapotranspiration coefficient, maximum infiltration rates
and Se discharge coefficient are higher in the control than in the rewetted site, and Sr and Sm discharge coefficients are
higher in the rewetted site than in the control one (Tab. 3). These differences are reflected in the water balance of each 8 Hydrol. Earth Syst. Sci. Discuss., https://doi.org/10.5194/hess-2017-578
Manuscript under review for journal Hydrol. Earth Syst. Sci. Discussion started: 1 November 2017
c⃝Author(s) 2017. CC BY 4.0 License. Hydrol. Earth Syst. Sci. Discuss., https://doi.org/10.5194/hess-2017-578
Manuscript under review for journal Hydrol. Earth Syst. Sci. Discussion started: 1 November 2017
c⃝Author(s) 2017. CC BY 4.0 License. location with a higher evapotranspiration and slow deep drainage in the control than in the rewetted location, and a higher
fast superficial drainage in the rewetted than in the control one (Tab. 2). Sensitivity analysis indicates that the model is the
most sensitive to the evapotranspiration coefficient in the growing season and the Se discharge coefficient, and the least
sensitive to the evapotranspiration coefficient in the dormant period and the Sr discharge coefficient for both locations (Tab. 4). 5 location with a higher evapotranspiration and slow deep drainage in the control than in the rewetted location, and a higher
fast superficial drainage in the rewetted than in the control one (Tab. 2). 3.2 Hydrological modeling
25 Sensitivity analysis indicates that the model is the
most sensitive to the evapotranspiration coefficient in the growing season and the Se discharge coefficient, and the least
sensitive to the evapotranspiration coefficient in the dormant period and the Sr discharge coefficient for both locations (Tab. 4). 5 3.3 DOC dynamics modeling Overall, DOC production, loss and exports were similar and in the same order of magnitude for each
location. Nevertheless, a difference can be observed for the partitioning between exports from the Se and Sm reservoirs. While all the exports are driven by Se in the control site, exports from Se only account for 36% of the total DOC exported in
the rewetted site (Tab. 2). Fig. 6 gives the temporal dynamics of simulated DOC exports for each location, showing that different between rewetted and control areas (Tab. 6). The model can compute the DOC balance for each location which is
15
shown in Tab. 2. Overall, DOC production, loss and exports were similar and in the same order of magnitude for each
location. Nevertheless, a difference can be observed for the partitioning between exports from the Se and Sm reservoirs. While all the exports are driven by Se in the control site, exports from Se only account for 36% of the total DOC exported in
the rewetted site (Tab. 2). Fig. 6 gives the temporal dynamics of simulated DOC exports for each location, showing that DOC exports are less variable in Se than in Sm where large peaks of DOC exports can be observed. 20 3.3 DOC dynamics modeling Simulated and observed pore water [DOC] are shown in Fig. 5. Overall, the model performed well considering its simplicity
(4 calibrated parameters), with RMSE < 6 mg L-1 for calibration and validation in both rewetted and control sites, and with
no systematic overestimations or underestimations. The model performed better for the control than for the rewetted site but
as opposed to the hydrological model simulations, the model performed similarly for the calibration and validation periods. 10
The model was able to reproduce [DOC] dynamics in both locations, especially the rising concentrations in the rewetted site
and the decreasing concentrations in the control site during summer 2015 (Fig. 5). The sensitivity analysis indicates that the
model was the most sensitive to parameters related to the Se reservoir and the least sensitive to parameters related to the Sm
reservoir for both locations (Tab. 5). Compared to the hydrological model, DOC related parameters were not greatly
different between rewetted and control areas (Tab. 6). The model can compute the DOC balance for each location which is
15
shown in Tab. 2. Overall, DOC production, loss and exports were similar and in the same order of magnitude for each
location. Nevertheless, a difference can be observed for the partitioning between exports from the Se and Sm reservoirs. Simulated and observed pore water [DOC] are shown in Fig. 5. Overall, the model performed well considering its simplicity
(4 calibrated parameters), with RMSE < 6 mg L-1 for calibration and validation in both rewetted and control sites, and with
no systematic overestimations or underestimations. The model performed better for the control than for the rewetted site but
as opposed to the hydrological model simulations, the model performed similarly for the calibration and validation periods. 10
The model was able to reproduce [DOC] dynamics in both locations, especially the rising concentrations in the rewetted site
and the decreasing concentrations in the control site during summer 2015 (Fig. 5). The sensitivity analysis indicates that the
model was the most sensitive to parameters related to the Se reservoir and the least sensitive to parameters related to the Sm
reservoir for both locations (Tab. 5). Compared to the hydrological model, DOC related parameters were not greatly different between rewetted and control areas (Tab. 6). The model can compute the DOC balance for each location which is
15
shown in Tab. 2. 4.1 Hydrological processes It enables deep
drainage dominated (control) and surface drainage dominated (rewetted) systems to be identified within the same peatland,
in relation with hydrological restoration work. slow deep discharge can also be related to the restoration work, since the blockage of the drain could have reduced the deep
15
drainage and increased the amount of surface drainage in the rewetted area. Therefore, the model helps to characterize the
impact of restoration as seen in the water balance and evapotranspiration and deep drainage coefficients. It enables deep
drainage dominated (control) and surface drainage dominated (rewetted) systems to be identified within the same peatland,
in relation with hydrological restoration work. 4.2.1 Model results A module simulating DOC production and loss was added to the hydrological model in order to better understand DOC
dynamics in the two peatland locations. Considering the simplicity of the model structure, it gave satisfactory results, with
RMSE always smaller than 6 mg L-1. However, the quality of the results is more difficult to assess than for the hydrological
model because few data were available for the calibration and validation steps. Nevertheless, it is noteworthy that the model,
25
based on only 4 calibrated parameters, is able to capture the two different dynamics recorded in each location, i.e. rising
[DOC] in the downstream location in summer 2015 and a decreasing [DOC] in the upstream location in the same period. 4.1 Hydrological processes In this study, observed water table dynamics were used to better understand the dominant hydrological processes taking
place in two locations of a restored peatland (rewetted and control), by calibrating a conceptual model. Though simple (6
calibrated parameters), the model was able to correctly reproduce the specific water table dynamics in each location of the
25
studied area, using the same input data (precipitation and potential evapotranspiration). This difference in observed water
table dynamics (24 cm of difference for the maximum water table drawdown) is reflected in the calibrated parameter values
for each location (Tab. 3). In addition, and in order to better assess the dominant processes, a sensitivity analysis of the
model was performed for each location (Tab. 4). The results indicate that the most sensitive parameters are Kcg and αp which are related to the evapotranspiration during the growing season and the deep drainage of the retention reservoir (Se),
30
respectively, meaning that these processes are the most important ones to explain the peatland hydrology. Both parameters 9 Hydrol. Earth Syst. Sci. Discuss., https://doi.org/10.5194/hess-2017-578
Manuscript under review for journal Hydrol. Earth Syst. Sci. Discussion started: 1 November 2017
c⃝Author(s) 2017. CC BY 4.0 License. are the highest for the control location and these differences can explain the dissimilarity in the severity of the water table
drawdown observed in the two locations. Kcg is 0.54 and 0.22 for control and rewetted locations, respectively. The value of
the coefficient for the control location is consistent with the values reported by Lafleur et al. (2005) for a shrub covered bog
in Canada (0.517). However, the value of the parameter for the rewetted location is lower than the range of commonly
observed values which is between 0.3 and 0.8 (Lafleur et al., 2005). This difference between the two locations is particularly
5
important considering that the vegetation in both is similar. In this case, low Kcg values can reflect lateral water
redistributions which are rapid lateral exchanges between inundated and non-inundated regions of the peatland, as suggested
by Mclaughlin and Cohen (2014). This is likely linked with the restoration work that created an inundated area in its vicinity. It is also in agreement with the results of Wilson et al. (2010) indicating that the frequency of full saturation of the peat
increases markedly after a drain blocking operation. 4.1 Hydrological processes The discharge coefficient of the retention reservoir indicates the
10
intensity of the slow deep drainage of the peatland. This coefficient is higher in the control part than in the rewetted part and
this is reflected in the water balance through the partitioning of the total discharge in the two locations. Deep drainage
represents the quasi-totality of the total discharge upstream while it accounts for 18% of the total discharge downstream. Similarly to the difference in evapotranspiration coefficients, this difference in the partitioning between fast superficial and
slow deep discharge can also be related to the restoration work, since the blockage of the drain could have reduced the deep
15
drainage and increased the amount of surface drainage in the rewetted area. Therefore, the model helps to characterize the observed values which is between 0.3 and 0.8 (Lafleur et al., 2005). This difference between the two locations is particularly
5
important considering that the vegetation in both is similar. In this case, low Kcg values can reflect lateral water
redistributions which are rapid lateral exchanges between inundated and non-inundated regions of the peatland, as suggested
by Mclaughlin and Cohen (2014). This is likely linked with the restoration work that created an inundated area in its vicinity. It is also in agreement with the results of Wilson et al. (2010) indicating that the frequency of full saturation of the peat
increases markedly after a drain blocking operation. The discharge coefficient of the retention reservoir indicates the
10
intensity of the slow deep drainage of the peatland. This coefficient is higher in the control part than in the rewetted part and
this is reflected in the water balance through the partitioning of the total discharge in the two locations. Deep drainage
represents the quasi-totality of the total discharge upstream while it accounts for 18% of the total discharge downstream. Similarly to the difference in evapotranspiration coefficients, this difference in the partitioning between fast superficial and 5 slow deep discharge can also be related to the restoration work, since the blockage of the drain could have reduced the deep
15
drainage and increased the amount of surface drainage in the rewetted area. Therefore, the model helps to characterize the
impact of restoration as seen in the water balance and evapotranspiration and deep drainage coefficients. 4.2.2 DOC concentrations and control factors Long-term studies have reported decreasing pore water [DOC] more than 10 years after a restoration operation took place
(Höll et al., 2009; Wallage et al., 2006), while others observed increasing [DOC] after restoration (Hribljan et al., 2014;
30
Strack et al., 2015). Glatzel et al. (2003) observed an increase in pore water [DOC] following a drain blocking operation but 10 predicted a decrease in [DOC] with time due to a depletion of easily decomposable organic matter in the peat. In this study,
the results indicate that, during the three years following a restoration operation, [DOC] were higher in the rewetted than in
the control location during the dry period (from 1st of June to 30th of November), while they were similar during the wet
period. In addition, the difference in [DOC] dynamics is also reflected in DOM quality inferred from its fluorescence
properties, with a greater increase in low molecular weight compounds (component M) and fewer aromatic high molecular
5
weight compounds (component C) in the rewetted location during the dry season compared to the control area. These
findings are in agreement with the studies by Höll et al. (2009), Hribljan et al. (2014) and Strack et al. (2015) who observed
that wetter sites would result in a pore water with smaller and fewer aromatic dissolved organic molecules (likely sourced
from inputs of fresh litter from growing vegetation) than the sites with a lower water table. predicted a decrease in [DOC] with time due to a depletion of easily decomposable organic matter in the peat. In this study,
the results indicate that, during the three years following a restoration operation, [DOC] were higher in the rewetted than in
the control location during the dry period (from 1st of June to 30th of November), while they were similar during the wet
period. In addition, the difference in [DOC] dynamics is also reflected in DOM quality inferred from its fluorescence
properties, with a greater increase in low molecular weight compounds (component M) and fewer aromatic high molecular
5
weight compounds (component C) in the rewetted location during the dry season compared to the control area. These
findings are in agreement with the studies by Höll et al. (2009), Hribljan et al. (2014) and Strack et al. 4.2.2 DOC concentrations and control factors The ability of the model to
reproduce pore water [DOC] dynamics can be attributed to its consideration of the water table drawdown which is expressed
in the model through the use of soil moisture (based on water level in the Sm and Se reservoirs) as a production rate
modifier. enhancing DOC release into the water column, as suggested by Hribljan et al. (2014). However the latter hypothesis is the
25
least probable in our case since no ponding water is observed in summer in the study area. The ability of the model to
reproduce pore water [DOC] dynamics can be attributed to its consideration of the water table drawdown which is expressed
in the model through the use of soil moisture (based on water level in the Sm and Se reservoirs) as a production rate
modifier. 4.2.2 DOC concentrations and control factors Indeed, the higher WTD in the dry period
in the rewetted site is related with a higher [DOC] than in the control site where the WTD is lower. A larger proportion of
low aromatic DOC is also observed during the same period in the rewetted than in the control site. Therefore, we propose to
explain the differences in [DOC] by the difference in water table drawdown in the dry period. When the water table drawdown is small (high water table), more DOC is produced from the top peat layer containing more recent and easily
20
biodegradable organic matter than when the water table drawdown is more severe (low water table). In addition, anaerobic
conditions in the rewetted site would lead to less efficient decomposition of organic matter, increasing the production of
water-soluble intermediate metabolites (Kalbitz et al., 2000; Strack et al., 2008). An increase in [DOC] in the rewetted
location can also be explained by an increase in the photic zone, potentially supporting algae photosynthate production drawdown is small (high water table), more DOC is produced from the top peat layer containing more recent and easily
20
biodegradable organic matter than when the water table drawdown is more severe (low water table). In addition, anaerobic
conditions in the rewetted site would lead to less efficient decomposition of organic matter, increasing the production of
water-soluble intermediate metabolites (Kalbitz et al., 2000; Strack et al., 2008). An increase in [DOC] in the rewetted
location can also be explained by an increase in the photic zone, potentially supporting algae photosynthate production drawdown is small (high water table), more DOC is produced from the top peat layer containing more recent and easily
20
biodegradable organic matter than when the water table drawdown is more severe (low water table). In addition, anaerobic
conditions in the rewetted site would lead to less efficient decomposition of organic matter, increasing the production of
water-soluble intermediate metabolites (Kalbitz et al., 2000; Strack et al., 2008). An increase in [DOC] in the rewetted
location can also be explained by an increase in the photic zone, potentially supporting algae photosynthate production enhancing DOC release into the water column, as suggested by Hribljan et al. (2014). However the latter hypothesis is the
25
least probable in our case since no ponding water is observed in summer in the study area. 4.2.2 DOC concentrations and control factors (2015) who observed
that wetter sites would result in a pore water with smaller and fewer aromatic dissolved organic molecules (likely sourced
from inputs of fresh litter from growing vegetation) than the sites with a lower water table. 5 The most sensitive parameters (production and loss rates in Se) of the DOC model do not differ between the control and
10
rewetted areas. There are differences in constant rates related to the Sm reservoir but these parameters are the least sensitive,
meaning that they would not greatly impact [DOC]. This means that the model is able to explain the difference in [DOC]
between the two locations with no differences in production and/or loss rates, suggesting that other factors control DOC
dynamics in the peatland. The main difference in [DOC] is observed during the dry period, when the water table dynamics is The most sensitive parameters (production and loss rates in Se) of the DOC model do not differ between the control and
10
rewetted areas. There are differences in constant rates related to the Sm reservoir but these parameters are the least sensitive,
meaning that they would not greatly impact [DOC]. This means that the model is able to explain the difference in [DOC]
between the two locations with no differences in production and/or loss rates, suggesting that other factors control DOC
dynamics in the peatland. The main difference in [DOC] is observed during the dry period, when the water table dynamics is
different between the two locations. This would confirm that hydrology, and especially the magnitude of the water table
15
drawdown, might be a major factor controlling [DOC] dynamics in the peatland. Indeed, the higher WTD in the dry period
in the rewetted site is related with a higher [DOC] than in the control site where the WTD is lower. A larger proportion of
low aromatic DOC is also observed during the same period in the rewetted than in the control site. Therefore, we propose to
explain the differences in [DOC] by the difference in water table drawdown in the dry period. When the water table
drawdown is small (high water table), more DOC is produced from the top peat layer containing more recent and easily
20
biodegradable organic matter than when the water table drawdown is more severe (low water table). The model enables DOC exports to be estimated for each location. The results are in the range reported in the literature
(from 4.2 to 18.9 g-C m2 yr-1, Birkel et al., 2014, 2017 and Jager et al., 2009). DOC exported from the control site is slightly
higher than that from the rewetted one but in the same order of magnitude. However, considering the simplicity of the 4.2.2 DOC concentrations and control factors In addition, anaerobic
conditions in the rewetted site would lead to less efficient decomposition of organic matter, increasing the production of
water-soluble intermediate metabolites (Kalbitz et al., 2000; Strack et al., 2008). An increase in [DOC] in the rewetted
location can also be explained by an increase in the photic zone, potentially supporting algae photosynthate production
enhancing DOC release into the water column, as suggested by Hribljan et al. (2014). However the latter hypothesis is the
25
least probable in our case since no ponding water is observed in summer in the study area. The ability of the model to
reproduce pore water [DOC] dynamics can be attributed to its consideration of the water table drawdown which is expressed
in the model through the use of soil moisture (based on water level in the Sm and Se reservoirs) as a production rate
modifier. different between the two locations. This would confirm that hydrology, and especially the magnitude of the water table
15
drawdown, might be a major factor controlling [DOC] dynamics in the peatland. Indeed, the higher WTD in the dry period
in the rewetted site is related with a higher [DOC] than in the control site where the WTD is lower. A larger proportion of
low aromatic DOC is also observed during the same period in the rewetted than in the control site. Therefore, we propose to
explain the differences in [DOC] by the difference in water table drawdown in the dry period. When the water table different between the two locations. This would confirm that hydrology, and especially the magnitude of the water table
15
drawdown, might be a major factor controlling [DOC] dynamics in the peatland. Indeed, the higher WTD in the dry period
in the rewetted site is related with a higher [DOC] than in the control site where the WTD is lower. A larger proportion of
low aromatic DOC is also observed during the same period in the rewetted than in the control site. Therefore, we propose to
explain the differences in [DOC] by the difference in water table drawdown in the dry period. When the water table different between the two locations. This would confirm that hydrology, and especially the magnitude of the water table
15
drawdown, might be a major factor controlling [DOC] dynamics in the peatland. 4.2.3 DOC exports
30 However, in the control site, DOC exports are more constant than in the rewetted one following the slow but regular deep drainage of the Se reservoir. These results suggest that hydrology has a major impact on DOC load
10
dynamics, since it is the partitioning between superficial quick flow and slow deep drainage that controls the temporal
dynamics of DOC exports. This hydrological control on DOC fluxes also affects the source of DOC exported from the
peatland, in relation with the difference in DOM composition observed with the fluorescence analysis. Therefore, in the
rewetted area the DOC exported will exhibit characteristics of top peat layer recent organic matter (less aromatic) while it is likely derived from older and deeper organic matter (more aromatic) in the control area. These findings indicate that, while
15
its impact on DOC loads can be negligible, restoration work might have an impact on stream quality by releasing a great
amount of DOC during rainfall events. However, this is valid for a three-year period following the restoration and might be
different for the future, underlining the need for long term monitoring to correctly assess the impact of hydrological
restoration on DOC dynamics. 20 4.2.3 DOC exports
30 This hydrological control on DOC fluxes also affects the source of DOC exported from the
peatland, in relation with the difference in DOM composition observed with the fluorescence analysis. Therefore, in the
rewetted area the DOC exported will exhibit characteristics of top peat layer recent organic matter (less aromatic) while it is
likely derived from older and deeper organic matter (more aromatic) in the control area. These findings indicate that, while
15
its impact on DOC loads can be negligible, restoration work might have an impact on stream quality by releasing a great
amount of DOC during rainfall events. However, this is valid for a three-year period following the restoration and might be
different for the future, underlining the need for long term monitoring to correctly assess the impact of hydrological
restoration on DOC dynamics. 20 model, it is difficult to affirm that this difference is significant and that more DOC is exported from the control than from the
rewetted site. Nevertheless, the partitioning between DOC exports from the two production reservoirs is clearly different for
each location. According to the water balance, DOC exports are only driven by the deep drainage from the Se reservoir in
the control site while the amount of DOC exported through deep drainage and runoff is more balanced in the rewetted site. model, it is difficult to affirm that this difference is significant and that more DOC is exported from the control than from the
rewetted site. Nevertheless, the partitioning between DOC exports from the two production reservoirs is clearly different for
each location. According to the water balance, DOC exports are only driven by the deep drainage from the Se reservoir in
the control site while the amount of DOC exported through deep drainage and runoff is more balanced in the rewetted site. 5 This clearly reflects the dominant hydrological processes in each location and can be seen in the temporal variability in DOC
5
exports (Fig. 6). DOC exports are more episodic in the rewetted site, with 67% of the DOC load coming from the Sm
reservoir and representing only 22% of the total simulated period length. These results are consistent with the results of
Birkel et al. (2017) who reported that 60% of the DOC was exported in 30% of the time in a small peat catchment through
rapid near-surface runoff. 4.2.3 DOC exports
30 The model enables DOC exports to be estimated for each location. The results are in the range reported in the literature
(from 4.2 to 18.9 g-C m2 yr-1, Birkel et al., 2014, 2017 and Jager et al., 2009). DOC exported from the control site is slightly
higher than that from the rewetted one but in the same order of magnitude. However, considering the simplicity of the The model enables DOC exports to be estimated for each location. The results are in the range reported in the literature
(from 4.2 to 18.9 g-C m2 yr-1, Birkel et al., 2014, 2017 and Jager et al., 2009). DOC exported from the control site is slightly
higher than that from the rewetted one but in the same order of magnitude. However, considering the simplicity of the 11 Hydrol. Earth Syst. Sci. Discuss., https://doi.org/10.5194/hess-2017-578
Manuscript under review for journal Hydrol. Earth Syst. Sci. Discussion started: 1 November 2017
c⃝Author(s) 2017. CC BY 4.0 License. model, it is difficult to affirm that this difference is significant and that more DOC is exported from the control than from the
rewetted site. Nevertheless, the partitioning between DOC exports from the two production reservoirs is clearly different for
each location. According to the water balance, DOC exports are only driven by the deep drainage from the Se reservoir in
the control site while the amount of DOC exported through deep drainage and runoff is more balanced in the rewetted site. This clearly reflects the dominant hydrological processes in each location and can be seen in the temporal variability in DOC
5
exports (Fig. 6). DOC exports are more episodic in the rewetted site, with 67% of the DOC load coming from the Sm
reservoir and representing only 22% of the total simulated period length. These results are consistent with the results of
Birkel et al. (2017) who reported that 60% of the DOC was exported in 30% of the time in a small peat catchment through
rapid near-surface runoff. However, in the control site, DOC exports are more constant than in the rewetted one following
the slow but regular deep drainage of the Se reservoir. These results suggest that hydrology has a major impact on DOC load
10
dynamics, since it is the partitioning between superficial quick flow and slow deep drainage that controls the temporal
dynamics of DOC exports. 4.3 Perspectives for application of the model The model developed in this study follows a parsimonious coupled hydrology-biogeochemistry model philosophy (Birkel et
al., 2014, 2017; Lessels et al., 2015). By keeping parametrization to a minimum, it was able to identify factors controlling
WTD and DOC dynamics in the two contrasted sites of the studied peatland with a relatively low requirement in input data
(precipitation, potential evapotranspiration and temperature). Contrary to similar models, hydrology is here calibrated on
25
WTD instead of on stream discharge. This way, the model proves to be a relevant tool to explore the hydrology of areas
located within the same peatland and to highlight the impact of hydrological restoration on hydrology and DOC dynamics
that would have been difficult to study with models calibrated on stream discharge and applicable at the catchment scale
only. In addition, the DOC model developed in this study has shown good results in modeling pore water [DOC] dynamics, y
p
y
q
p
(precipitation, potential evapotranspiration and temperature). Contrary to similar models, hydrology is here calibrated on
25
WTD instead of on stream discharge. This way, the model proves to be a relevant tool to explore the hydrology of areas
located within the same peatland and to highlight the impact of hydrological restoration on hydrology and DOC dynamics
that would have been difficult to study with models calibrated on stream discharge and applicable at the catchment scale
only. In addition, the DOC model developed in this study has shown good results in modeling pore water [DOC] dynamics, (precipitation, potential evapotranspiration and temperature). Contrary to similar models, hydrology is here calibrated on
25
WTD instead of on stream discharge. This way, the model proves to be a relevant tool to explore the hydrology of areas
located within the same peatland and to highlight the impact of hydrological restoration on hydrology and DOC dynamics
that would have been difficult to study with models calibrated on stream discharge and applicable at the catchment scale
only. In addition, the DOC model developed in this study has shown good results in modeling pore water [DOC] dynamics, meaning that the formulation of the 4 calibrated parameters model is adapted to peatland ecosystems. However, as this
30
model is calibrated on pore water [DOC], DOC export results have to be interpreted with care. 4.3 Perspectives for application of the model In order to improve its
significance, the model should be compared with a discharge calibrated model on a study site where the outlet is well
defined and monitored. Therefore, if applied to several WTD time series, it could provide spatial information by identifying meaning that the formulation of the 4 calibrated parameters model is adapted to peatland ecosystems. However, as this
30
model is calibrated on pore water [DOC], DOC export results have to be interpreted with care. In order to improve its
significance, the model should be compared with a discharge calibrated model on a study site where the outlet is well
defined and monitored. Therefore, if applied to several WTD time series, it could provide spatial information by identifying 12 Hydrol. Earth Syst. Sci. Discuss., https://doi.org/10.5194/hess-2017-578
Manuscript under review for journal Hydrol. Earth Syst. Sci. Discussion started: 1 November 2017
c⃝Author(s) 2017. CC BY 4.0 License. Hydrol. Earth Syst. Sci. Discuss., https://doi.org/10.5194/hess-2017-578
Manuscript under review for journal Hydrol. Earth Syst. Sci. Discussion started: 1 November 2017
c⃝Author(s) 2017. CC BY 4.0 License. the main areas of DOC production within a peatland. This model could also be applied to longer time series and different
study sites to assess the effect of hydrological restoration over longer periods and the dominant controlling factors in
peatlands with different settings. the main areas of DOC production within a peatland. This model could also be applied to longer time series and different
study sites to assess the effect of hydrological restoration over longer periods and the dominant controlling factors in
peatlands with different settings. 5 Conclusions A conceptual hydrological model, especially developed for peatland and calibrated on WTD, has been combined with a
5
simple DOC production/loss model and applied to two locations of a peatland, one of them affected by hydrological
restoration. The application of this model has shown the following: A conceptual hydrological model, especially developed for peatland and calibrated on WTD, has been combined with a
5
simple DOC production/loss model and applied to two locations of a peatland, one of them affected by hydrological
restoration. The application of this model has shown the following:
The hydrological restoration was found to impact water balance, by increasing fast superficial drainage compared to
slow deep drainage. 10 10
The intensity of the maximum water table drawdown was found to be the main factor controlling pore water [DOC]
dynamics in the peatland.
Higher [DOC] in the rewetted location was linked to differences in DOM composition
Simulated DOC exports are in the same order of magnitude for rewetted and control locations, in a short-term
period (3 years).
Water partitioning between fast superficial drainage and slow deep drainage controls DOC sources as well as the
temporal dynamics of DOC exports These results suggest that hydrological restoration does not affect short term DOC fluxes in peatland. In addition, this study
has shown that the proposed conceptual hydrological and biogeochemical model can provide relevant information about
water balance and the factors controlling element cycling processes in peatlands. The application of a WTD based model is a These results suggest that hydrological restoration does not affect short term DOC fluxes in peatland. In addition, this study
has shown that the proposed conceptual hydrological and biogeochemical model can provide relevant information about
water balance and the factors controlling element cycling processes in peatlands. The application of a WTD based model is a
relevant alternative to a discharge calibrated catchment model when the outlet is not easily identifiable or when seeking for
20
within-peatland spatial information. relevant alternative to a discharge calibrated catchment model when the outlet is not easily identifiable or when seeking for
20
within-peatland spatial information. Author contributions
FLa, SG and SB designed the study site restoration and monitoring. LBJ, SG, FLa, FLe and LP helped with instrumentation and data collection. Author contributions FLa, SG and SB designed the study site restoration and monitoring. LBJ, SG, FLa, FLe and LP helped with instrumentation and data collection. 25
CD, NJ and RZ helped with fluorescence analysis and data interpretation
LBJ and SB developed the model
LBJ performed simulations and data analysis
LBJ prepared the draft of the manuscript FLe, SG, CD, NJ, FLa and SB helped improve the final manuscript
30 5 Conclusions 25
CD, NJ and RZ helped with fluorescence analysis and data interpretation
LBJ and SB developed the model
LBJ performed simulations and data analysis
LBJ prepared the draft of the manuscript
FLe, SG, CD, NJ, FLa and SB helped improve the final manuscript
30
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c⃝Author(s) 2017. CC BY 4.0 License. Hydrol. Earth Syst. Sci. Discuss., https://doi.org/10.5194/hess-2017-578
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c⃝Author(s) 2017. CC BY 4.0 License. Hydrol. Earth Syst. Sci. Discuss., https://doi.org/10.5194/hess-2017-578
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c⃝Author(s) 2017. CC BY 4.0 License. This paper is a contribution of the Labex VOLTAIRE (ANR-10-LABX-100- 01) and of the PIVOTS project (ARD 2020 of
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k
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0 3 3 2014 Yu, Z. C.: Northern peatland carbon stocks and dynamics: A review, Biogeosciences, 9(10), 4071–4085, doi:10.5194/bg-9-
4071-2012, 2012. Zambrano-Bigiarini, M. and Rojas, R.: hydroPSO: Particle swarm optimisation, with focus on environmental models, URL
http//www. rforge. net/hydroTSM/, http//cran. r-project. org/web/packages/hydroTSM/. R Packag. version 0.3-3, 2014. Zambrano-Bigiarini, M. and Rojas, R.: hydroPSO: Particle swarm optimisation, with focus on environmental models, URL
http//www. rforge. net/hydroTSM/, http//cran. r-project. org/web/packages/hydroTSM/. R Packag. version 0.3-3, 2014. 5 17 17 17 Figures
Figure 1: Location and settings of the study area. Locations of control and rewetted monitoring are indicated. Hydrol. Earth Syst. Sci. Discuss., https://doi.org/10.5194/hess-2017-578
Manuscript under review for journal Hydrol. Earth Syst. Sci. Discussion started: 1 November 2017
c⃝Author(s) 2017. CC BY 4.0 License. Hydrol. Earth Syst. Sci. Discuss., https://doi.org/10.5194/hess-2017-578
Manuscript under review for journal Hydrol. Earth Syst. Sci. Discussion started: 1 November 2017
c⃝Author(s) 2017. CC BY 4.0 License. References Figures
Figure 1: Location and settings of the study area. Locations of control and rewetted monitoring are indicated. Figure 1: Location and settings of the study area. Locations of control and rewetted monitoring are indicated. Figure 2: Structure of the hydrological model, composed of three reservoirs, surface (Sr), macroporosity (Sm) and retention (Se). 5
The different fluxes are indicated in italics, P (precipitation), ET (evapotranspiration), ISm (infiltration from Sr to Sm), ISe
(infiltration from Sm to Se), D (deep drainage from Se), R (runoff from Sm), O (overland flow from Sr). Total discharge Q
corresponds to the sum of D, R and O. Note that given parameters are written in red and calibrated parameters associated to each
flux in blue, see description in Tab. 1. Figure 2: Structure of the hydrological model, composed of three reservoirs, surface (Sr), macroporosity (Sm) and retention (Se). 5
The different fluxes are indicated in italics, P (precipitation), ET (evapotranspiration), ISm (infiltration from Sr to Sm), ISe
(infiltration from Sm to Se), D (deep drainage from Se), R (runoff from Sm), O (overland flow from Sr). Total discharge Q
corresponds to the sum of D, R and O. Note that given parameters are written in red and calibrated parameters associated to each
flux in blue, see description in Tab. 1. 18 Hydrol. Earth Syst. Sci. Discuss., https://doi.org/10.5194/hess-2017-578
Manuscript under review for journal Hydrol. Earth Syst. Sci. Discussion started: 1 November 2017
c⃝Author(s) 2017. CC BY 4.0 License. Hydrol. Earth Syst. Sci. Discuss., https://doi.org/10.5194/hess-2017-578
Manuscript under review for journal Hydrol. Earth Syst. Sci. Discussion started: 1 November 2017
c⃝Author(s) 2017. CC BY 4.0 License. Figure 3: (a) Time series of meteorological data (PET, potential evapotranspiration and P, precipitation) used as input data in the
hydrological model, (b) simulated and observed WTD in the rewetted site and (c) simulated and observed WTD in the control site. Calibration and validation periods are also indicated. Manuscript under review for journal Hydrol. Earth Syst. Sci. Discussion started: 1 November 2017
c⃝Author(s) 2017. CC BY 4.0 License. Figure 3: (a) Time series of meteorological data (PET, potential evapotranspiration and P, precipitation) used as input data in the
hydrological model, (b) simulated and observed WTD in the rewetted site and (c) simulated and observed WTD in the control site. Calibration and validation periods are also indicated. References Figure 3: (a) Time series of meteorological data (PET, potential evapotranspiration and P, precipitation) used as input data in the
hydrological model, (b) simulated and observed WTD in the rewetted site and (c) simulated and observed WTD in the control site. Calibration and validation periods are also indicated. 19 Figure 4: (a) DOC concentrations in control and rewetted sites for dry (1st of June to 30th of November, n=7) and wet periods (1st of
December to 31st of May, n=6). ). (b) Excitation-emission matrices for the identified PARAFAC components (see the text for
details). (c) Ratio of contribution of component C over component A for dry and wet conditions in control and rewetted sites (n=4). (d) Ratio of contribution of component M over component A for dry and wet conditions in control and rewetted sites (n=4). The
letter above the bar indicates significant differences across different conditions (Tukey’s p<0.01). (b) Excitation-emission matrices
for the identified PARAFAC components (see the text for details). Hydrol. Earth Syst. Sci. Discuss., https://doi.org/10.5194/hess-2017-578
Manuscript under review for journal Hydrol. Earth Syst. Sci. Discussion started: 1 November 2017
c⃝Author(s) 2017. CC BY 4.0 License. Figure 4: (a) DOC concentrations in control and rewetted sites for dry (1st of June to 30th of November, n=7) and wet periods (1st of
December to 31st of May, n=6). ). (b) Excitation-emission matrices for the identified PARAFAC components (see the text for
details). (c) Ratio of contribution of component C over component A for dry and wet conditions in control and rewetted sites (n=4). (d) Ratio of contribution of component M over component A for dry and wet conditions in control and rewetted sites (n=4). The
letter above the bar indicates significant differences across different conditions (Tukey’s p<0.01). (b) Excitation-emission matrices
for the identified PARAFAC components (see the text for details). Figure 5: Simulated and observed pore water [DOC] in control and rewetted sites. Observations are the average of 4 samples for
each sampling date. Error bars indicate standard deviation. 10 Figure 5: Simulated and observed pore water [DOC] in control and rewetted sites. Observations are the average of 4 samples for
each sampling date. Error bars indicate standard deviation. 0 Figure 5: Simulated and observed pore water [DOC] in control and rewetted sites. Observations are the average of 4 samples for
each sampling date. Error bars indicate standard deviation. References 0 10 20 Figure 6: Simulated DOC exports for control and rewetted sites. Hydrol. Earth Syst. Sci. Discuss., https://doi.org/10.5194/hess-2017-578
Manuscript under review for journal Hydrol. Earth Syst. Sci. Discussion started: 1 November 2017
c⃝Author(s) 2017. CC BY 4.0 License. Figure 6: Simulated DOC exports for control and rewetted sites. 21 21 Tables
Table 1: List of the parameters used in the hydrological and in the DOC model. The hydrological flux associated to each
parameter is in parenthesis. Calibrated parameters are indicated. Hydrol. Earth Syst. Sci. Discuss., https://doi.org/10.5194/hess-2017-578
Manuscript under review for journal Hydrol. Earth Syst. Sci. Discussion started: 1 November 2017
c⃝Author(s) 2017. CC BY 4.0 License. Tables
Table 1: List of the parameters used in the hydrological and in the DOC model. The hydrological flux associated to each
parameter is in parenthesis. Calibrated parameters are indicated. Symbol
Process
Description
Units
Calibrated
Hydrological
model
Hmax
WTD-moisture relation
Peat depth
mm
no
ϴmin
WTD-moisture relation
Porosity at maximum depth
m3.m-3
no
ϴmax
WTD-moisture relation
Porosity at the surface
m3.m-3
no
Kcd
Evapotranspiration (ET)
Crop coefficient for dormant season
-
yes
Kcg
Evapotranspiration (ET)
Crop coefficient for growing season
-
yes
Imax
Infiltration Sm to Se (ISe)
Maximum infiltration rates in Se
mm
yes
αp
Se discharge (D)
Discharge coefficient of Se
day-1
yes
αr
Sm discharge (R)
Discharge coefficient of Sr
day-1
yes
αo
Sr discharge (O)
Discharge coefficient of So
day-1
yes
DOC model
SOC
DOC module
Mass of TOC per height of peat
mgC mm-1
no
DOCrain
DOC module
DOC concentration in rain water
mg L-1
no
kprodSe
DOC module
DOC production coefficient in Se
day-1
yes
klossSe
DOC module
DOC loss coefficient in Se
day-1
yes
kprodSm
DOC module
DOC production coefficient in Sm
day-1
yes
klossSm
DOC module
DOC loss coefficient in Sm
day-1
yes
Table 2: Water and DOC balance computed from 01/04/2015 to 01/04/2017 in rewetted and control areas. P is precipitation, ET is
t
i
ti
Q i t t l di
h
O i
l
d fl
R i
it
ff D i d
d
i
PDOC i th
t f
Hydrol. Earth Syst. Sci. Discuss., https://doi.org/10.5194/hess-2017-578
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c⃝Author(s) 2017. CC BY 4.0 License. Hydrol. Earth Syst. Sci. Discuss., https://doi.org/10.5194/hess-2017-578
Manuscript under review for journal Hydrol. Earth Syst. Sci. Discussion started: 1 November 2017
c⃝Author(s) 2017. CC BY 4.0 License. References Table 1: List of the parameters used in the hydrological and in the DOC model. The hydrological flux associated to each
parameter is in parenthesis. Calibrated parameters are indicated. p
p
p
Symbol
Process
Description
Units
Calibrated
Hydrological
model
Hmax
WTD-moisture relation
Peat depth
mm
no
ϴmin
WTD-moisture relation
Porosity at maximum depth
m3.m-3
no
ϴmax
WTD-moisture relation
Porosity at the surface
m3.m-3
no
Kcd
Evapotranspiration (ET)
Crop coefficient for dormant season
-
yes
Kcg
Evapotranspiration (ET)
Crop coefficient for growing season
-
yes
Imax
Infiltration Sm to Se (ISe)
Maximum infiltration rates in Se
mm
yes
αp
Se discharge (D)
Discharge coefficient of Se
day-1
yes
αr
Sm discharge (R)
Discharge coefficient of Sr
day-1
yes
αo
Sr discharge (O)
Discharge coefficient of So
day-1
yes
DOC model
SOC
DOC module
Mass of TOC per height of peat
mgC mm-1
no
DOCrain
DOC module
DOC concentration in rain water
mg L-1
no
kprodSe
DOC module
DOC production coefficient in Se
day-1
yes
klossSe
DOC module
DOC loss coefficient in Se
day-1
yes
kprodSm
DOC module
DOC production coefficient in Sm
day-1
yes
klossSm
DOC module
DOC loss coefficient in Sm
day-1
yes Table 2: Water and DOC balance computed from 01/04/2015 to 01/04/2017 in rewetted and control areas. P is precipitation, ET is
5
evapotranspiration, Q is total discharge, O is overland flow, R is macroporosity runoff, D is deep drainage, PDOC is the amount of
DOC produced, LDOC is the amount of DOC loss and DOC exports is the amount of DOC exported. Number in brackets
represents the DOC balance for Se and Sm reservoirs. Table 2: Water and DOC balance computed from 01/04/2015 to 01/04/2017 in rewetted and control areas. P is precipitation, ET is
5
evapotranspiration, Q is total discharge, O is overland flow, R is macroporosity runoff, D is deep drainage, PDOC is the amount of
DOC produced, LDOC is the amount of DOC loss and DOC exports is the amount of DOC exported. Number in brackets
represents the DOC balance for Se and Sm reservoirs. Table 2: Water and DOC balance computed from 01/04/2015 to 01/04/2017 in rewetted and control areas. References P is precipitation, ET is
5
evapotranspiration, Q is total discharge, O is overland flow, R is macroporosity runoff, D is deep drainage, PDOC is the amount of
DOC produced, LDOC is the amount of DOC loss and DOC exports is the amount of DOC exported. Number in brackets
represents the DOC balance for Se and Sm reservoirs. 01/04/2015 to 01/04/2017
rewetted
control
P (mm yr-1)
821
821
ET (mm yr-1)
160
494
Q (mm yr-1)
653
330
O (mm yr-1)
243
1
R (mm yr-1)
295
1
D (mm yr-1)
115
329
PDOC [Se – Sm] (g-C m-2 yr-1)
79.2 [73.7-5.5]
80.8 [80.4-0.4]
LDOC [Se – Sm] (g-C m-2 yr-1)
74.0 [74.0-0]
74.6 [74.6-0]
DOC exports [Se – Sm] (g-C m-2 yr-1)
6.7 [2.4-4.3]
8.2 [8.2-0] 22 Hydrol. Earth Syst. Sci. Discuss., https://doi.org/10.5194/hess-2017-578
Manuscript under review for journal Hydrol. Earth Syst. Sci. Discussion started: 1 November 2017
c⃝Author(s) 2017. CC BY 4.0 License. Hydrol. Earth Syst. Sci. Discuss., https://doi.org/10.5194/hess-2017-578
Manuscript under review for journal Hydrol. Earth Syst. Sci. Discussion started: 1 November 2017
c⃝Author(s) 2017. CC BY 4.0 License. Hydrol. Earth Syst. Sci. Discuss., https://doi.org/10.5194/hess-2017-578
Manuscript under review for journal Hydrol. Earth Syst. Sci. Discussion started: 1 November 2017
c⃝Author(s) 2017. CC BY 4.0 License. Table 3: Calibrated parameters and efficiency of the hydrological model
Parameter
Units
rewetted
control
Kcd
-
0.0120
0.500
Kcg
-
0.221
0.540
Imax
mm
0.955
2.88
αp
day-1
1.13E-3
4.21E-3
αr
day-1
0.422
0.001
αo
day-1
0.258
0.001
NS calibration
-
0.80
0.86
NS validation
-
0.33
0.57 Table 3: Calibrated parameters and efficiency of the hydrological model Table 4: Sensitivity rank of the parameters of the hydrological model Table 4: Sensitivity rank of the parameters of the hydrological model Parameter
Sensitivity rank
rewetted
control
Kcg
1
1
αp
2
2
αr
4
3
Imax
3
4
αo
6
5
Kcd
5
6
5
Table 5: Sensitivity rank of the parameters of the DOC model
Parameter
Sensitivity rank
rewetted
control
kprodSe
2
1
klossSe
1
2
kprodSm
3
3
klossSm
4
4 Parameter
Sensitivity rank
rewetted
control
Kcg
1
1
αp
2
2
αr
4
3
Imax
3
4
αo
6
5
Kcd
5
6
5 Table 5: Sensitivity rank of the parameters of the DOC model Parameter
Sensitivity rank
rewetted
control
kprodSe
2
1
klossSe
1
2
kprodSm
3
3
klossSm
4
4 23 Hydrol. Earth Syst. Sci. Discuss., https://doi.org/10.5194/hess-2017-578
Manuscript under review for journal Hydrol. Earth Syst. Sci. Discussion started: 1 November 2017
c⃝Author(s) 2017. CC BY 4.0 License. Hydrol. Earth Syst. Sci. Discuss., https://doi.org/10.5194/hess-2017-578
Manuscript under review for journal Hydrol. Earth Syst. Sci. Discussion started: 1 November 2017
c⃝Author(s) 2017. CC BY 4.0 License. Table 6: Calibrated parameters and efficiency of the DOC model
Parameter
Units
rewetted
control
kprodSe
day-1
1E-6
1.7E-6
klossSe
day-1
3.7E-2
3.9E-2
kprodSm
day-1
7.2E-8
1E-8
klossSm
day-1
1E-6
1E-5
RMSE calib
mg L-1
5.7
3.3
RMSE valid
mg L-1
5.3
3.9 Table 6: Calibrated parameters and efficiency of the DOC model 24 24 24
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https://openalex.org/W4220905064
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http://journal.eahn.org/article/id/8286/download/pdf/
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English
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"Housewives and Architects": Marie-Elisabeth Lüders’ Management of the New Architecture From Pot-Lid to Siedlung
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Architectural histories
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cc-by
| 10,369
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Meister, A-M. 2022. ‘Housewives and Architects’:
Marie-Elisabeth Lüders’ Management of the New
Architecture from Pot Lid to Siedlung. Architectural
Histories, 10(1): pp. 1–24. DOI: https://doi.
org/10.16995/ah.8286 $UFKLWHFWXUDO
+LVWRULHV $UFKLWHFWXUDO
+LVWRULHV $UFKLWHFWXUDO
+LVWRULHV Meister, A-M. 2022. ‘Housewives and Architects’:
Marie-Elisabeth Lüders’ Management of the New
Architecture from Pot Lid to Siedlung. Architectural
Histories, 10(1): pp. 1–24. DOI: https://doi. org/10.16995/ah.8286 ‘Housewives and Architects’: Marie-Elisabeth Lüders’
Management of the New Architecture from Pot Lid
to Siedlung Anna-Maria Meister, Department of Architecture, Architecture Theory and Science, Technical University of Darmstadt,
Germany, meister@atw.tu-darmstadt.de In 1926, women’s rights activist Marie-Elisabeth Lüders (1878-1966) gave a speech at the annual
meeting of the German Institute for Norms (DIN), right after a talk by architect Walter Gropius
(1883–1969), on ‘norming and housing shortage’. Claiming the improvement of household regimes
as essential for conquering the pressing post–WWI housing shortage and impending economic
catastrophe, Lüders saw the mission at hand to be one of an ‘urgent collaboration’ between ‘producers,
traders, housewives and architects, one just like the DIN strives toward’. Her task list named the
standardization of pots and pans alongside that of architectural elements such as doors, windows and
stairs, rendering the improvement of the household (hence, of female labor) a decidedly architectural
challenge — even necessity. As a founding member of the Reichsforschungsgesellschaft (a research
committee for cost-efficient building) alongside Gropius, Lüders steered what became known as the
modernist Siedlung into existence: not as architect, but as managerial expert. This article aims to extend
the techno-scientific (and male) histories of both standardization and the New Architecture with a
reframing of what constituted ‘architectural elements’ from the viewpoint of the very ‘housewives‘
who shaped modern architecture from the pot-lid outward. Architectural Histories is a peer-reviewed open access journal published by the Open Library of Humanities. © 2022 The
Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International
License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original
author and source are credited. See http://creativecommons.org/licenses/by/4.0/. OPEN ACCESS Architectural Histories is a peer-reviewed open access journal published by the Open Library of Humanities. © 2022 The
Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International
License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original
author and source are credited. See http://creativecommons.org/licenses/by/4.0/. OPEN ACCESS Architectural Histories is a peer-reviewed open access journal published by the Open Library of Humanities. © 2022 The
Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International
License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original
author and source are credited. See http://creativecommons.org/licenses/by/4.0/. ‘Housewives and Architects’: Marie-Elisabeth Lüders’
Management of the New Architecture from Pot Lid
to Siedlung OPEN ACCESS 2 2 An “Unknown Army”: The Rise of Housewives as Experts
In her 1936 book, Das unbekannte Heer (The Unknown Army), Marie-Elisabeth Lüders
(1878–1966) defined the fundamental shift that had occurred during WWI, when men
left to fight on the front and women took over many formerly ‘male’ preserves (Fig. 1)
(Lüders 1936). Lüders sketched a dramatic picture of the ‘women’s army’ in a steel
foundry: The red-hot wire rods rattled and hissed closer along the roller line. At its end, a
platoon of most robust women in men’s clothes and leather aprons stood ready to
catch them with mighty tongs. A tough grip, the sparking snake was caught and flew Figure 1: Cover of Marie-Elisabeth Lüders, Das unbekannte Heer [The Unknown Army] (1936),
displaying photographs of women working in factories during WWI. Figure 1: Cover of Marie-Elisabeth Lüders, Das unbekannte Heer [The Unknown Army] (1936),
displaying photographs of women working in factories during WWI. 3 3 to the next rattling track … Calmly, women sailed back and forth between the dark
scrap piles and glowing furnaces …. Earlier, the woman had sewn coats and jackets,
hunched over the sewing machine, eyes on the presser foot, hands fixed on the fab
ric. Today, her gaze extends far and wide, stretching vast spaces, across great dis
tances and slagheaps.1 (Lüders 1936: 172–73) Where the economic contribution of women had (at least in most histories) been
limited mainly to the textile industry,2 now they handled ‘red-hot wire rods’ and iron
claws. Their scope had shifted from needlework to ironwork, from confined tasks and
small-scale tools to ‘mighty tongs’. This change — a material process directly linked
to the wartime reorganization of the female workforce — effectively redrew the limits
of women’s expertise. For Lüders, women had not simply ventured into male territory,
they had started to exercise supervision and control. Women were now mastering
the glowing metal, forming a ‘platoon’, ready for anything that might come their
way. But this physically taxing and dangerous work was not depicted as stressful or
exhausting; rather, it was conducted ‘calmly’, despite the many risks involved. Women
had conquered male domains, and they were doing just fine. Where the economic contribution of women had (at least in most histories) been
limited mainly to the textile industry,2 now they handled ‘red-hot wire rods’ and iron
claws. ‘Housewives and Architects’: Marie-Elisabeth Lüders’
Management of the New Architecture from Pot Lid
to Siedlung Their scope had shifted from needlework to ironwork, from confined tasks and
small-scale tools to ‘mighty tongs’. This change — a material process directly linked
to the wartime reorganization of the female workforce — effectively redrew the limits
of women’s expertise. For Lüders, women had not simply ventured into male territory,
they had started to exercise supervision and control. Women were now mastering
the glowing metal, forming a ‘platoon’, ready for anything that might come their
way. But this physically taxing and dangerous work was not depicted as stressful or
exhausting; rather, it was conducted ‘calmly’, despite the many risks involved. Women
had conquered male domains, and they were doing just fine. The only daughter of a Prussian government official, Lüders trained as a teacher
before studying political science in Berlin. She enrolled in 1909, a year after Prussian
universities were finally opened to female students, and was the first woman in
Germany to receive a doctorate in political science. A founding member of the German
Democratic Party (DDP), she was also among the first cohort of female representatives
in the Weimar National Assembly in 1919 — one year after German women were
granted the right to vote — and would continue her position in the Reichstag until
1930 (Fig. 2). For Lüders, these pursuits did not run counter to her activities as a self-
proclaimed ‘housewife’. In fact, it was the knowledge, experience and tasks of the
so-called housewives of 1920s Germany that informed her professional and political
engagement. As Lüders made clear in an article from 1921, the ‘housewife’ had a distinct
vocation (Lüders 1921). Moreover, rather than being a separate sphere, the ‘household’
(Haushalt) encompassed a specific expertise that she deemed instrumental to the
economic make-up of the German nation-state. Thus, although she never studied
architecture or planning, Lüders actively shaped the appearance and perception
of modern architecture as a cofounder of the Reichsforschungsgesellschaft für
Wirtschaftlichkeit im Bau- und Wohnungswesen (RfG), the German Reich Society for
Economic Efficiency in Building and Housing (an institution financing the construction
of housing estates), as the first female member of the Committee of German Engineers
(VDI), as a committee member of the German Institute for Norms, as a legislator and as
a collaborator of Walter Gropius (1883–1969) (Fig. 3).3 4 Figure 2: Vote of the penal law committee in the Reichstag, chaired by Prof. Dr. Wilhelm Kahl. ‘Housewives and Architects’: Marie-Elisabeth Lüders’
Management of the New Architecture from Pot Lid
to Siedlung Included in the photo are Prof. Dr. Wilhelm Kahl (Volkspartei) (middle); Justice Minister a.D. Erich
Emminger (left); and Marie[-Elisabeth] Lüders (right). Photograph by Erich Salomon, 1930. Erich
Salomon Archive, Berlinische Galerie. Figure 2: Vote of the penal law committee in the Reichstag, chaired by Prof. Dr. Wilhelm Kahl. Included in the photo are Prof. Dr. Wilhelm Kahl (Volkspartei) (middle); Justice Minister a.D. Erich
Emminger (left); and Marie[-Elisabeth] Lüders (right). Photograph by Erich Salomon, 1930. Erich
Salomon Archive, Berlinische Galerie. Figure 3: Invitation of the Reichsforschungsgesellschaft to experience ‘Das neue Wohnen’
[The New Housing], featuring Lüders as expert, 1929. Bundesarchiv BArch, N 1151/45,
Reichsforschungsgesellschaft. Figure 3: Invitation of the Reichsforschungsgesellschaft to experience ‘Das neue Wohnen’
[The New Housing], featuring Lüders as expert, 1929. Bundesarchiv BArch, N 1151/45,
Reichsforschungsgesellschaft. 5 This essay will investigate how a growing recognition of the value of women’s
work — an equation of the experience of the (female) housewife with the expertise
of the (mostly male) architect — came to play a formative role in the creation of
modern architecture. The history of the kitchen is deeply embedded in that of women’s
movements, and the impact of Taylorism on modern architecture, the technologization
of the domestic sphere, and modernism’s drive toward rationalization have all been
extensively studied (McLeod 1983; Hanisch and Widrich, 1999). As Susan Henderson
details in her book Building Culture, many modern architects forged alliances with
female experts: figures like the economist Erna Meyer and the architect Margarete
Schütte-Lihotzky feature prominently in the literature on ‘scientific management’
(Henderson 2013: 143–202). In fact, accounts of the rationalization of the domestic
mark the usual entry point for women into histories of modern architecture, which
for a long time regarded architects and engineers, the vast majority of them men, as
the sole experts in the field. Meyer became known as an expert on household reform,
Schütte-Lihotzky primarily as a kitchen designer. What Lüders brought to the making
of modern architecture was another kind of expertise: that of managerial supervision. In the spirit of Lüders, this article sets out to reframe modern architecture’s
fascination with standardization and rationalization from the viewpoint of the
household. It approaches domestic labor, not as a set of ‘unprofessional’ practices but
as the seed for the reform of a devastated national economy. ‘Housewives and Architects’: Marie-Elisabeth Lüders’
Management of the New Architecture from Pot Lid
to Siedlung A re-evaluation of female
work in a male-dominated society opened the way for ‘laypeople’ to become involved
in the shaping of modern architecture. In turn, the domestic labor carried out by women
and their expertise in managing ‘household concerns’ reshaped the male domain of
architectural design. In Lüders’ book, it was no longer just Gropius wielding his pencil
but women like herself who were defining the lines of the new architecture. The DIN: Rationalizing Architecture after WWI The German Institute for Norms (DIN), founded by engineers and bureaucrats in 1917 to
optimize the production of military equipment, soon turned its attention to architecture
in the postwar period.4 Along with standardizing industrial production across different
scales, the rapid construction of affordable mass housing for the population was seen
as essential to the nation’s economic recovery. The major obstacle to achieving this
was the shortage of construction materials, such as timber and steel, resulting from
reparation payments and the loss of industrial capacity. In this moment of crisis,
standardization became a means to saving scarce resources. For instance, Waldemar
Hellmich, the DIN’s first director, repeatedly condemned wasteful construction
practices as ‘immoral’, and architects such as Ludwig Hilberseimer argued against
wasteful building practices (Hellmich [n.d.]; Hilberseimer 1927: 3). Architecture was 6 now decomposed into thousands of different parts — staircases, door handles, ceiling
beams, window frames — each item regulated on a norm sheet that precisely prescribed
the conditions for its mass production (Meister 2018). No element was thought to be
too small, no savings too inconsequential. As the DIN’s standardization efforts expanded in the 1920s to take in more and
more objects and professions, a group of ‘housewives’ entered the fray (Reicke 1984). Not just unpaid minders of children or housekeepers, these particular housewives were
experts in spatial organization and ergonomics, decidedly upper middle class, well off
and well educated. The DIN and the RfG, as well as various women’s organizations,
were organized as ‘registered societies’ (eingtragene Vereine) working for the ‘greater
good’ of society.5 As others have noted, the early women’s movements were decidedly
class based (Nolan 1990). For women of the bourgeoisie, volunteer work for this kind
of society became a path toward expertise and professional work. Lüders herself had
worked full-time for five years as a volunteer ‘house carer’ (Wohnungspflegerin) in
Charlottenburg before studying political science. For Lüders, such volunteer work was
a building block of human society, inculcating a sense of responsibility. The DIN: Rationalizing Architecture after WWI And anyone
who dared to think of shirking that responsibility was warned that then they may step outside the circle of civic society, they will no longer be ‘alive’ then they may step outside the circle of civic society, they will no longer be ‘alive’
— they will merely exist as a number in the files of the local residents’ registration
office.6 (Lüders 1961: 15) y
y
p
y,
y
g
— they will merely exist as a number in the files of the local residents’ registration
office.6 (Lüders 1961: 15) Volunteer work, then, was a moral and civic duty. But it also trained the women in
administrative work, fundraising, managing finances, organizing committees, and
teaching other volunteers — all abilities that would prove crucial in making modern
architecture in Germany a success (and an icon that would later be exported around the
world). These housewives were also, it could be said, material feminists in the sense of
Dolores Hayden’s definition: they insisted on the immediate impact on women’s lives
of a redesign of their material working conditions (Hayden 1981). Lüders understood
the improvement and standardization of the domestic realm as a means to reconfigure
women’s spatial, economic and political conditions. Herself a housewife and a
professional, she saw the role of the housewife as extending beyond the household. Occupying two expert roles, that of domestic organizer and of established economist,
she linked the oikos back to oiko-nomia in her work. For Lüders, the household run by
housewives was not qualitatively different from the national scale of the economy run
by politicians. Lüders had pushed for the ‘recruitment of women’ to increase Germany’s industrial production during the war. As she reflected in Das unbekannte Heer, ‘All
thoughts and actions were directed toward a single purpose: the defense of the
Vaterland … the men with weapons — but [what about] us — the women?’ (Lüders 1936: 2).7
Now, with the war over, and Germany on course for a major economic depression,
she saw the continued training and mass employment of women as essential for the
country’s survival. Where the Neues Bauen (New Architecture) aimed to give Germans a new home,
the Neue Haushalt told them how to live most efficiently in it (Meyer 1932). The DIN: Rationalizing Architecture after WWI In 1926,
Lüders (1927) gave a speech, ‘Norming and Household’, at the annual meeting of the
DIN, right after a talk called ‘Norming and the Housing Shortage’ by Walter Gropius
(1927), an early collaborator of the institute, who was then working on his Dessau-
Törten housing estate, conceived as a model for cost-effective mass housing based on
standardized parts and industrial construction processes (Figs. 4 and 5). Figure 4: Walter Gropius’s article ‘Normung und Wohnungsnot’ [Standardization and Housing
Shortage] in Technik und Wirtschaft (1927: 8). Figure 4: Walter Gropius’s article ‘Normung und Wohnungsnot’ [Standardization and Housing
Shortage] in Technik und Wirtschaft (1927: 8). 8 article, ‘Normung und Haushalt’ [Standardization and
Gropius’s article in Technik und Wirtschaft (1927: 10). mung und Haushalt’ [Standardizat
ticle in Technik und Wirtschaft (192 Figure 5: Marie-Elisabeth Lüders’s article, ‘Normung und Haushalt’ [Standardization and
Household], appears directly after Gropius’s article in Technik und Wirtschaft (1927: 10). Figure 5: Marie-Elisabeth Lüders’s article, ‘Normung und Haushalt’ [Standardization and
Household], appears directly after Gropius’s article in Technik und Wirtschaft (1927: 10). Figure 5: Marie-Elisabeth Lüders’s article, ‘Normung und Haushalt’ [Standardization and
Household], appears directly after Gropius’s article in Technik und Wirtschaft (1927: 10). For Lüders, the most prominent female member of the norm committee working
with the German Engineering Association (VDI), it was just as important to establish
DIN norms for pots and cookware as it was to standardize the construction elements —
stairs, doors or windows — that Gropius had talked about. Household objects were not a
separate sphere but an integral part of the larger focus on apartments (Kleinwohnungen)
as a solution to the housing crisis. As she put it, The simplification and improvement of the household starts with the apartment: the
situation, arrangement and configuration of the rooms, but also the type of stairs,
doors, windows, the equipping of the service spaces, particularly the kitchen … To
reach satisfactory solutions for all these tasks, we urgently need the kind of collab
oration between producers, retailers, housewives and architects that DIN is trying to
achieve.8 (‘Jahresversammlung’, 1926) 9 According to historian Nicholas Bullock, while architects like Bruno Taut called for the
need of collaborative reform of German dwellings, it was Lüders who in 1926 urged the
DIN to convene a committee on housing typologies (Typisierung der Wohngebäude)
that actively bridged these two spheres: the design of household items and the making
of architecture (Bullock 1988: 177, 184–185). According to historian Nicholas Bullock, while architects like Bruno Taut called for the
need of collaborative reform of German dwellings, it was Lüders who in 1926 urged the
DIN to convene a committee on housing typologies (Typisierung der Wohngebäude)
that actively bridged these two spheres: the design of household items and the making
of architecture (Bullock 1988: 177, 184–185). The reforms pursued by modern architects and the DIN related to both the built
environment and the domestic life that took place within it. Soon after regulating
the architectural elements itemized by Lüders, the DIN undertook to standardize
household objects such as jars and cookware. Since it was not a government agency but
depended on (largely voluntary) market compliance, it commissioned advertisements
depicting its vision of a DIN-ordered universe. One ad showed a shelf with a diverse
array of Weck jars, each with its own lid size; proclaiming ‘Unification saves Time and
Money’, it demonstrated how the introduction of a new norm, where ‘one lid fits all’,
would liberate the housewife from the hassle of going to several shops to find a suitable
fit (Fig. 6) (Deutscher Normenausschuss 1927). Figure 6: Advertisement for standardized Weck jars by the DIN [then NDI], 1926. Bundesarchiv
BArch, N 1151/43. Figure 6: Advertisement for standardized Weck jars by the DIN [then NDI], 1926. Bundesarchiv
BArch, N 1151/43. The stories of the DIN and the modern Siedlung are often told as histories of men —
engineers, architects and bureaucrats — standardizing objects, convening in committees
and founding institutions, strategizing the systematization of the world. When women 10 enter the story, it is as contributors to what architectural history long categorized as
‘female’ domains: textile design, for example, or culinary skills, both located in the
domestic sphere. The assumed gender separation of competence and authority was
deployed by the men in power both discriminatorily, of course, but also strategically. While this separation was not necessarily contested by the women’s movement of
early-1920s Germany, it was precisely the longstanding trust in women’s ‘spheres of
competence’ that made them powerful players in the larger project of reforming the
nation. The same influence can be seen behind the widespread take-up of Fordism and
Taylorism, the success of which ‘gave engineers a new role in the organization and
administration of larger industrial firms’ (Rabinbach 1990: 276). Male authors such as
the German economist Friedrich von Gottl-Ottlilienfeld wrote glowing endorsements
of Fordism and technological reason (1926), but the generation of architects who
were contemporaries of Margaret Schütte-Lihotzky were influenced more by books
such as The New Housekeeping: Efficiency Studies in Home Management (1913) and
Household Engineering: Scientific Management in the Home (1919) by the American home
economist Christine Frederick, which were quickly translated into German and found
their counterpart in Erna Meyer’s bestselling volume, Der neue Haushalt (1926). Meyer
even reviewed the model housing estates of the RfG in the organization’s own journal
(Reichsforschungsgesellschaft 1929: 34; Frederick 1919a; Frederick 1919b; Meyer 1926). While (male) engineers seemed to draw the plans, it was women who could — albeit in
very different ways — extend their reach beyond the expert audience. As Mary Nolan notes, the ‘liberation’ of women seems to have provided the
middle classes with scope to reform working class life — an approach that sometimes
‘conflicted with working class realities’ (Nolan 1990: 551). Susan Henderson links the
increasing criticism of the women’s movement to its insistence on freeing the housewife
from ‘drudgery’. Such liberation was contingent on the woman staying within her
given domestic boundaries, where she would be ‘freed for brief moments through the
application of techniques invented by authorities in the professional world’ — in other
words, it was only a temporary respite (Henderson 2013: 159). Lüders, however, was
less interested in giving women a break. Her ambitions went further: Women should not be liberated from the scrub bucket and duster because they are
lazy. They should be liberated from the burden and the ‘treacherous object’ in their
housework to liberate intellectual, spiritual and cultural values and to enhance the
fulfilment of their duties as mothers and citizens.9 (Lüders 1927: 11; Nolan 1990: 568) Lüders was herself a product of the 19th-century Bildungsbürgertum, the educated
upper middle class. As she saw it, the emancipatory potential of education could only 11 be fully realized by taking on new roles outside the household. In this context, the
desired collaboration between ‘housewives and architects’ was ‘not just an economic,
but a political and cultural task of the utmost importance’ (Lüders 1927: 10).10 It was
imperative to find solutions, otherwise ‘culture will disappear in the scrub bucket and
humanity will get caught up in the duster’ (‘Jahresversammlung’, 1926).11 Rather than getting caught up in the duster, the goal of the expert housewife was
to eliminate all the obstacles, unnecessary work and material excess that undermined
those essential intellectual, spiritual and cultural values — and the nation’s economy. After all, for Lüders ‘home economics is macro-economics!’ (Lüders 1927: 13):
‘housewives’ were not just informed consumers of standardized goods (and therefore
participants in the national economy), but culture-makers on a par with architects or
politicians. Hence, their work was as important for the norm effort as that of the DIN
engineers. While acknowledging that ‘up to now, a large part of the German population
has not conceived of home economics as part of the national economy’, Lüders pushed
the DIN’s agents to develop norms for homes not only in relation to architecture but
in domestic terms (Lüders 1927: 10).12 And the DIN followed up (Fig. 7). A report of a
meeting on 5 March 1927 to discuss norms for the Kleinwohnung lists as participants
‘representatives of the architecture profession, housewife committees, furniture
sellers, houseware stores and the furniture industry’ (‘Mitteilungen des Deutschen
Werkbundes’ 1927).13 Figure 7: Advertisement for standardized cooking pots by the DIN [then DNA], 1928. Bundesarchiv BArch, N 1151/43. Figure 7: Advertisement for standardized cooking pots by the DIN [then DNA], 1928. Bundesarchiv BArch, N 1151/43. 12 The RfG: Managing the Modern Housing Estates Lüders’ contribution to reconfiguring domestic life in a time of economic and
political crisis went beyond her involvement in the DIN’s norming of parts. In 1927,
together with the architects Walter Gropius and Otto Bartning, Lüders founded
the influential Reichsforschungsgesellschaft für Wirtschaftlichkeit im Bau- und
Wohnungswesenwhich, or RfG, aiming to foster economic efficiency in housing and
the building industry (Fleckner 1993). An embodiment of the desired ‘collaboration
between housewives and architects’, the institution helped to finance modernist icons
such as the Weißenhofsiedlung in Stuttgart. Its scope reached across all scales: as
Lüders put it, the RfG was founded to investigate all elements that have a bearing on costs and rent; from road planning,
utility connections, organization of the plan and the standardization of construction
elements to the interior fittings and the various facilities that lighten the workload
of the housewife and mother (laundry room, kindergarten, green spaces and play
areas).14 (Lüders 1963: 114) Addressing the Reichstag in 1927, Lüders raised the issue of the misuse of public
funds and corruption in the development of some prototype housing estates. The
RfG, she said, would exercise a much tighter control, ‘closely observing the entire
construction process’. In fact, she continued, ‘I do not think it too bold to say that
if we were only to calculate the actual building costs — [that is,] if everyone were to
forgo their “silent shares” — then private capital would be in a position to re-engage
with the housing market today’ (Fleckner 1993: 28).15 To produce these new, efficient,
family-friendly and corruption-free modern estates, the RfG would not commission
the developments directly but would act as what we would now call a project manager
(Reichsforschungsgesellschaft 1927).16 Approaching modernist mass housing from the
viewpoint of managerial oversight — a key function of fundraising and volunteer work
— Lüders was the one who connected the work of the architects with everyday reality
by ensuring financial structures, timelines and a key focus. In this way, she formed the
crucial link between designers and politics, between ideas and their translation into
built form. But rather than acting as a distant patron — a fundraiser for a modernist
pipe-dream — Lüders cooperated with the architects as much as they cooperated with
her. The RfG: Managing the Modern Housing Estates As a public institution, the RfG’s aim was to completely overhaul social housing,
both its form and its financing, and it saw modern architecture as a means by which to
realize this vision. The stated goals of the RfG were to Addressing the Reichstag in 1927, Lüders raised the issue of the misuse of public
funds and corruption in the development of some prototype housing estates. The
RfG, she said, would exercise a much tighter control, ‘closely observing the entire
construction process’. In fact, she continued, ‘I do not think it too bold to say that
if we were only to calculate the actual building costs — [that is,] if everyone were to
forgo their “silent shares” — then private capital would be in a position to re-engage
with the housing market today’ (Fleckner 1993: 28).15 To produce these new, efficient,
family-friendly and corruption-free modern estates, the RfG would not commission
the developments directly but would act as what we would now call a project manager
(Reichsforschungsgesellschaft 1927).16 Approaching modernist mass housing from the
viewpoint of managerial oversight — a key function of fundraising and volunteer work — Lüders was the one who connected the work of the architects with everyday reality
by ensuring financial structures, timelines and a key focus. In this way, she formed the
crucial link between designers and politics, between ideas and their translation into
built form. But rather than acting as a distant patron — a fundraiser for a modernist
pipe-dream — Lüders cooperated with the architects as much as they cooperated with
her. As a public institution, the RfG’s aim was to completely overhaul social housing,
both its form and its financing, and it saw modern architecture as a means by which to
realize this vision. The stated goals of the RfG were to — Lüders was the one who connected the work of the architects with everyday reality
by ensuring financial structures, timelines and a key focus. In this way, she formed the
crucial link between designers and politics, between ideas and their translation into
built form. But rather than acting as a distant patron — a fundraiser for a modernist
pipe-dream — Lüders cooperated with the architects as much as they cooperated with
her. The RfG: Managing the Modern Housing Estates As a public institution, the RfG’s aim was to completely overhaul social housing,
both its form and its financing, and it saw modern architecture as a means by which to
realize this vision. The stated goals of the RfG were to 13 develop and disseminate economic forms of construction elements, apartments and
houses, economic methods of site preparation, cost calculation, construction pro
cesses and, more generally, the highest possible economic efficiency in the housing
and construction industry.17 (Reichsforschungsgesellschaft 1927) To achieve this, Lüders secured funding — 10 million Reichsmark — from the Reichstag,
which also granted the RfG tax benefits as an association (Verein) whose work was seen
to be of ‘public utility’ (Fleckner 1993: 28). To achieve this, Lüders secured funding — 10 million Reichsmark — from the Reichstag,
which also granted the RfG tax benefits as an association (Verein) whose work was seen
to be of ‘public utility’ (Fleckner 1993: 28). Lüders would continue to stress the parallels between domestic labor and the
construction industry, referring to households as ‘domestic businesses’ and pointing
out the economic power of the ‘19 million housewives’ in the Weimar Republic. In a
speech in 1928, reported in the journal of the Deutscher Städtetag (German Association
of Cities and Towns), Lüders used ‘examples of the huge values transferred to domestic
consumption and usage’ to emphasize ‘the immense importance of household
economies for the construction industry and the national economy as a whole’. The
report continues: [Lüders] asked for homes that would fulfill their economic and cultural purpose,
namely the economic running of the household and the physical, spiritual and emo
tional nurturing of the family. This requirement could only be satisfied if the apart
ments were consciously and intentionally built from the inside out, if the buildings
were constructed and equipped from the usage of the apartment, ‘from the cooking
pot to the facade’, so to speak.18 (Die Rationalisierung 1928: 531) For Lüders, modern architecture was to be constructed from the inside out, in accordance
with the needs of the family and the labor of women. As such, her modernism was
centered around domestic labor. In contrast to more famous examples, such as Erna
Meyer’s books, this domestic labor was not only defined ergonomically but was itself a
determinant of the form of the architecture. The RfG: Managing the Modern Housing Estates For Lüders, modern architecture was to be constructed from the inside out, in accordance
with the needs of the family and the labor of women. As such, her modernism was
centered around domestic labor. In contrast to more famous examples, such as Erna
Meyer’s books, this domestic labor was not only defined ergonomically but was itself a
determinant of the form of the architecture. In its brief four-year existence, the RfG assessed and co-financed housing
developments such as Gropius’s Dessau-Törten estate (350,800 Reichsmark in credits
and subsidies) and the Weißenhofsiedlung in Stuttgart (150,000 Reichsmark) (Fleckner
1993: 52–53). It organized a ‘technical symposium’ to present research findings;
speakers included Gropius, Bartning, Meyer and Schütte-Lihotzky, among many
others. The RfG also published brochures detailing its recommendations for improving
the efficiency of housing construction, always with the ideals of the affordable 14 Kleinwohnung and standardization through DIN norms in mind. However, the most
ambitious project, intended to provide insight into the ‘entire building process’
(Fleckner 1993: 28), from the very first sketch to the final balancing of the books, was
Spandau-Haselhorst, a prototype housing estate for 10,000 people. The competition
for its design, which attracted 221 entries, was won by Gropius, an insider of sorts, in
collaboration with Stephan Fischer. Gropius provided the essential plans and initially
acted as head of the group of architects. However, the built scheme was, after much
criticism, designed by Paul Mebes, Fred Forbát and Paul Emmerich (Fig. 8). Figure 8: The ‘Reichsforschungssiedlung’ Haselhorst (here the section designed by Paul Mebes),
under construction. Photographer unknown, undated [ca. 1931]. Architekturmuseum TUM. Figure 8: The ‘Reichsforschungssiedlung’ Haselhorst (here the section designed by Paul Mebes),
under construction. Photographer unknown, undated [ca. 1931]. Architekturmuseum TUM. Wanting to ensure the objectivity of the research, the RfG did not engage directly with
the project as a client, but instead commissioned Heimag (Gemeinnützige Heimstätten
AG) — a subsidiary of a company with experience in rationalized construction processes
— to act as the developer. However, this splitting of responsibilities led to friction during
the planning process. Even Bartning, a staunch supporter of the attempt to research the
entire process of development, argued that this task was effectively made impossible 15 by the lack of direct oversight from the commissioning body. Heimag’s financing
models were also questioned by, among others, the architect Martin Wagner in his role
as building commissioner (Stadtbaurat) of Berlin. The RfG: Managing the Modern Housing Estates RfG ended up canceling its contract
with Heimag, and the firm went on to build the estate without supplying the research
material that the RfG had hoped for (Fleckner 1993: 62). Even the advertisement for
the project’s development prospectus, featuring a collage by none other than László
Moholy-Nagy, presented the project as a lush garden oasis without any hint of the
modernist architecture that the RfG wanted to test (Fig. 9). Figure 9: László Moholy-Nagy, cover design for Spandau-Haselhorst Housing Development
Prospectus, about 1928–1929. Gelatin silver print, 42.7 × 49.4 cm. The J. Paul Getty Museum,
Los Angeles. © Estate of László Moholy-Nagy/Artists Rights Society (ARS), New York. Figure 9: László Moholy-Nagy, cover design for Spandau-Haselhorst Housing Development
Prospectus, about 1928–1929. Gelatin silver print, 42.7 × 49.4 cm. The J. Paul Getty Museum,
Los Angeles. © Estate of László Moholy-Nagy/Artists Rights Society (ARS), New York. Housewives: A Managerial Profession Lüders was unconvinced by architects’ attempts to resolve the housing crisis without
the insights of the housewife. Disappointed at how the Haselhorst project had played
out, she pinned some of the blame on the architects, and especially on Gropius, who 16 painfully combined ‘unfinished technical work with pompous art-speak and pushy
salesmanship’ (Fleckner 1993: 61).19 It was unseemly, she thought, that Gropius should
behave in this way when he held such a prominent role in the RfG and was actively
engaged in building for the association. The ‘salesmanship’ she detected in Gropius
was a far cry from the managerial expertise she attributed to the housewife. But others had a different take on the travails of the RfG. In a 1929 issue of Die Form,
Ludwig Hilberseimer leapt to the defense of the male architects: Many women, Marie-Elisabeth Lüders among them, tend to blame the architects
for the badly designed apartments. They forget that housing used to be a matter for
[speculative developers] before the war … It was only after the war that architects
were called on to design housing.20 (Hilberseimer 1929: 295) While Hilberseimer did not dispute that there were problems — qualitative and
quantitative — with postwar housing, he simply did not see how housewives could
meaningfully contribute to its improvement. ‘After all’, he insisted, ‘housewives
managed to live before the war without finding it necessary to address the damage done
by speculative housing developments’ (Hilberseimer 1929: 295).21 As Hilberseimer saw
it, the RfG had misconstrued its core purpose: rather than the construction of houses,
the committee ought to be focusing on their use: It is not the role of the RfG to build mass housing; rather, its role is to test new
apartment floor plans for their usability, alongside the constructive, thermal,
hygienic and spatial-economic issues; in that way, the experience of the inhabitants
would build an important foundation for further work. For such experiments it is
more practical to build a smaller number of apartments each year, but to make them
as different from each other as possible.22 (Hilberseimer 1929: 295; emphasis mine) In short, Hilberseimer was assigning to Lüders and her team at the RfG precisely the
role that women still performed for architects (and historians) in the making of modern
architecture: that of the housewife optimizing the use of the kitchen. Housewives: A Managerial Profession In the process,
he was inadvertently making her more responsible (read: to blame) for the RfG’s
failings than his architect colleague Gropius. But instead of experimenting formally,
Lüders wanted to experiment managerially, applying the oversight of that new female
profession: the housewife. Rather than being limited to commissioning a housing
project or optimizing domestic space, she saw the RfG’s task in terms of testing the
entire building process, from the preparatory work of investigating users’ needs to the
post-construction evaluation of the resources used (both financial and material). 17 So, who benefited most from the collaboration between this new female profession
and the established male expert, the architect? Some say that architects only ventured
into the domestic sphere in the wake of its post–WWI industrialization; others argue
that, with the urgent need for affordable mass housing in the 1920s, the women’s
movement focused minds on the idea of the ‘New Household’, which architects then
used as an opportunity to rationalize the domestic sphere — and housing as a whole
(Bullock 1988: 190). In 1924, the architect Bruno Taut defined the collaboration between
housewife and architect as ‘the architect thinks, the housewife steers’ (1924: 104).23
While the catchy phrase has been criticized as dismissive, it has been interpreted by
others as an indication of the housewife’s influence on the shaping of the modernist
mass housing (Bullock 1988: 177). I would argue, instead, that Taut’s formulation
points to an essential truth: the increasing influence of the housewife was to be gauged
less in terms of conventional creative practice and more in terms of a managerial
(literally, steering) function. The New ‘Insiders’ of Modern Architecture As historians of architecture have shown time and again, architecture is shaped not
only by a diverse array of people but by financial streams and bureaucratic processes
(Abramson 2016; Aggregate 2012; Stevens 2016). When read within that context,
Lüders’ attempt to liberate women ‘from the mass of objects’ and ‘small household
tasks’ was intended to allow them to ‘work on larger issues that go beyond the
narrow frame of the house and the family’ (Lüders 1927: 14).24 For Lüders, seeing
the micro-scale of the cooking pot as inherently linked to questions of national
economy was key to bringing about societal and economic change. In fact, she
claimed that it is an abuse of women’s power for family and state if we stay within our four walls,
scrubbing and polishing, thinking with false pride ‘my home, my world’. Instead, we
should see our home in the world.25 (Lüders 1927: 11; emphasis mine) As Lüders saw it, the ‘larger’, more important role of women was to contribute their
expertise to the conception and execution of modern architecture’s forms and façades,
and also, through this, to work toward the liberation of women as politically active,
civic agents. So, what were Lüders’ real aims in joining the DIN and co-founding the RfG — and
did the RfG indeed end in ‘failure’, as historian Sigurd Fleckner puts it in the subtitle of
his account (‘Entwicklung und Scheitern’)? Lüders had suggested in 1929 that the RfG 18 should evolve into a research institute — a proposal that was ultimately declined — and
the association was dissolved in 1931, only four years after its foundation. One of the
reasons for its premature demise was undoubtedly historical contingency, as Fleckner
explains: at a time of rising unemployment, it was no longer so essential to save labor
resources (Fleckner 1993: 108). But while this may have diminished the urgency of the
RfG’s task, other organizations, such as the DIN, continued to thrive — and the DIN’s
whole remit was to conserve material and energy resources through rationalization. The magazine Bauwelt suggested a more mundane problem at the heart of the RfG: ‘the
conflict of interests, emblematic of contemporary economic life’. More precisely, the
project had started with great gusto, but it was dominated by a handful of architects,
and soon those who felt excluded from the work began to complain (Fleckner 1993:
111). The New ‘Insiders’ of Modern Architecture One might see this as a problem of the privilege and aesthetic dominance of the
so-called avant-garde. And yet the lack of formal experimentation was one of the
areas, according to Hilberseimer, in which ‘the RfG has completely failed. It has largely
been satisfied with existing models and shied away from addressing new requirements
and possibilities’ (Hilberseimer 1929: 295).26 Mass producing such ‘existing models’,
however, did not yield the economic benefits Lüders and Gropius had hoped for. Just as
the construction costs for the Dessau-Törten housing skyrocketed, so the costs for the
appliances and fittings recommended by the RfG were not as affordable as Lüders had
claimed. The disconnect between an ideally planned future for the masses and the material
and lived reality is an increasingly forceful strand in the narrative of modern
architecture. In the case of the RfG, the misalignment of aesthetic promise and actual
cost was perhaps also grounded in Lüders’ own failure to grasp what ‘affordable’ might
look like for the working class: as a bourgeois, well-educated woman, she planned for
housewives of a different kind (Lüders 1927: 14; Lüders 1961: 5; Bullock 1988: 190). Lüders’ story in modern architecture is not a simple narrative of radical rescue or
long-overdue liberation. In fact, much of her approach was not aimed at fundamentally
redefining women’s roles (not least because, as was discussed above, women seemed
to be doing just fine when it came to taking on new roles, as Lüders acknowledged);
rather, it sought to recognize and scale up women’s collective power. The aggregation
of individual households would mean that every small change to the domestic sphere
had a real effect on the national economy. And yet, what Lüders really proposed — and brought into being — was the expansion
of a field: the extension of the female gaze to oversee a male discipline. Lüders, one
could say, did not just highlight a shift in women’s expertise but reconfigured the
whole sequence of designing modern architecture. For her, ‘form followed function’ 19 — only form, in this instance, was not an abstract functionalist aesthetic (which she
criticized) but the very logic of a new domestic sphere that defined the architecture. By
asking women to not only give form to kitchens but to shape housing developments
and, ultimately, even the national economy, Lüders extended the role of the housewife. Notes Notes 1 ‘Rasselnd und zischend sausten glühende Drahtschlangen auf der Walzenstrasse heran. Eine Kolonne kräftigster Frauen
stand in Männerkleidung und Lederschurz fangbereit mit riesigen Zangen am Ende der Straße. Ein harter Griff, die
funkensprühende Schlange war gepackt und flog mit einem mächtigen sicheren Schwung auf die nächste ratternde
Bahn … Die Frau segelte ruhig hin und her zwischen dunklen Schrottbergen und leuchtenden Ofengräbern. … Früher
nähte die Frau in dem schwebenden Haus Jacken und Mäntel, über die Maschine gebückt, die Augen auf den Tretfuß,
die Hände an den Stoff gebannt. Heute gleitet ihr Blick hin und her über weite Räume, über lange Strecken und Halden’. 2 In architectural history, this categorization became most evident (and later disputed) in relation to the Bauhaus weaving
workshop, for example, and remained historiographically fixed until the early 1990s, when Sigrid Wortmann Weltge
published Bauhaus Textiles: Women Artists and the Weaving Workshop. Since then, there have been monographs on the
Bauhaus weaving workshop master Gunta Stölzl and an investigation of gender in the school by Anja Baumhoff. Virginia
Gardner Troy’s monograph on Anni Albers has examined the interest of Bauhaus weavers (and modern German artists
generally) in ancient textile artifacts and techniques from South America. Most recently, T’ai Smith and Leah Dickerman
have published on the Bauhaus weaving workshops, countering those assumptions. Anja Baumhoff, The Gendered World
of the Bauhaus: The Politics of Power at the Weimar Republic’s Premier Art Institute, 1919–1932 (Frankfurt am Main: Peter
Lang, 2001); T’ai Smith, Bauhaus Weaving Theory: From Feminine Craft to Mode of Design (Minneapolis: University of Min
nesota Press, 2014); Monika Stadler, Gunta Stölzl: Bauhaus Master (New York: Hatje Cantz, 2009); Virginia Gardner Troy,
Anni Albers and Ancient American Textiles: From Bauhaus to Black Mountain (Burlington, VT: Ashgate, 2002); Sigrid Wort
mann Weltge, Bauhaus Textiles: Women Artists and the Weaving Workshop, new edition (London: Thames & Hudson, 1998). 3 The term ‘collaborator’ is an increasingly contested one for its downplaying of the agency and contributions of those
who had worked with and for Gropius. For example, recent scholarship shows that Adolf Meyer and Carl Fieger, whom
Gropius (and, for a long time, historians) called collaborators or employees, were crucial not just for drawing projects in
the Atelier Gropius but for designing and conceptualizing them. Notes Where Winfried Nerdinger still stresses Gropius’s posi
tion as the master of his designs ‘by verbal communication’ (seeing how Gropius almost never drew himself), scholars like
Bernd Polster and others are uncovering increasing evidence to the contrary. See Fiona MacCarthy, Gropius: The Man Who
Built the Bauhaus (Cambridge: Harvard University Press, 2019); Winfried Nerdinger, Walter Gropius: Architekt der Moderne
1883–1969 (Munich: C.H. Beck, 2019); Bernd Polster, Walter Gropius: der Architekt seines Ruhms (Munich: Carl Hanser,
2019). 4 After its founding in 1917 as Normenausschuß der deutschen Industrie, in 1926 this committee was named Deutscher
Normenausschuß (DNA) and only later renamed Deutsches Institut für Normung (DIN). The latter is the far-better-
known denomination and hence used throughout this piece. Conventionally Norm is translated into English as ‘stand
ard’ for the name of the organization, but as I explain in this article, the association with ‘norm’ is crucial. 5 This was, of course, not just a female construct: the DIN Institute, for example, was (and still is) a ‘Verein’ and consisted of
almost exclusively men. But overall, volunteer work was a female occupation. 5 This was, of course, not just a female construct: the DIN Institute, for example, was (and still is) a ‘Verein’ and consisted of
almost exclusively men. But overall, volunteer work was a female occupation. 6 ‘[dann] stell sich ein jeder außerhalb des Kreises der bürgerlichen Gemeinschaft, er ‘lebt’ nicht mehr — er ist nur noch eine
Nummer in der Kartei des Einwohnermeldeamtes’. 6 ‘[dann] stell sich ein jeder außerhalb des Kreises der bürgerlichen Gemeinschaft, er ‘lebt’ nicht mehr — er ist nur noch eine
Nummer in der Kartei des Einwohnermeldeamtes’. 7 ‘Alles Denken, Wollen und Handeln hatte nur ein Ziel: Verteidigung des Vaterlandes … Die Männer mit der Waffe — und
wir — die Frauen?’ 7 ‘Alles Denken, Wollen und Handeln hatte nur ein Ziel: Verteidigung des Vaterlandes … Die Männer mit der Waffe — und
wir — die Frauen?’ 8 ‘Die Vereinfachung und Verbesserung des Haushalts beginnt bei der Wohnung: Lage, Einteilung und Gestaltung der
Räume, Art der Treppen, Türen und Fenster, Ausgestaltung der Wirtschaftsräume, vor allem der Küche. … Zu befriedi
genden Lösungen aller dieser Aufgaben ist Zusammenarbeit zwischen Erzeugern, Händlern, Hausfrauen und Architekten,
wie sie der Normenausschuß erstrebt, dringend erforderlich’. The New ‘Insiders’ of Modern Architecture Beyond a ‘new household’ and through ‘Neues Bauen’, Lüders tilted a housewife’s
tasks, skills and duties toward a new understanding of domestic expertise: women
were no longer simply inside, but insiders. — only form, in this instance, was not an abstract functionalist aesthetic (which she
criticized) but the very logic of a new domestic sphere that defined the architecture. By
asking women to not only give form to kitchens but to shape housing developments
and, ultimately, even the national economy, Lüders extended the role of the housewife. Beyond a ‘new household’ and through ‘Neues Bauen’, Lüders tilted a housewife’s
tasks, skills and duties toward a new understanding of domestic expertise: women
were no longer simply inside, but insiders. 20 Notes Notes 15 ‘Wenn wir uns hier einmal gemeinsam mit dem RAM zusammentun, um durch die genaue Beobachtung des ganzen
Bauvorgangs, des verwendeten Materials, der Vorkalkulation, der Nachkalkulation, der Preisgestaltung für die Mieten
und für alles, was mit dem ganzen Bau zusammenhängt, einwandfreie Unterlagen herauszukriegen, so glaube ich, es
wird keine zu kühne Behauptung sein, daß, wenn wirklich nur die tatsächlichen Baukosten aufgerechnet werden, die
entstanden sind, auch heute schon das Privatkapital im weiten Maße in der Lage wäre, sich wieder auf dem Wohnungs
markt zu betätigen, wenn jeder auf “stille Verdienste” verzichtet’. 15 ‘Wenn wir uns hier einmal gemeinsam mit dem RAM zusammentun, um durch die genaue Beobachtung des ganzen
Bauvorgangs, des verwendeten Materials, der Vorkalkulation, der Nachkalkulation, der Preisgestaltung für die Mieten
und für alles, was mit dem ganzen Bau zusammenhängt, einwandfreie Unterlagen herauszukriegen, so glaube ich, es
wird keine zu kühne Behauptung sein, daß, wenn wirklich nur die tatsächlichen Baukosten aufgerechnet werden, die
entstanden sind, auch heute schon das Privatkapital im weiten Maße in der Lage wäre, sich wieder auf dem Wohnungs
markt zu betätigen, wenn jeder auf “stille Verdienste” verzichtet’. 16 ‘Die RfG tritt nicht selbst als Bauherr oder Bauunternehmer auf’. 16 ‘Die RfG tritt nicht selbst als Bauherr oder Bauunternehmer auf’. 17 ‘wirtschaftliche Formen von Bauteilen, Wohnungen und Wohnhäusern, wirtschaftliche Verfahren für Geländeer
schließung, Kostenberechnung, Bauausführung, überhaupt die höchste Wirtschaftlichkeit im Bau- und Wohnungswesen
zu ermitteln und zu verbreiten’. 17 ‘wirtschaftliche Formen von Bauteilen, Wohnungen und Wohnhäusern, wirtschaftliche Verfahren für Geländeer
schließung, Kostenberechnung, Bauausführung, überhaupt die höchste Wirtschaftlichkeit im Bau- und Wohnungswesen
zu ermitteln und zu verbreiten’. 18 ‘Die Rednerin betonte die engen, bisher meist übersehenen Zusammenhänge zwischen Haus- und Bauwirtschaft und
knüpfte an die Tatsache an, dass die von der Bauwirtschaft zu errichtenden Wohnungen den Arbeitsplatz für die mate
rielle Bedürfnisbefriedigungen der Menschen und der Schauplatz auch für den kulturellen und ideellen Inhalt des Fami
lienlebens sein sollen und dass in ihnen 19 Millionen Hausfrauen für 12 Millionen hauswirtschaftlicher Betriebe diesen
Forderungen gerecht werden sollen … Sie verlangte, dass die zu errichtenden Wohnbauten den einfachen praktischen
Forderungen entsprechen, die zu Erfüllung der wirtschaftlichen und kulturellen Wohnfunktion notwendig sind, d.h. zur
wirtschaftlichen Führung des Haushalts und zur körperlichen, geistigen und seelischen Pflege der Familie. Notes Dieser Forde
rung kann nur genügt werden, wenn endlich bewusst und gewollt von innen nach außen gebaut wird, wenn die Bauten
aus dem Verwendungszweck der Wohnung heraus konstruiert und ausgestatte werden, sozusagen “Vom Kochtopf zur
Fassade”’. 18 ‘Die Rednerin betonte die engen, bisher meist übersehenen Zusammenhänge zwischen Haus- und Bauwirtschaft und
knüpfte an die Tatsache an, dass die von der Bauwirtschaft zu errichtenden Wohnungen den Arbeitsplatz für die mate
rielle Bedürfnisbefriedigungen der Menschen und der Schauplatz auch für den kulturellen und ideellen Inhalt des Fami
lienlebens sein sollen und dass in ihnen 19 Millionen Hausfrauen für 12 Millionen hauswirtschaftlicher Betriebe diesen
Forderungen gerecht werden sollen … Sie verlangte, dass die zu errichtenden Wohnbauten den einfachen praktischen
Forderungen entsprechen, die zu Erfüllung der wirtschaftlichen und kulturellen Wohnfunktion notwendig sind, d.h. zur
wirtschaftlichen Führung des Haushalts und zur körperlichen, geistigen und seelischen Pflege der Familie. Dieser Forde
rung kann nur genügt werden, wenn endlich bewusst und gewollt von innen nach außen gebaut wird, wenn die Bauten
aus dem Verwendungszweck der Wohnung heraus konstruiert und ausgestatte werden, sozusagen “Vom Kochtopf zur
Fassade”’. 19 ‘unreife technische Arbeiten mit volltönenden künstlerischen Redewendungen und einer recht weitgehenden Geschäfts
tüchtigkeit in peinlicher Weise verbanden’. 19 ‘unreife technische Arbeiten mit volltönenden künstlerischen Redewendungen und einer recht weitgehenden Geschäfts
tüchtigkeit in peinlicher Weise verbanden’. 20 ‘Mit Maria Elisabeth Lüders sind viele Frauen sehr geneigt, die Architekten für die schlechten Wohnungen verantwortlich
zu machen. Sie vergessen, daß der Wohnungsbau vor dem Kriege eine Spekulationsangelegenheit … Erst in der Nach
kriegszeit wurden die Architekten zum Wohnungsbau herangezogen’. 20 ‘Mit Maria Elisabeth Lüders sind viele Frauen sehr geneigt, die Architekten für die schlechten Wohnungen verantwortlich
zu machen. Sie vergessen, daß der Wohnungsbau vor dem Kriege eine Spekulationsangelegenheit … Erst in der Nach
kriegszeit wurden die Architekten zum Wohnungsbau herangezogen’. 21 ‘Außerdem lebten die Hausfrauen ja auch schon vor dem Kriege, ohne daß sie es damals für nötig fanden, sich um die mit
dem Spekulationswohnungsbau zusammenhängenden Schäden zu kümmern’. 21 ‘Außerdem lebten die Hausfrauen ja auch schon vor dem Kriege, ohne daß sie es damals für nötig fanden, sich um die mit
dem Spekulationswohnungsbau zusammenhängenden Schäden zu kümmern’. Notes 22 ‘Es ist nicht Aufgabe der Reichsforschungsgesellschaft, in großem Ausmaß Wohnungen zu bauen, ihre Aufgabe ist viel
mehr neben konstruktiven, wärmetechnischen, hygienischen und raumwirtschaftlichen Problemen neue Wohnungs
grundrisse auf ihre Brauchbarkeit hin auszuprobieren, wobei die Erfahrungen der Bewohner dieser Wohnungen eine
wichtige Grundlage für die Weiterarbeit bilden. Für diese Versuche ist es praktischer, jährlich weniger aber dafür mög
lichst verschiedenartige Wohnungen bauen zu lassen’. 22 ‘Es ist nicht Aufgabe der Reichsforschungsgesellschaft, in großem Ausmaß Wohnungen zu bauen, ihre Aufgabe ist viel
mehr neben konstruktiven, wärmetechnischen, hygienischen und raumwirtschaftlichen Problemen neue Wohnungs
grundrisse auf ihre Brauchbarkeit hin auszuprobieren, wobei die Erfahrungen der Bewohner dieser Wohnungen eine
wichtige Grundlage für die Weiterarbeit bilden. Für diese Versuche ist es praktischer, jährlich weniger aber dafür mög
lichst verschiedenartige Wohnungen bauen zu lassen’. 23 ‘Der Architekt denkt — die Hausfrau lenkt’. 23 ‘Der Architekt denkt — die Hausfrau lenkt’. 24 ‘Befreien wollen wir die Frauen von der Masse der Objekte, um den Menschen besser dienen zu können … Befreiung vom
Kleinen im Haushalt zum Zweck der Arbeit im Großen auch über den engsten Rahmen des Hauses und der Familie hinaus’. 24 ‘Befreien wollen wir die Frauen von der Masse der Objekte, um den Menschen besser dienen zu können … Befreiung vom
Kleinen im Haushalt zum Zweck der Arbeit im Großen auch über den engsten Rahmen des Hauses und der Familie hinaus’. 25 ‘Nicht weil wir aus dem Hause hinausdrängen aus Abneigung gegen das Haus und die Hauswirtschaft, sondern weil wir
meinen, daß es ‘mißbrauchte Frauenkraft’ für Familie und Staat ist, wenn wir scheuernd und putzend auf die vier Wände 24 ‘Befreien wollen wir die Frauen von der Masse der Objekte, um den Menschen besser dienen zu können … Befreiung vom
Kleinen im Haushalt zum Zweck der Arbeit im Großen auch über den engsten Rahmen des Hauses und der Familie hinaus’. 25 ‘Nicht weil wir aus dem Hause hinausdrängen aus Abneigung gegen das Haus und die Hauswirtschaft, sondern weil wir
meinen, daß es ‘mißbrauchte Frauenkraft’ für Familie und Staat ist, wenn wir scheuernd und putzend auf die vier Wände
beschränkt bleiben mit dem mißverstandenen Stolz ‘Mein Haus, meine Welt’, anstatt auch in der Welt unser Haus zu
sehen. Dazu aber müssen wir weit mehr als heute von dem Haus befreit werden’. Notes 8 ‘Die Vereinfachung und Verbesserung des Haushalts beginnt bei der Wohnung: Lage, Einteilung und Gestaltung der
Räume, Art der Treppen, Türen und Fenster, Ausgestaltung der Wirtschaftsräume, vor allem der Küche. … Zu befriedi
genden Lösungen aller dieser Aufgaben ist Zusammenarbeit zwischen Erzeugern, Händlern, Hausfrauen und Architekten,
wie sie der Normenausschuß erstrebt, dringend erforderlich’. 9 ‘Die Frauen sollen nicht aus Faulheit vom Scheuereimer und Staubtuch frei werden, sie sollen frei werden von der Last und
“Tücke des Objekts” in der Hauswirtschaft, um des Freiwerdens geistiger, seelischer, kultureller Werte, um der erhöhten
Erfüllung mütterlicher und staatsbürgerlicher Pflichten willen’. Translation Nolan’s. 9 ‘Die Frauen sollen nicht aus Faulheit vom Scheuereimer und Staubtuch frei werden, sie sollen frei werden von der Last und
“Tücke des Objekts” in der Hauswirtschaft, um des Freiwerdens geistiger, seelischer, kultureller Werte, um der erhöhten
Erfüllung mütterlicher und staatsbürgerlicher Pflichten willen’. Translation Nolan’s. 10 ‘Die Neugestaltung des Hauswesens auf der Grundlage der vom Deutschen Normenausschuß [sic] begonnenen Verein
heitlichung ist nicht nur eine wirtschaftliche, sondern auch eine staatspolitische und kulturelle Aufgabe von allergrößter
Bedeutung’. 10 ‘Die Neugestaltung des Hauswesens auf der Grundlage der vom Deutschen Normenausschuß [sic] begonnenen Verein
heitlichung ist nicht nur eine wirtschaftliche, sondern auch eine staatspolitische und kulturelle Aufgabe von allergrößter
Bedeutung’. 11 ‘Sonst versinkt Kultur im Scheuereimer und Menschentum verfängt sich im Staubtuch’. 11 ‘Sonst versinkt Kultur im Scheuereimer und Menschentum verfängt sich im Staubtuch’. 21 12 ‘Die Hauswirtschaft wird bislang in der Vorstellung eines beträchtlichen Teiles der deutschen Bevölkerung nicht als ein Teil
der Volkswirtschaft empfunden’. 12 ‘Die Hauswirtschaft wird bislang in der Vorstellung eines beträchtlichen Teiles der deutschen Bevölkerung nicht als ein T
der Volkswirtschaft empfunden’. 13 ‘Vertretern der Architektenschaft, der Hausfrauenverbände, der Möbelhändler, Haushaltungsgeschäfte und Möbelindus
trie’. 13 ‘Vertretern der Architektenschaft, der Hausfrauenverbände, der Möbelhändler, Haushaltungsgeschäfte und Möbelindus
trie’. 14 ‘Diese sollte alle für die Preis- und Mietgestaltung irgendwie maßgeblichen Elemente untersuchen; beginnend bei der
Straßenführung, den Anliegerkosten, der Grundrißgestaltung und der Normung von Bauteilen bis hin zur Innenausstat
tung und verschiedenen, die Arbeit der Hausfrau und Mutter erleichternden Anlagen (Waschhaus, Kindergarten, Grün-
und Spielflächen)’. 14 ‘Diese sollte alle für die Preis- und Mietgestaltung irgendwie maßgeblichen Elemente untersuchen; beginnend bei der
Straßenführung, den Anliegerkosten, der Grundrißgestaltung und der Normung von Bauteilen bis hin zur Innenausstat
tung und verschiedenen, die Arbeit der Hausfrau und Mutter erleichternden Anlagen (Waschhaus, Kindergarten, Grün-
und Spielflächen)’. Notes 25 ‘Nicht weil wir aus dem Hause hinausdrängen aus Abneigung gegen das Haus und die Hauswirtschaft, sondern weil wir
meinen, daß es ‘mißbrauchte Frauenkraft’ für Familie und Staat ist, wenn wir scheuernd und putzend auf die vier Wände
beschränkt bleiben mit dem mißverstandenen Stolz ‘Mein Haus, meine Welt’, anstatt auch in der Welt unser Haus zu
sehen. Dazu aber müssen wir weit mehr als heute von dem Haus befreit werden’. 26 ‘Das ist einer der Punkte, wo die Reichsforschungsgesellschaft vollkommen versagt hat. Man begnügte sich im wesent
lichen [sic] mit dem Bestehenden und scheute sich vor einer Auseinandersetzung mit neuen Forderungen und Möglich
keiten’. 26 ‘Das ist einer der Punkte, wo die Reichsforschungsgesellschaft vollkommen versagt hat. Man begnügte sich im wesent
lichen [sic] mit dem Bestehenden und scheute sich vor einer Auseinandersetzung mit neuen Forderungen und Möglich
keiten’. 22 Author’s Note I want to thank SE Eisterer and Erin Eckhold Sassin for putting together this Special Issue with critical
care, and the editors of Architectural Histories for their facilitation and help. I am grateful to Mary
McLeod for her thoughtful comments on earlier drafts and the reviewers for their insightful queries;
a presentation at SAH and the GTA invites at ETH Zurich of some of the material sharpened the
argument. Pamela Johnston helped clarify the intention and flow of the piece immensely. All remaining
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Böhlau Verlag. pp. 17–48. Hayden, D. 1981. The Grand Domestic Revolution: A History of Feminist Designs for American Homes,
Neighborhoods, and Cities. Cambridge: MIT Press. Henderson, SR. 2013. Building Culture: Ernst May and the New Frankfurt am Main Initiative,
1926–1931. New York: Lang. DOI: https://doi.org/10.1093/gerhis/ghu070 Hilberseimer, L. 1927. Großstadtarchitektur. Stuttgart: Hoffmann. Hilberseimer, L. 1927. Großstadtarchitektur. Stuttgart: Hoffmann. Hilberseimer, L. 1929. Städtebau und Wohnungsbau auf der Technischen Tagung der
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10–14. Lüders, M-E. 1936. Das unbekannte Heer; Frauen kämpfen für Deutschland, 1914–1918. Berlin: ES
Mittler & Sohn. Lüders, M-E. 1961. Die Frau im modernen demokratischen Staat. Bochum: Schacht. Lüders, M-E. 1963. Fürchte dich nicht: persönliches und politisches aus mehr als 80 Jahren 1878–1962. Köln: Westdeutscher Verlag. DOI: https://doi.org/10.1007/978-3-322-98441-8 MacCarthy, F. 2019. Gropius: The Man Who Built the Bauhaus. Cambridge: Harvard University Press. Abramson, DM. 2016. Obsolescence: An Architectural History. Chicago: University of Chicago Pres
DOI: https://doi.org/10.7208/chicago/9780226313597.001.0001 DOI: https://doi.org/10.4159/9780674239890 McLeod, M. 1983. ‘Architecture or Revolution’: Taylorism, Technocracy, and Social Change. Art
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https://doi.org/10.17104/9783406741333 Nolan, M. 1990. ‘Housework Made Easy’: The Taylorized Housewife in Weimar Germany’s
Rationalized Economy. Feminist Studies, 16: 549–77. DOI: https://doi.org/10.2307/3178019 Polster, B. 2019. Walter Gropius: der Architekt seines Ruhms. Munich: Carl Hanser Verlag. Rabinbach, A. 1990. The Human Motor: Energy, Fatigue, and the Origins of Modernity. New York:
Basic Books. Reichsforschungsgesellschaft für Wirtschaftlichkeit im Bau- und Wohnungswesen [Preprint]. 1927. Mitteilungen des Deutschen Städtetages. 24 Reichsforschungsgesellschaft für Wirtschaftlichkeit im Bau- und Wohnungswesen. 1929. Auswertung der Versuchssiedlungen in hauswirtschaftlicher Hinsicht. 2 Jahre Bauforschung: 34. Reicke, I. 1984. Die großen Frauen der Weimarer Republik: Erlebnisse im ‘Berliner Frühling’. Freiburg im
Breisgau: Herder. Smith, T. 2014. Bauhaus Weaving Theory: From Feminine Craft to Mode of Design. Minneapolis: University
of Minnesota Press. DOI: https://doi.org/10.5749/minnesota/9780816687237.001.0001 mith, T. 2014. Bauhaus Weaving Theory: From Feminine Craft to Mode of Design. Minneapolis: University
Minnesota Press. DOI: https://doi.org/10.5749/minnesota/9780816687237.001.0001 Stadler, M. 2009. Gunta Stölzl: Bauhaus Master. New York: Hatje Cantz Verlag. Stevens, S. 2016. Developing Expertise: Architecture and Real Estate in Metropolitan America. New
Haven: Yale University Press. Taut, B. 1924. Die neue Wohnung. Die Frau als Schöpferin. Leipzig: Klinkhardt & Biermann. Troy, VG. 2002. Anni Albers and Ancient American Textiles: From Bauhaus to Black Mountain. Burlington, VT: Ashgate. Weltge-Wortmann, S, and Weltge, S. 1998. Bauhaus Textiles: Women Artists and the Weaving
Workshop. London: Thames & Hudson Ltd.
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Critical Hope or Principled Infidelity? How an Urban Secondary School in an Area of Sustained Poverty in England Continues to Improve
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Urban Rev (2016) 48:560–578
DOI 10.1007/s11256-016-0368-7 1
London Centre for Leadership in Education, UCL Institute of Education, 20 Bedford Way,
London WC1H 0AL, England, UK & Trevor Male
t.male@ucl.ac.uk
Ioanna Palaiologou
ioannapad@icloud.com & Trevor Male
t.male@ucl.ac.uk Keywords Urban education Public school School leadership Critical hope Introduction This is the examination of how an urban secondary school in England not only made
the transition from failure to success ‘‘against the odds’’ (Ofsted 2009), but
sustained their outstanding status despite changes in local demographics which
resulted in the school serving a significantly different ethnic community than was
evident at the start of their journey of improvement in 1997. The Robert Clack High School is a state maintained comprehensive school
situated in the Becontree Estate based in the London Borough of Barking and
Dagenham, an area of England’s capital city that has consistently featured as one
of poverty. At the time of writing it ranked amongst the four lowest London
boroughs in 10 of 21 social indicators and is considered to be getting ‘slightly
worse’ according to the data presented by the National Policy Institute (2013). In
other words the socio-economic indicators of the locality had not improved since
the beginning of their journey and, unlike some other schools which changed their
student population in search of success, the school continued to serve its
immediate local community which compromised almost exclusively of working
class families. Critical Hope or Principled Infidelity? How an Urban
Secondary School in an Area of Sustained Poverty
in England Continues to Improve Ioanna Palaiologou1 • Trevor Male1 Published online: 28 July 2016 Published online: 28 July 2016 Published online: 28 July 2016
The Author(s) 2016. This article is published with open access at Springerlink.com y
The Author(s) 2016. This article is published with open access at Springerlink.com Abstract This is the examination of how a secondary school in England, the Robert
Clack High School, not only made the transition from failure to success ‘‘against the
odds’’, but sustained and enhanced that status despite changes in local demographics
resulting in the school serving a significantly different ethnic community than was
evident at the start of their improvement journey. The school is situated in an area of
London that has consistently featured as one of poverty. Unlike some other schools
which changed their student population in search of success, the school continues to
serve its immediate local community which compromises almost mainly working
class families. Although empirical research undertaken in the school demonstrates
some evidence of ‘principled infidelity’ (seeming to follow external policy diktats
whilst pursuing their own agenda) we conclude that the ethos underpinning the
school’s sustained improvement is the concept of ‘critical hope’. This is an
approach where the needs of young people in disadvantaged communities are
recognised and addressed in order to provide them with the opportunity to control
their destiny. The consequence is that the school has exceeded its prescripted
expectations, continues to improve and has lifted both the students and the com-
munity’s aspirations exponentially. Keywords Urban education Public school School leadership Critical hope 123 Urban Rev (2016) 48:560–578 561 The Local Context The borough of Barking and Dagenham is to the east of London with a population
of close to 200,000 and is most well known for being the ‘home’ of the Ford
motor company in the UK which at one time in the early 1950s employed over
40,000 people. The principal reason for expansion of the borough, however, was
the building of a huge council estate (Becontree) in the early 1920s in order to
relocate skilled workers from the slums of Inner London after the First World
War. Sashin (2011) reports that whilst many people still believe that the Becontree
Estate was built to accommodate the huge numbers of Ford workers, the
development was actually planned well before Ford shifted production to
Dagenham from Manchester in 1931. Initially Ford workers were not allowed
to rent property on the estate, but when the London County Council later
struggled to keep East Enders in their new homes the rules were relaxed to
accommodate car workers, a development which started the complementary
relationship between the two monolithic cornerstones of the borough’s identity—
Becontree and Ford. This history is significant as the population of the borough was almost
exclusively white working class, a situation that continued until the beginning of
the new millennium in 2000. Consequently the school in which Paul had worked
as a teacher since 1990 and took over as headteacher in 1997 was a reflection of
that local population and he was often invited, as the school’s success became
noticed more widely, to contribute to debates and initiatives about white working
class young people. Typical of such invitations were the two seminars run jointly
by the National Union of Teachers (NUT) and the National College for School
Leadership (NCSL) in 2008 and 2009 where he and others were asked to provide
views and experiences of how to succeed with such groups of young people. Subsequently the reports from these seminars were reported to Parliament in 2010. Paul’s offerings to such events were interesting in that he did not draw attention to
the ethnicity of the students for whom he was responsible, instead highlighting the
impact of a legacy of poverty and inequality which was endemic to the borough
by this time. The Story of Success Begins When Paul Grant was appointed as headteacher in 1997 he was taking on a school
that was in extremely challenging circumstances. An adverse inspection report
conducted by the Office for Standards in Education (Ofsted), the national
inspection service, in 1995 was a fair reflection of how far the school had slipped
in terms of reputation and student attainment in comparison to previous decades
where it had been considered a ‘good’ school by the local authority and the
community (Haydn 1998). The story of the school since his appointment has been
one of constant improvement with widespread recognition of success, however,
including the award of a knighthood to Paul in 2009. The school has moved from
being one where 21 % of students left the school with no academic qualifications,
and stories of student disruption and appalling behaviour were common, to a
situation in 2013 where all students achieved a qualification, with over 80 %
getting five good grade GCSEs at Key Stage 4 (including Mathematics and
English) and 87 students in the sixth form having been offered places at university
for year 2013–2014. This paper is not an account of that success, however, which
has been and continues to be reported widely elsewhere (e.g. Haydn 1998, 2010;
Woodhead 2002; Hopkins 2007; Ofsted 2009; Mongon and Chapman 2012). Instead this is an exploration of how and why the school has sustained that
success and continued improvement since those early days despite changes in
local demographics which have led to significant changes in the ethnicity of the
student population. This exploration begins with a portrait of the local context
before moving on to investigate how and why leadership behaviour in the school
has managed to achieve and consistently sustain high levels of success. 12 123 562 Urban Rev (2016) 48:560–578 Changing the School: Part #1 Paul was and remains an unusual artefact of the state maintained system in that he
moved directly to headship from the classroom, probably the last person to be able
to do that in England as new professional qualifications for headship had just started
to appear in 1997 when he was appointed. After 7 years in the school as Head of
Humanities he and his team of teachers had achieved good very good results whilst
the school was seemingly in terminal decline. The school at that time was judged as
having serious problems, including low pupil attainment, poor pupil behaviour and
motivation, weak staff morale, falling rolls, budgetary deficits and staff recruitment
and retention problems (Haydn 1998). In an interview undertaken in 2012 with the
Deputy Headteacher (who was himself a pupil of the school at the time of that
inspection) he reported that the reputation of the school ‘‘was rock bottom, I mean,
it was terrible, and the school went through its really bad phase in the 3 years before
Paul was appointed as headteacher’’. Whilst Paul was not the only teacher getting
good results despite the environment, the results from his faculty were outstanding
and came to the attention of the governors and the local authority, both of which
encouraged him to make his application to take over as headteacher following the
retirement of the previous postholder. He was appointed and took up post in May,
1997. After a spirited talk on his first day to the staff on both buildings of the split-site
school he set about the task of restoring order to the working environment. This was
a combination of student discipline, school reputation management and a close
focus on enhancing the learning environment, particularly through improving
teaching. Central to this strategy was regaining adult control epitomised by the
exclusion of 246 students in the first 2 weeks. The work associated with this
dramatic gesture is legendary with Paul describing the action laconically as ‘‘being
the only way I could get to talk to the parents’’. After marathon sessions of
discussions and appeals eventually 10 students were permanently excluded in
November 1997, with a new atmosphere of discipline emerging that was
unremitting in its resolve. The Local Context Changed circumstances have led to Ford reducing its output to engines rather
than car assembly, however, with a concomitant reduction in job availability
(down to just 4000 employees by 2010) which means the company was no
longer the main employer in the area. The locality had become to be recognised
as one where there was poverty and a need for social welfare, but with a
population that was largely white. It was easy at the time, therefore, to see Paul
as a champion of improving the life chances of groups of disadvantaged young
people and with limited insight assume that such students were only white. His
approach was not ethnically focused, however, as will be discussed more fully
later in this paper. 123 Urban Rev (2016) 48:560–578 563 Changing the School: Part #1 In one respect Paul was fortunate with the timing of
his appointment in that he had several weeks at the end of the school year to set
expectations for the next academic year. The planned introduction of school
uniform, for example, was eased by the astute use (an attribute later to be applied to
many key decisions) of some of the more challenging students as walking models
during the last weeks of their school life, thus popularising the new uniform which
was to become obligatory in the next school year. The reputation of the school was further enhanced through engaging and
supporting parents and the local community. Paul and his staff made a point of
being highly visible not only in and around the school, but also in the wider
community. As Haydn (2010: 426) reports this included Paul getting on local public
buses and apologising to drivers for past pupil atrocities and giving a number to
contact in case of future misdemeanours, spelling out the new rules, and standing up
to pupils and parents who were not aware of the ‘new rules’. The regaining of adult
control was thus the first step that allowed teachers to focus on their teaching and 12 3 564 Urban Rev (2016) 48:560–578 within 2 years success was noted by the Ofsted (1999) report which was almost
unremittingly positive and complimentary. The percentage of lessons where
teaching was satisfactory or better had risen from 73 to 95 %, and was adjudged to
be ‘good to very good’ in 65 % of lessons. The school ethos was described as
‘excellent’ (and the behaviour and attitude to learning of students also evinced
praise) with the report noting that the school provided ‘a calm and orderly learning
environment’ (Haydn 2010: 423). At this point, therefore, the school had been
turned around and a new regime established of teacher and student discipline to
replace the chaos and disorder evidenced just a few years before. I think it probably took possibly less than a year for the community to think I think it probably took possibly less than a year for the community to think
‘This is happening […] kids are behaving, the lunatics aren’t in control of the
asylum any more, there’s some order here, and fairness. Changing the School: Part #1 (Parent Governor—in
post from 1992) We consider, however, creating the necessary change is not that difficult or
complicated; it is sustaining that level of improvement that is the hard task. In many
ways, we suggest, the intervention strategies employed in the first stage of Paul’s
headship were not novel if judged solely on the intention to stabilise the school,
ensure a safe working environment and to provide planned learning opportunities
that at least matched basic expectations of government inspection agencies. They
are myriad examples of headteachers who have effected ‘turn-round’ strategies, but
very few examples where they have continued to build on that initial intervention to
sustain and extend the improvement process. What has been seen since those early
days suggest that a more focused effort was required to move the level of student
attainment and achievement beyond ‘satisfactory’ to the high and still improving
levels witnessed today. 123 Sustaining the New Levels of Success 1 Student outcomes for GCSE results 1996–2012 a sixth form of 380 students in 2012, giving a total population of 1852 students and
making it one of the biggest schools in the country. The school has become
extremely popular as a result if its success and has received over 900 statements of
preference (combined first and second choices) from parents and guardians which
compares dramatically with the popularity of the school in 1997 when they received
only 109 such preferences. Admission criteria are so strict, however, that apart from
having a sibling in the school the only other realistic chance of getting admitted is
proximity. In other words the school has not recruited more widely and accepted
students from higher socio-economic backgrounds, it has achieved its success
though the actions that have been taken in concert with the local community. This is
a key point of principle in that the school is inclusive and recognises success in
many ways, not just through student attainment on national tests. The student population is thus drawn from a community which has had and
continues to have high levels of poverty, but is one which has changed significantly
in terms of ethnicity during the current century. As can be seen from Table 1 the
White British population fell from 81 % to less than 50 % during the period leading
up to the national census of 2011, with large increases in the proportion of Other
White (typically East European) and other ethnic groups. Interestingly the ethnic
mix of the local population now brings it much more in line with the rest of London,
but the difference is dramatic in terms of the local community. In explaining the reasons for increased diversity in the local population one of the
governors, also a local authority planning officer, describes how the borough has
gone from having one of the highest proportions of people in their 80s to a much
younger population. In many instances their council tenancy has disappeared into
the buy-to-let market which, he calculates, means some 25–30 % of properties on
the local council estate which serves the school are now privately owned leaseholds. This appears to be a growing market with many owners of previous council
properties (which they were allowed to buy under a government policy of the 1980s)
taking the opportunity to sell and make a healthy profit. Sustaining the New Levels of Success The school continued to improve beyond what could be reasonably expected from
the stabilisation achieved from the early interventions in the late 1990s with student
outcomes on standard examinations continuing to rise and to a level far above
performance normally anticipated from the student profile on entry to the school
(see Fig. 1). When judging against GCSE results at the end of compulsory schooling
(i.e. at age 16 years) it can be seen, for example, that all students had achieved at
least one pass by the year 2000 and by 2011 there was universal success in five
subjects. Ofsted inspections during the first decade of the century graded the school
as outstanding and by 2009 the school was featured as one which was ‘‘chosen from
the small number nationally that have been judged outstanding in two or more
inspections, which serve disadvantaged communities and which have exceptionally
good results’’ (Ofsted 2009). What is intriguing, however, is that during the same decade the school population
changed dramatically and by the national census of 2011 was no longer populated
almost entirely by students from white working class backgrounds. With a
catchment area of just 1.3 square miles the school has an intake of 300 per year and 123 Urban Rev (2016) 48:560–578 565 a sixth form of 380 students in 2012, giving a total population of 1852 students and
making it one of the biggest schools in the country. The school has become
extremely popular as a result if its success and has received over 900 statements of
preference (combined first and second choices) from parents and guardians which
compares dramatically with the popularity of the school in 1997 when they received
only 109 such preferences. Admission criteria are so strict, however, that apart from
having a sibling in the school the only other realistic chance of getting admitted is
proximity. In other words the school has not recruited more widely and accepted
students from higher socio-economic backgrounds, it has achieved its success
though the actions that have been taken in concert with the local community. This is
a key point of principle in that the school is inclusive and recognises success in
many ways, not just through student attainment on national tests. Fig. 1 Student outcomes for GCSE results 1996–2012 Fig. 1 Student outcomes for GCSE results 1996–2012 Fig. 1 Student outcomes for GCSE results 1996–2012 Fig. Sustaining the New Levels of Success As the area is one of the 12 12 3 3 Urban Rev (2016) 48:560–578 566 Table 1 London Borough of Barking and Dagenham 2011 census key statistics
2011 census data
LBBD %
increase/
decrease
LBBD %
population
LBBD %
population
London %
population
England
%
population
Ethnic Groups
2011
2001
2011
2011
White British
-30.64
49.46
80.86
44.89
79.75
White Other
234.06
7.81
2.65
12.65
4.58
Mixed/multiple ethnic groups: White
and Black Caribbean
87.96
1.44
0.87
1.46
0.78
Mixed/multiple ethnic groups: White
and Black African
272.03
1.14
0.35
0.80
0.30
Mixed/multiple ethnic groups: White
and Asian
133.33
0.67
0.33
1.24
0.63
Mixed/multiple ethnic groups: Other
mixed
233.64
0.99
0.34
1.45
0.53
Asian/Asian British: Indian
102.01
4.00
2.25
6.64
2.63
Asian/Asian British: Pakistani
162.09
4.31
1.86
2.74
2.10
Asian/Asian British: Bangladeshi
1044.28
4.14
0.41
2.72
0.82
Asian/Asian British: Chinese
69.68
0.71
0.47
1.52
0.72
Asian/Asian British: Other Asian
485.52
2.76
0.53
4.88
1.55
Black/African/Caribbean/Black
British: African
293.81
15.43
4.44
7.02
1.84
Black/African/Caribbean/Black
British: Caribbean
52.21
2.81
2.09
4.22
1.11
Black/African/Caribbean/Black
British: Other Black
347.09
1.74
0.44
2.08
0.52
Other Ethnic group: Arab
0.52
n/a
1.30
0.42
Other Ethnic Group: Any other ethnic
group
177.14
1.04
0.43
2.14
0.62 Table 1 London Borough of Barking and Dagenham 2011 census key statistics cheapest places to live in London there has not only been a migration into the
borough from Inner London, but there has also been much immigration into the
country of refugees who have located into the area. The consequence, he states, is to
see a startling change in the local population: cheapest places to live in London there has not only been a migration into the
borough from Inner London, but there has also been much immigration into the
country of refugees who have located into the area. The consequence, he states, is to
see a startling change in the local population: With people coming in from civil war conflicts in Africa and the Balkans
together with the churn that started to occur on buy to let, there started to be a
ready market of people who wanted rented property. The number of properties
that have become buy-to-let has increased consistently year on year on and
people just come here from a wider range of different places, a little rush here,
a little rush there. Sustaining the New Levels of Success Unlike most of the London boroughs where there tends to be
one dominating group from an immigration point, either because they all came
at one point or people who came in the first settlement and then other people
joined them, here it is a bit more diversified. We have got people from all over
the place. Obviously, more recently, people from Eastern Europe [due to 12 23 Urban Rev (2016) 48:560–578 567 economic migration within the European Union], but there’s a lot of people
came here in 1990 from the Congo and Angola and lots of people from Bosnia
Herzegovina, Croatia and Kosovo who came around the time of the Balkan
War. Consequently there isn’t one dominating group, it is quite a mix of
people from all over the place. (Local Authority Governor) As can be seen from Tables 2 and 3 the changes in local population are reflected
in the school with not only sharp increases in the ethnic mix, but also with a general
rising trend of students who are eligible for free school meals (the most common
indicator of poverty in compulsory education). There has also been a notable in-
crease in the proportion of students for whom English is not their first language. By the time we started our research on the school in 2012, therefore, the ethnicity
of the school population was significantly different from the one evident at the start
of their journey to success in 1997 and yet the school was even more successful. This we argue has been through a combination of principled leadership coupled with
a determination to provide a success culture for children from challenging socio-
economic circumstances. The school has thus transcended its anticipated station
without devaluing the attributes of the local community. A careful balance has been
maintained between the demands of the larger society (such as continued
examination success) and an unmitigated desire to support the development of
students who build on, rather than dispense with, their cultural heritage. This is not
the story of school leadership in a white working class community, therefore, but a
story of generating educational success irrespective of gender, ethnicity, creed or
social status. It is an approach we will argue that can be replicated in alternative
environments providing the underlying principle of equal opportunity is evident. Changing the School: Pedagogical Leadership From an examination of the data we collected from over 50 interviews between
2012 and 2014 in the school with Sir Paul, as headteacher, other senior leaders,
governors, teaching staff, support staff, students, local authority officers and parents
together with reviews of documentation such as Ofsted reports, internal documen-
tation, press cuttings and correspondence between a parent governor and the
previous headteacher we have identified leadership behaviours which have
sustained and extended the impact made by the initial intervention strategies. During that investigation the issue of ethnicity has only ever come up once when
a parent governor, also employed as a student counsellor in the school and himself
Black British, talked about the zero tolerance policy the school has to gangs (which
in London are typically associated with Black youths). His description of the
approach is: One of my other roles is gang prevention, which is unusual. I don’t know of
any other school that does exactly what I do. Most schools don’t have
someone like me, but when kids get in trouble outside with the police we get
told about it and if it’s gang-related we’ll isolate them, take out of the normal
programme and they do their lessons with me. You can go to some of the 12 3 568 Urban Rev (2016) 48:560–578 Table 2 Basic Characteristics by National Curriculum Year Group. Source: Ofsted (2013): RAISEonline 2013 Summary Report
NC year group
Number
on roll
% Boy/girl
% Free school
meals*
% Minority
Ethnic Group
% 1st language
not English
% Special
education needs
Children
looked after
7
301
48.5/51.15
44.9
59.3
36.9
15.3
3
8
298
50.0/50.0
47.7
45.9
29.2
17.1
3
9
296
51.7/48.3
43.6
44.9
29.3
17.9
2
10
295
54.6/45.4
40.0
38.1
24.5
24.1
0
11
282
55.7/44.3
33.3
33.0
25.3
23.8
1
Post-compulsory
380
50.8/49.2
–
30.8
22.4
23.2
0
* The categorisation of pupils eligible for free school meals (FSM) changed in 2012. Pupils are classed as FSM if they have been eligible for and claiming FSM at any time
in the last 6 years 123 569 Urban Rev (2016) 48:560–578 Table 3 Ethnic Groups and English as a first language. Changing the School: Pedagogical Leadership Source: Ofsted (2013): RAISEonline 2013
Summary Report
School %
National %
Ethnic Group
2011
2012
2013
2013
White
British
69.1
64.3
56.9
72.7
Irish
0.1
0.3
0.2
0.3
Traveller of Irish heritage
0.0
0.0
0.0
0.1
Romany or Gypsy
0.1
0.0
0.0
0.2
Any other white background
4.3
6.5
8.4
4.3
Mixed
White and Black Caribbean
1.8
2.2
1.9
1.4
White and Black African
0.5
0.6
0.4
0.5
White and Asian
0.2
0.2
0.4
1.0
Any other mixed background
0.9
1.0
1.2
1.6
Asian or Asian British
Indian
1.1
1.3
1.7
2.6
Pakistani
0.7
1.0
1.7
3.9
Bangladeshi
1.8
2.5
2.7
1.6
Any other Asian background
0.7
1.0
1.2
1.6
Black or Black British
Caribbean
2.7
2.4
2.5
1.3
African
13.1
13.5
15.3
3.3
Any other Black background
1.8
2.0
2.0
0.6
Chinese
0.1
0.2
0.2
0.4
Any other ethnic group
0.7
0.5
1.0
1.5
Parent/Pupil preferred not to say
0.1
0.0
0.0
0.5
Ethnicity not known
0.3
0.5
2.3
0.4
First language
English
78.4
76.2
70.1
83.9
Other
21.0
23.3
26.9
16.9
Unclassified
0.5
0.5
3.0
0.2 Table 3 Ethnic Groups and English as a first language. Source: Ofsted (2013): RAISEonline 20
Summary Report schools and see evidence of gangs because even if they’re in uniform they’ll
have the same haircut, or they’ll have a tick shaved into their hair. In our
school if there’s any ticks we order them to cut it out and tell them ‘‘If you
can’t cut it out you’re in isolation ‘til it’s done’’. It seems really extreme but it
works. In some other some other schools they’ve all got a tick and it’s
intimidating. I’ve been to other schools and thought, ‘‘Don’t you notice that?’’
and teachers don’t know what I’m talking about. It’s, ‘‘As long as it doesn’t
happen in school it’s nothing to do with us’’. We take the view ‘‘Well actually
we’re a community and as long as you come to the school what you do outside schools and see evidence of gangs because even if they’re in uniform they’ll
have the same haircut, or they’ll have a tick shaved into their hair. In our
school if there’s any ticks we order them to cut it out and tell them ‘‘If you
can’t cut it out you’re in isolation ‘til it’s done’’. It seems really extreme but it
works. Changing the School: Pedagogical Leadership In some other some other schools they’ve all got a tick and it’s
intimidating. I’ve been to other schools and thought, ‘‘Don’t you notice that?’’
and teachers don’t know what I’m talking about. It’s, ‘‘As long as it doesn’t
happen in school it’s nothing to do with us’’. We take the view ‘‘Well actually
we’re a community and as long as you come to the school what you do outside 12 3 570 Urban Rev (2016) 48:560–578 affects the school and the reputation of the school’’, so we’re interested in
everything. (Parent Governor and Student Support Officer) affects the school and the reputation of the school’’, so we’re interested in
everything. (Parent Governor and Student Support Officer) This was the only evidence we saw, however, of the school seeking to
disassociate the internal environment from the mores of the wider local community. In general the culture of the school was to value all members of the student
population and to celebrate their success, regardless of gender, ethnicity, creed or
social status. This is a key element of our subsequent analysis of how the school
continues with its success, despite moving from a mono- to multi-cultural
population. In other words their success was not based on their ability to
communicate with and motivate a white working class population, their success
appears to lie in the ability to raise the aspirations of the school population and the
local community. We consider the approach used within the school corresponds to the ideals of
pedagogical leadership we outline below (and more fully in previous papers:
Alameen et al. 2015; Male and Palaiologou 2015). Pedagogical leadership, we
claim, is an extension of ideas pertaining to other constructs of educational
leadership which focus on enhancing the learning environment, variously called
Instructional, Learning-Centred and Learner-Centred. Where pedagogical leader-
ship differs from other approaches is that we consider the process is more than just
supporting teaching and learning in order to match external demands for improved
test scores. We see pedagogical leaders as being responsive to the local community
as well as to larger society, with their actions being relevant to both situation and
context and with an expectation that actions should not be pre-determined, but
adaptable and flexible. 123 Sustaining the ‘Outstanding’ School One of the key ways in which the leadership team of the Robert Clack High School
has been able to sustain the levels of improvement already evident by 1999 has been
consistency of purpose in relation to the effective implementation of core (and
shared) values. The school can be considered as inclusive in the truest sense of the
word, based on sound principles and practice that transcend issues solely related to
white working class young people and with attributes that are transferable to other
cultures and ethnicities. Earlier research work where educational leadership had made a difference
identified similar behaviours amongst some headteachers who were labelled
‘maverick’, yet nevertheless achieved results that were beyond expectations (Hay
Group 2002). In a later work Mongon and Chapman (2012) extended their work on
white working class young people to schools they considered as demonstrating ‘high
leverage’ leadership and applied the three ‘intelligences’ they had identified in 2008
which were needed to enculture success: contextual, professional and social
intelligence: 1. Contextual intelligence These leaders show a profound respect for the social
context they are working in without ever patronising it. They have deliberately
chosen to work in these places. 1. Contextual intelligence These leaders show a profound respect for the social
context they are working in without ever patronising it. They have deliberately
chosen to work in these places. 2. Professional intelligence These leaders are very good at their core business:
leadership and management to nurture the teamwork on which the school’s
excellent standards of teaching and learning are dependent. 2. Professional intelligence These leaders are very good at their core business:
leadership and management to nurture the teamwork on which the school’s
excellent standards of teaching and learning are dependent. 3. Social intelligence These leaders appear to be sensitive to the emotional state of
their pupils and colleagues and use that to guide their own thoughts and actions. In turn, they are deeply admired across their staff and student body (Mongon
and Chapman 2008: 10). Perhaps not surprisingly the Robert Clack High School was one of the study
schools considered to be exhibiting ‘high leverage leadership’, an approach to
school leadership which draws on the analogy offered by Archimedes that with a
long enough lever, and a place to stand, he could move the earth. Changing the School: Pedagogical Leadership Such an approach to leadership thus extends the simplistic
definition of pedagogy as the relationship between teacher and learner in order to
recognise the needs of the learner within the environment which we labelled
internal and external pedagogical (social) axes: Internal axes (values, beliefs, culture, religion, customs and local economy), and •
Internal axes (values, beliefs, culture, religion, customs and local econom •
External axes (societal values, global economy, mass media, social networking,
information communication technologies, national curriculum, the ‘academic
press’ of student test scores). •
External axes (societal values, global economy, mass media, social networking,
information communication technologies, national curriculum, the ‘academic
press’ of student test scores). Consequently we determined that pedagogical leadership sought to deal with
these competing demands by exploring how: … the centrality of interactions and relationships among learners, teachers,
family and community (i.e. their values, beliefs, culture, religion, customs and
economic circumstances) interact with external elements (such as the global
economy, climate and social phenomena that additionally influence the life of
the community) in order to jointly construct knowledge. (Male and
Palaiologou 2015: 219) … the centrality of interactions and relationships among learners, teachers,
family and community (i.e. their values, beliefs, culture, religion, customs and
economic circumstances) interact with external elements (such as the global
economy, climate and social phenomena that additionally influence the life of
the community) in order to jointly construct knowledge. (Male and
Palaiologou 2015: 219) From this perspective pedagogical leadership goes beyond the immediacy of the
school buildings to recognise and deal with the tension between the needs and
desires of larger society and those of the learner within their local community. In 123 Urban Rev (2016) 48:560–578 571 such a context we would expect leaders to take decisions and actions that were
informed by both sets of pedagogical axes and to exhibit behaviours that support
those ambitions, particularly in regard to sustaining internal axes. such a context we would expect leaders to take decisions and actions that were
informed by both sets of pedagogical axes and to exhibit behaviours that support
those ambitions, particularly in regard to sustaining internal axes. Sustaining the ‘Outstanding’ School Mongon and
Chapman (2012) argue that high-leverage leaders hypothetically might stand at a
distance and apply a massive effort against an inert weight—in their case, ‘the
school’—to not only address short term issues, but also to invest in long term
capability. The school leaders they examined were uncommonly powerful in this
respect and were frequently to be found in areas where there are a disproportionate
number of socio-economically disadvantaged families. The work of the Hay Group and of Mongon and Chapman thus highlight
important features required of headteachers and senior leaders when contemplating 12 3 572 Urban Rev (2016) 48:560–578 acting in a way that can be perceived as different from accepted norms in order to
achieve an effective environment for a specific school context. Their work
corresponds to the suggestion made by Hoyle and Wallace (2007) that there are
some school leaders who fashion their commitment to policy while maintaining a
steadfast focus on student interests. This, they argued, was a process of principled
infidelity by which they adapt policy to the needs of students whilst appearing to be
following policy to the letter. Whilst this may be true in part, we conclude that the
main underlying factor that supports continued success in this school, irrespective of
changing demographics, is an overwhelming desire to confront disadvantage. This
approach bears more resemblance to the concept of critical hope (Duncan-Andrade
2009) where the needs of young people in disadvantaged communities are
recognised and addressed in order to provide them with ‘‘control of destiny’’
whereby they learn ‘‘to deal with the forces that affect their lives, even if they
decide not to deal with them’’ (Syme 2004: 3). Confronting Disadvantage I won’t have the door locked, like it’s locked most of the time—you
can’t see the chief constable, you can’t see the director of education, you can’t
see the chief medical officer—but I was going to make damn sure they could
see the head teacher and they could have a piece of that head teacher, because
that’s very important for working class people. (Sir Paul Grant, Headteacher) This driving force moved him beyond strategic management of the school to
maximise learning outputs, which leads us to discount the notion of principled
infidelity being the prime reason for sustained success. Like in most other schools
the senior leaders and teachers in the Robert Clack High School are familiar with
the notion of ‘gaming’ whereby you can maximise outcomes on student test scores
through a number of practices that, whilst legitimate, are of doubtful moral
turpitude. It was clear from our research that had never been the means by which the
school was cultivating and sustaining success. Nor was there extensive evidence of
principled infidelity as the culture of the school was openness with literally
hundreds of visitors, including multiple inspections, many of whom were astute
enough to see through any attempt to disguise motives. The policy of the school was
transparent—it was a state maintained local authority school which sought to serve
the needs of the local community. The prime motivation we conclude is critical
hope. A key feature of the concept of critical hope is to recognise the ‘moral outrage’
caused to young people in disadvantaged communities and not to either defer hope
or to produce what Duncan-Andrade calls ‘‘hokey hope’’ (2009: 3). Too many
people working in schools, he argues, project aspirations for students to ‘‘to set their
sights on some temporally distant (and highly unlikely) future well-being’’, thus
deferring hope (2009: 5). ‘Hokey hope’ he sees meanwhile as the false promise of
some ‘‘multicultural, middle-class opportunity structure that is inaccessible to the
overwhelming majority of working-class, urban youth of color’’. Neither approach
is adequate to meet the needs of disadvantaged communities whereas critical hope is
based on a genuine belief that students can not only transcend their local
community, but help their community to also transcend their circumstances. In other
words it is not a case of educating students out of disadvantage, but improving the
circumstances for all members of that community. Confronting Disadvantage Our research within this school provided the opportunity for us to explore the way in
which members of the school’s leadership team, and in particular the headteacher,
created an environment of success which has enabled the school to exceed
expectations in relation to its social demographics. This school has resisted the
‘‘banking model’’ of education described by the work of Freire (1972). In such a
system he argues students are treated as ‘receptacles’ that are to be ‘filled’ with the
‘‘content of the teachers narration’’ and who then are expected to regurgitate
information given in class, on tests, quizzes, and anything that requires an answer
that is ‘‘word for word’’ what the teacher says (Freire 1972: 1). Instead our data
suggests there was ‘‘mutual humanization’’ whereby students and teachers became
partners in critical thinking and engaged with ‘‘problems of human beings in their
relations with the world’’ (Freire 1972: 5). This approach, we argue, was based more on the notion of critical hope than on
principled infidelity. We saw this in our first interview with Sir Paul makes when he
said ‘‘if these children have been let down that’s a disgrace, it’s outrageous’’. Coming from a very similar background himself he empathised with the local
community and students’ reduced chances of life success because of their low
socioeconomic status. In his case he did not even believe in his mother’s faith in him
to one day become a headteacher ‘‘because from where I was it looked like a closed
shop of middle class people who all knew each other’’. The fact that he had
overcome such a start to life himself to reach his current status resulted in him
aiming to create more opportunities for young people in similar circumstances to the
ones he faced in a part of Liverpool that was eerily similar to Barking and
Dagenham in terms of its socio-economic indicators. His determination to change
those circumstances is summed up in his desire to make himself available to the
local people: 123 573 Urban Rev (2016) 48:560–578 I’m working class and all these people are. I’ll be the first working class
person of a position of responsibility that they’ve probably ever seen in their
lives. Confronting Disadvantage That seems to us to be the fundamental driving force of the Robert Clack High
School in that all manner of achievement was celebrated, not just success on
measures of attainment generally represented by test scores. It seems akin to a
philosophy based on the work of Tupac Shakur, the American actor and rapper,
when he uses the analogy of growing roses in concrete. The results should be valued
as highly as any measure of beauty as the opportunity to grow was so hard: We wouldn’t ask why a rose that grew from the concrete for having damaged
petals, in turn, we would all celebrate its tenacity, we would all love its will to
reach the sun, well, we are the roses, this is the concrete and these are my
damaged petals, don’t ask me why, thank god, and ask me how. (Shakur 2006) 12 3 574 Urban Rev (2016) 48:560–578 The data suggest that there was little evidence of either ‘deferred’ or ‘hokey’
hope here, but instead a recognition by staff at the Robert Clack that it is a ‘‘false
binary that suggests we must choose between an academically rigorous pedagogy
and one geared toward social justice’’ and that the school was serious about hope by
connecting ‘‘our pedagogy to the harsh realities of poor, urban communities’’
(Duncan-Andrade 2009: 6). We consider that Sir Paul and his team were mainly exhibiting critical hope,
although principled infidelity cannot be ruled out completely. These artefacts of
success were manifested more by the use of ‘contextual’ intelligence, we suggest,
rather than ‘professional’ or ‘social’ intelligence. Clearly whilst there was ample
evidence to see the latter two intelligences in action within the school, in our view
these two intelligences can be seen in almost any well-run school and to that extent
the Robert Clack High School was not unusual. What makes it different is the way
in which decisions are context specific and based on the notion of critical hope. This
contextual intelligence can be seen in the early stages of change through the efforts
to regain adult control and providing effective teaching. The ‘Robert Clack Good
Lesson’, first introduced in the first days of Paul’s headship, was the basis of the
school’s transition to success. 123 Conclusions We have demonstrated that there has been a multi-faceted change to the community
in terms of economical, political and ethnic changes with movement from a mono-
to multi-cultural society. The area has shifted from a simple and trading economy
(Ford Motor Company) to a highly complex urban industrial order with a variety of
economies transforming the population of the locality into single specialities,
although the socio-economic indicators of poverty are still present. In recognising
these features we have taken a postmodernist approach to examine how learning
takes place in the school in order to be ‘‘democratic’’ and inclusive to all learners
despite their socio-economic backgrounds. The school has thus moved out of a relatively self-contained monocultural
neighbourhood into a loosely aggregated multicultural society marked by differ-
entiation of structure and function. This presented new challenges which required
close integration of parts and an ever increasing measure of cooperation with the
community, but above all a unified direction. The vision of the school was a simple
one in principle: all children are learners who can achieve. In reality, however, this
has been a complex vision to meet. In an era of individualism in the economy, standardisation of teaching and
learning and the ‘modelisation’ of leadership in schools to meet government
agendas (Male and Palaiologou 2015) what this school has demonstrated is to
operate in a collective way. Central to this was its dominating socio demographic
reality. Whilst this did not shape the school in terms of core principles, it did lead to
understanding the mechanical energy of the community and go beyond the
standardised ‘‘schooling’’ of compulsory education and achieve what Derrida calls
‘‘the only possible invention, the impossible invention’’ (1992:41). The school
challenged the unimagined potentialities of the community, not for the transfor-
mation of self, but to develop the transformation of that society to ensure survival. To a large extent his has been achieved by offering education that helped students to
not only transform as individuals, but also collectively as a community. Societies are not static and the history of the Robert Clack High School is an
example of this, but what lessons can be learnt from the school that is making these
choices? To retain the principles for education for all children, the school engaged
in the performance of educational functions that inevitably shaped the community. Confronting Disadvantage This
perhaps best summed up by a final year student on his way to take up a place at a
university in the USA in 2013: evolving scenario was, therefore, one of enhancing opportunity to transcend their
community personally without dispensing with local culture and history. This
perhaps best summed up by a final year student on his way to take up a place at a
university in the USA in 2013: The school instils a sense of sort of appreciation for the differences in society,
but also shows you a way forward in order to be successful in wider society,
not just academically, not just in traditional school ways. (Year 13 student) Confronting Disadvantage This is the provision of what Duncan-Andrade calls
‘material hope’ for which the first step is for teachers understanding that ‘‘quality
teaching is the most significant ‘material’ resource they have to offer youth’’ (2009:
6). From that position the ambitions of and for students at the school were limited
only by imagination. Consequently we found many examples of students being encouraged to value
who they were rather than aspire to social values not associated with their
community. One outstanding example was a former student, with a significant series
of misdemeanours culminating in a subsequent confrontation with Sir Paul,
returning to the school as a prospective teacher. Given a second chance in life she
made sure of such an opportunity and become a subject leader within a short time of
successfully completing her teaching qualification. Her words describe the way in
which one of her school friends perceived her transition: When I finally got my teaching qualification I spoke to my best friend who
was here in the dark days. We’ve stayed very close ever since and she’s gone
off and just had children and hasn’t really done anything with her life other
than the children. I phoned her to tell her that I’d got the job and I was going to
be working at Robert Clack and then I put the phone down. About 10 min later
she phoned me back crying and she said, ‘I can’t believe it, I can’t believe that
you, my friend, like, have become a teacher’. She couldn’t believe that
actually someone from our background, our area, was actually… and she says
she tells all of her friends and I think that’s what’s changed in the school for
the young people now. (Subject Leader) The Deputy Headteacher was keen to describe in his first interview with us how
the school will celebrate individual success equally for any aspect of the individual
student’s life irrespective of where that took place, a claim backed by a Year 12
student who said: ‘‘even if you’re not the best at the academic side of the school,
you will get a chance to shine and they will recognise what you’ve done’’. The 123 Urban Rev (2016) 48:560–578 575 evolving scenario was, therefore, one of enhancing opportunity to transcend their
community personally without dispensing with local culture and history. Conclusions While acknowledging formal government agenda driven targets, what Hallinger
calls the ‘‘academic press’’ (2005: 3), they resisted narrow approaches to education. 12 123 3 576 Urban Rev (2016) 48:560–578 Instead the school within its field of principles regarded education as a central tenet
of the society in the process of change, as exemplified by one teacher who had been
at the school for some 25 years and had witnessed the school before, during and
after its transition: All elements of achievement are recognised, but underlying all that is that
unless you come out of here with decent academic qualifications you are going
to have your life chances limited. So what we do is to encourage all young
people to do as well as they can in all areas (Teacher since 1990) Consequently the school exceeded its prescripted expected limits because they
saw the purpose of the school through the eyes of the community rather than from
the perspective of government targets. They transcended leaders, teachers and
students from what Bernstein describes in the Code of Language from a ‘middle
class’ delivery of the curriculum to one set within the specific code of community. To that extent the local community is mirrored in teaching and leadership in just the
way it is mirrored in them. This was not an accidental anomaly, but was the result of
a deep connection to the community. The school thus reflected and extended the
laws and customs of the local social system and rejected the singularity of the
national curriculum and the standards by offering education to that sustained the
integrity of the community. Education can move human beings away from their own
roots and anchor them more in the cosmos. By rooting itself in the community the
Robert Clack High School anchored learners within their original social context, as
indicated to us by Sir Paul when stating: ‘‘the best interests of the school was to
continue to be tied in with the community, with the Council and with the Local
Authority’’, a view echoed by a student from the School Council who pointed out
that ‘‘we were voted like one of the worst places to live in the UK, it doesn’t take
away from how well this school does in trying to address those social issues’’. Closing Thoughts In an era where politicians may claim that the basis of a ‘‘new world’’ is education
of all children, despite their socioeconomic and ethnical background, this means
nothing so long as this imperative does not derive from the actual individuals and
the communities to which they belong. This means that it is only when a school
values the right of the learners to be part of a community and participate in it rather
than submit to an authority of order such as targets and external inspection that the
school can transcend its prescripted expectations. This, in our view, is because
creative cooperation, which is now at the heart of the Robert Clack High School,
lies in the hearts of people rather than in politicians’ statements, antipathies and
sympathies. We do not suggest anarchy with schools breaking away from national
agendas, but we do suggest a concept of anarchoautonomy of schools. In such a
model of school government the immediate community would be valued and their
right ‘‘to be’’ honoured, creating a path of coexistence that moves away from the
banking education and hope that is deferred or ‘hokey’. In that sense we use the
term anarchoautnomous school to describe any school that despite the odds
transcend itself to an education for children and the community without distancing
themselves completely from government agendas, as the Robert Clack High School
has done, by filtering them with the lenses of valued education for students and the
community. Whilst you do need a measure of principled infidelity to create such
schools, to become the impossible invention it is the concept of ‘‘critical hope’’
which drives which them on. 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, dis-
tribution, 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. Conclusions In essence the school has created a formula based on archetypal roots, the
concept being that we are part of a whole, but our actions depend not merely on
what we do for ourselves, but also on what we do for the whole. This ideal that is
embedded in the organisational ethos of the Robert Clack High School and has led
the school as an institution to transmit to the students higher values than those
encompassed within narrow confines of the academic press. One of the things that has formed the basis of appreciation to the Robert Clack
High School from the community is the way is has promoted self-understanding of
its place in the micro-cosmos as well as the macro-cosmos and ultimately achieved
the principles of ‘‘critical hope’’ for a renewal of community interest in the school
and in education through the achievement of their children. This awareness endows
the community with the capacity to support and believe in the school and is
mirrored in the emergent pedagogy, summed up in 2012 by a member of the school
leadership team who had started his teaching career at the school just a few years
before: There are high standards and I would call it ‘supportive accountability’. Everybody knows as a teacher what’s expected of them, they know they’re
expected to deliver for the pupils and so everybody is accountable for their 12 123 577 Urban Rev (2016) 48:560–578 result. But that is done in a supportive way, it’s not ruling with an iron first like
fear of somebody walking into your room and seeing what you’re doing. So
the ethos is incredibly supportive, incredibly high standard of the pupils and of
the teaching staff. (Assistant Headteacher) result. But that is done in a supportive way, it’s not ruling with an iron first like
fear of somebody walking into your room and seeing what you’re doing. So
the ethos is incredibly supportive, incredibly high standard of the pupils and of
the teaching staff. (Assistant Headteacher) 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, dis-
tribution, 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
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Highly chemo-, enantio-, and diastereoselective [4 + 2] cycloaddition of 5<i>H</i>-thiazol-4-ones with <i>N</i>-itaconimides
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Beilstein journal of organic chemistry
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cc-by
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Email: * Corresponding author Keywords:
[4 + 2] annulation; asymmetric organocatalysis; dipeptide-based
Brønsted bases; 5H-thiazol-4-ones; N-itaconimides Keywords: Abstract A dipeptide-based urea-amide tertiary amine (DP-UAA) was shown to be an effective Brønsted base catalyst for the first asym-
metric annulation reaction between 5H-thiazol-4-ones and N-itaconimides. High levels of enantioselectivity (up to 99% ee) and dia-
stereoselectivity (>19:1 dr) were obtained for a series of spirocyclic 1,4-sulfur-bridged piperidinone-based succinimides. Highly chemo-, enantio-, and diastereoselective [4 + 2] cyclo-
addition of 5H-thiazol-4-ones with N-itaconimides
Shuai Qiu1, Choon-Hong Tan*2 and Zhiyong Jiang*1 Full Research Paper
Open Access
Address:
1Key Laboratory of Natural Medicine and Immuno-Engineering of
Henan Province, Henan University, Kaifeng, 475004, Henan, P. R. China and 2Division of Chemistry and Biological Chemistry, Nanyang
Technological University, 21 Nanyang Link, 637371, Singapore
Email:
Choon-Hong Tan* - choonhong@ntu.edu.sg; Zhiyong Jiang* -
chmjzy@henu.edu.cn
* Corresponding author
Keywords:
[4 + 2] annulation; asymmetric organocatalysis; dipeptide-based
Brønsted bases; 5H-thiazol-4-ones; N-itaconimides
Beilstein J. Org. Chem. 2016, 12, 2293–2297. doi:10.3762/bjoc.12.222
Received: 21 August 2016
Accepted: 19 October 2016
Published: 01 November 2016
This article is part of the Thematic Series "Strategies in asymmetric
catalysis". Guest Editor: T. P. Yoon
© 2016 Qiu et al.; licensee Beilstein-Institut. License and terms: see end of document. Open Access
Beilstein J. Org. Chem. 2016, 12, 2293–2297. doi:10.3762/bjoc.12.222
Received: 21 August 2016
Accepted: 19 October 2016
Published: 01 November 2016
This article is part of the Thematic Series "Strategies in asymmetric
catalysis". Guest Editor: T. P. Yoon
© 2016 Qiu et al.; licensee Beilstein-Institut. License and terms: see end of document. Full Research Paper Full Research Paper
Address:
1Key Laboratory of Natural Medicine and Immuno-Engineering of
Henan Province, Henan University, Kaifeng, 475004, Henan, P. R. China and 2Division of Chemistry and Biological Chemistry, Nanyang
Technological University, 21 Nanyang Link, 637371, Singapore
Email:
Choon-Hong Tan* - choonhong@ntu.edu.sg; Zhiyong Jiang* -
chmjzy@henu.edu.cn
* Corresponding author
Keywords:
[4 + 2] annulation; asymmetric organocatalysis; dipeptide-based
Brønsted bases; 5H-thiazol-4-ones; N-itaconimides Introduction metric variants using 5H-thiazol-4-ones as nucleophiles have
been disclosed; such as amination [6], allylation [7], conjugate
addition to enones [8], and γ-addition with allenoates [9]. All
these examples focused on nucleophilic addition reactions of
the C5 atom of 5H-thiazol-4-ones. Sulfur-containing tetrasubstituted carbon stereocenters are
widely present in a number of natural and non-natural products
with significant biological activities [1]. In the past few
decades, diverse competent asymmetric strategies have been
established to access these chiral entities [1-4]. Among them,
catalytic asymmetric functionalization of S-containing prochiral
carbon centers can be considered to be one of the most efficient
and expedient approach [1-4]. The development of novel S-con-
taining substrates has therefore attracted the attention of
chemists [1-4]. For example in 2013, Palomo and co-workers
introduced 5H-thiazol-4-ones as a new class of sulfur-contain-
ing pro-nucleophiles in a highly enantio- and diastereoselective
conjugate addition to nitroalkenes, providing α,α-disubstituted
α-mercapto carboxylic acids [5]. Since then, several asym- Recently, we described an organocatalytic asymmetric [4 + 2]
cyclization of 5H-thiazol-4-ones with a series of activated
alkenes, including nitroalkenes, 4-oxo-4-arylbutenones, 4-oxo-
4-arylbutenoates and methyleneindolinones [10]. This work elu-
cidated the feasibility of the electrophilic addition to the C2 po-
sition of 5H-thiazol-4-ones. More importantly, it provided a
direct and convenient approach to synthesize three kinds of bio-
logically important chiral 1,4-sulfur-bridged piperidinones and 2293
2293 Beilstein J. Org. Chem. 2016, 12, 2293–2297. demonstrated in a series of asymmetric reactions [18]. It was
found that the reaction was completed after 48 hours, affording
the desired [4 + 2] annulation adduct 3a in 55% yield with 64%
ee. A significant amount of conjugate addition adduct led to the
unsatisfactory chemoselectivity and thus the moderate yield. When the H-bond donor was changed from thiourea to urea
(catalyst II), it did not provide better results (Table 1, entry 2)
[17,19]. In the [4 + 2] annulation of 5H-thiazol-4-ones with
nitroalkenes, dipeptide-based thiourea−amide−tertiary amine
III (DP-TAA) was devised and demonstrated as a competent
catalyst to furnish excellent chemo- and stereoselectivity [10]. Therefore, we examined catalyst III for this reaction (Table 1,
entry 3); annulation adduct 3a was obtained in 60% yield with
70% ee. The increased in enantioselectivity indicates the poten-
tial of dipeptide-based tertiary amine for this type of reaction. By modifying the thiourea moiety of III to urea lead us to cata-
lyst DP-UAA IV, which could further increase the enantioselec-
tivity (Table 1, entry 4). Introduction Subsequently, we screened the solvent
effect with IV as the catalyst (Table 1, entries 5–7), and the
results revealed that chloroform was the best reaction medium
regarding the enantioselectivity (Table 1, entry 7). By changing
the reaction temperature (Table 1, entries 8 and 9), we found their related derivatives [10]. In order to develop novel chiral
S-containing polycyclic scaffolds, the development of [4 + 2]
annulations of 5H-thiazol-4-ones with unusual activated alkenes
still remains highly desirable. Succinimides are present in many biologically significant mole-
cules and are investigated as potential pharmacophores in the
research of drug discovery [11,12]. Our group has recently
devised N-itaconimides for the assembly of succinimide frame-
works [13-18]. As an extension of these works, herein, we
report an asymmetric [4 + 2] annulation reaction of 5H-thiazol-
4-ones with N-itaconimides. The method features excellent
chemo-, enantio, and diastereoselectivities, thus leading to a
series of chiral spirocyclic 1,4-sulfur-bridged piperidinone-
based succinimides with excellent results. By modifying the thiourea moiety of III to urea lead us to cata-
lyst DP-UAA IV, which could further increase the enantioselec-
tivity (Table 1, entry 4). Subsequently, we screened the solvent
effect with IV as the catalyst (Table 1, entries 5–7), and the
results revealed that chloroform was the best reaction medium
regarding the enantioselectivity (Table 1, entry 7). By changing
the reaction temperature (Table 1, entries 8 and 9), we found Results and Discussion Our studies were initiated by examining a model reaction be-
tween 5H-thiazol-4-one 1a and N-phenyl itaconimide 2a
(Table 1). The reaction was first evaluated in toluene at 25 °C
and using L-tert-leucine-based thiourea−tertiary amine I as the
catalyst (Table 1, entry 1), with excellent catalytic efficacy as Table 1: Optimization of reaction conditionsa. Entry
Catalyst
Solvent
T (°C)
t (h)
Yield (%)b
ee (%)c
1
I
toluene
25
48
55
64
2
II
toluene
25
48
50
62
3
III
toluene
25
48
60
70
4
IV
toluene
25
48
62
74
5
IV
CH2Cl2
25
48
68
77
6
IV
Et2O
25
48
50
76
7
IV
CHCl3
25
48
72
86
8
IV
CHCl3
0
48
70
89
9
IV
CHCl3
−10
60
60
92
10
V
CHCl3
−10
18
98
93
aThe reaction was performed in a 0.05 mmol scale; byield was isolated by flash column; cee was determined by HPLC. Table 1: Optimization of reaction conditionsa. Entry
Catalyst
Solvent
T (°C)
t (h)
Yield (%)b
ee (%)c
1
I
toluene
25
48
55
64
2
II
toluene
25
48
50
62
3
III
toluene
25
48
60
70
4
IV
toluene
25
48
62
74
5
IV
CH2Cl2
25
48
68
77
6
IV
Et2O
25
48
50
76
7
IV
CHCl3
25
48
72
86
8
IV
CHCl3
0
48
70
89
9
IV
CHCl3
−10
60
60
92
10
V
CHCl3
−10
18
98
93
aThe reaction was performed in a 0.05 mmol scale; byield was isolated by flash column; cee was determined by HPLC. Table 1: Optimization of reaction conditionsa. Table 1: Optimization of reaction conditionsa. aThe reaction was performed in a 0.05 mmol scale; byield was isolated by flash column; cee was determined by HPLC. 2294 Beilstein J. Org. Chem. 2016, 12, 2293–2297. Scheme 1: Substrate scope of the [4 + 2] annulation. Reaction conditions: 1 (0.1 mmol), 2 (0.15 mmol), V (0.01 mmol), CHCl3 (1.0 mL) at −10 °C. All
drs are >20:1; ees were determined via chiral HPLC analysis. a20 mol % of V was used, T = 0 °C. Scheme 1: Substrate scope of the [4 + 2] annulation. Reaction conditions: 1 (0.1 mmol), 2 (0.15 mmol), V (0.01 mmol), CHCl3 (1.0 mL) at −10 °C. All
drs are >20:1; ees were determined via chiral HPLC analysis. a20 mol % of V was used, T = 0 °C. Supporting Information Supporting Information File 1
Experimental information and spectroscopic data. [http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-12-222-S1.pdf] Supporting Information File 1
Experimental information and spectroscopic data. [http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-12-222-S1.pdf] Acknowledgements Financial support from the National Science Foundation of
China (Nos. 21672052, 21072044), the Program for Innovative
Research Team from the University of Henan Province
(14IRTSTHN006), and Henan University is greatly appreciated. To extend the usefulness of this reaction, we demonstrate that
the adduct can be efficiently reduced with borane. As shown in
Scheme 2, 3a was readily reduced to afford a spirocyclic sulfur-
bridged N-heterocycle 4 in 70% yield and without compro-
mising its ee value. References 1. Yu, J.-S.; Huang, H.-M.; Ding, P.-G.; Hu, X.-S.; Zhou, F.; Zhou, J. ACS Catal. 2016, 6, 5319–5344. doi:10.1021/acscatal.6b01496 Results and Discussion 2295 Beilstein J. Org. Chem. 2016, 12, 2293–2297. Scheme 2: Transformation of adduct. that the yield of 3a was decreased to 60% but the ee value was
increased to 92% at −10 °C (Table 1, entry 9). To improve the
chemoselectivity, we synthesized a series of DP-UAAs through
tuning of the substituent groups of the urea. We were pleased to
find that the reaction rate could be tremendously increased by
utilizing DP-UAA V as the catalyst, and 3a was obtainable with
high enantioselectivity, high chemoselectivity and excellent
yield of 98% (Table 1, entry 10). Scheme 2: Transformation of adduct. With the optimal reaction conditions in hand, we examined the
substrate scope of the enantioselective [4 + 2] cycloaddition be-
tween 5H-thiazol-4-ones 1 and N-itaconimides 2, catalyzed by
DP-UAA V (Scheme 1). Firstly, with 1a as the model
5H-thiazol-4-one substrate, a series of N-itaconimides contain-
ing various N-aryl groups were transformed to the correspond-
ing [4 + 2] annulation adducts 3a–i in 82−98% yield with
90−96% ee. We then investigated substrates with diverse aro-
matic (3j–o) and heteroaromatic (3p–t) groups on the 2-posi-
tion of 5H-thiazol-4-ones, and found the reactions completed
within 12−72 hours, affording the corresponding annulation
adducts 3j–t in 80−98% yield with 90−99% ee. Altering the R
group on 5H-thiazol-4-ones for a benzyl (3u) or phenyl (3v)
substituent also presented 3u and 3v in high yields and excel-
lent enantioselectivities. The absolute configurations of annula-
tion adducts 3 were assigned based on X-ray crystallographic
analysis of a single crystal of 3r [20]. yields (up to 98%) and excellent enantio- and diastereoselectiv-
ities (up to 99% ee and >19:1 dr). Further investigations involv-
ing new [4 + 2] annulation of 5H-thiazol-4-ones using
DP-TAAs and DP UAAs are currently ongoing and will be re-
ported in due course. Experimental Representative procedure for the synthesis of 3a: N-Phenyl
itaconimide 2a (0.15 mmol, 1.5 equiv) and catalyst V
(0.01 mmol, 0.1 equiv) were dissolved in chloroform (1.0 mL)
and stirred at −10 °C for 10 min. This is followed by the addi-
tion of 5H-thiazol-4-one 1a (0.1 mmol, 1.0 equiv). The reaction
mixture was stirred at −10 °C and monitored by TLC. Upon
complete consumption of 1a, the reaction mixture was directly
loaded onto a short silica gel column, followed by gradient
elution with DCM/MeOH mixture (500:1−200:1 ratio). Removing the solvent in vacuo, afforded product 3a. Through an analysis of the absolute configuration of adduct 3, it
is proposed that a plausible reaction mechanism should be simi-
lar to the DP-TAA-catalyzed [4 + 2] annulation between
5H-thiazol-4-ones and nitroalkenes [10]. In this stepwise
process, the use of 3,5-dichlorophenyl as the substituent group
of the urea in catalyst V would remarkably increase the free
energy difference between R- and S-selection in the first
Michael addition step, and also decreases the free energy of the
second formal Mannich reaction, thus improving the reaction
rate and chemoselectivity of the [4 + 2] cycloaddition. Supporting Information
Supporting Information File 1
Experimental information and spectroscopic data. [http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-12-222-S1.pdf] 1. Yu, J.-S.; Huang, H.-M.; Ding, P.-G.; Hu, X.-S.; Zhou, F.; Zhou, J.
ACS Catal. 2016, 6, 5319–5344. doi:10.1021/acscatal.6b01496
2. Enders, D.; Lüttgen, K.; Narine, A. A. Synthesis 2007, 959–980.
doi:10.1055/s-2007-965968
3. Zhao, X.; Shen, J.; Jiang, Z. Mini-Rev. Org. Chem. 2014, 11, 424–431.
doi:10.2174/1570193X1104140926165906
4. Clayden, J.; MacLellan, P. Beilstein J. Org. Chem. 2011, 7, 582–595.
doi:10.3762/bjoc.7.68 doi:10.1002/anie.200801378 The license is subject to the Beilstein Journal of Organic
Chemistry terms and conditions:
(http://www.beilstein-journals.org/bjoc)
The definitive version of this article is the electronic one
which can be found at:
doi:10.3762/bjoc.12.222 License and Terms
This is an Open Access article under the terms of the
Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0), which
permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited. The license is subject to the Beilstein Journal of Organic
Chemistry terms and conditions:
(http://www.beilstein-journals.org/bjoc)
The definitive version of this article is the electronic one
which can be found at:
doi:10.3762/bjoc.12.222 Beilstein J. Org. Chem. 2016, 12, 2293–2297. doi:10.1002/anie.200801378 doi:10.1002/anie.200801378 14.Wang, J.; Liu, H.; Fan, Y.; Yang, Y.; Jiang, Z.; Tan, C.-H. 14.Wang, J.; Liu, H.; Fan, Y.; Yang, Y.; Jiang, Z.; Tan, C.-H. Chem. – Eur. J. 2010, 16, 12534–12537. doi:10.1002/chem.201002183 Chem. – Eur. J. 2010, 16, 12534–12537. doi:10.1002/chem.201002183 15.Yang, W.; Tan, D.; Li, L.; Han, Z.; Yan, L.; Huang, K.-W.; Tan, C.-H.; 15.Yang, W.; Tan, D.; Li, L.; Han, Z.; Yan, L.; Huang, K.-W.; Tan, C.-H.;
Jiang, Z. J. Org. Chem. 2012, 77, 6600–6607. doi:10.1021/jo3012539 Jiang, Z. J. Org. Chem. 2012, 77, 6600–6607. doi:10.1021/jo3012539 16.Liu, X.; Ye, X.; Bureš, F.; Liu, H.; Jiang, Z. Angew. Chem., Int. Ed. 2015, 54, 11443–11447. doi:10.1002/anie.201505193 17.Zhu, B.; Lee, R.; Li, J.; Ye, X.; Hong, S.-N.; Qiu, S.; Coote, M. L.;
Jiang, Z. Angew. Chem., Int. Ed. 2016, 55, 1299–1303. 17.Zhu, B.; Lee, R.; Li, J.; Ye, X.; Hong, S.-N.; Qiu, S.; Coote, M. L.;
Jiang, Z. Angew. Chem., Int. Ed. 2016, 55, 1299–1303. doi:10.1002/anie.201507796 Jiang, Z. Angew. Chem., Int. Ed. 2016, 55, 1299–1303. 18.Zhao, X.; Zhu, B.; Jiang, Z. Synlett 2015, 26, 2216–2230. doi:10.1055/s-0034-1378865 18.Zhao, X.; Zhu, B.; Jiang, Z. Synlett 2015, 26, 2216–2230. 18.Zhao, X.; Zhu, B.; Jiang, Z. Synlett 2015, 26, 2216–2230. doi:10.1055/s-0034-1378865 19.Jiao, L.; Zhao, X.; Liu, H.; Ye, X.; Li, Y.; Jiang, Z. Org. Chem. Front. 2016, 3, 470–474. doi:10.1039/C5QO00428D 19.Jiao, L.; Zhao, X.; Liu, H.; Ye, X.; Li, Y.; Jiang, Z. Org. Chem. Front. 2016, 3, 470–474. doi:10.1039/C5QO00428D 20.CCDC 1495751 (3r) contains the supplementary crystallographic data
for this paper. 20.CCDC 1495751 (3r) contains the supplementary crystallographic data
for this paper. 20.CCDC 1495751 (3r) contains the supplementary crystallographic data
for this paper. License and Terms
This is an Open Access article under the terms of the
Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0), which
permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited. The license is subject to the Beilstein Journal of Organic
Chemistry terms and conditions:
(http://www.beilstein-journals.org/bjoc)
The definitive version of this article is the electronic one
which can be found at:
doi:10.3762/bjoc.12.222 License and Terms
This is an Open Access article under the terms of the
Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0), which
permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited. Conclusion 2. Enders, D.; Lüttgen, K.; Narine, A. A. Synthesis 2007, 959–980. doi:10.1055/s-2007-965968 In conclusion, we have developed the first asymmetric reaction
of 5H-thiazol-4-ones with N-itaconimides. By employing a
DP-UAA catalyst, the reaction undergoes a [4 + 2] annulation
process with excellent chemoselectivity and with a broad sub-
strate scope, affording a series of valuable chiral spirocyclic
1,4-sulfur-bridged piperidinone-based succinimides in high 3. Zhao, X.; Shen, J.; Jiang, Z. Mini-Rev. Org. Chem. 2014, 11, 424–431. doi:10.2174/1570193X1104140926165906 3. Zhao, X.; Shen, J.; Jiang, Z. Mini-Rev. Org. Chem. 2014, 11, 424–431. doi:10.2174/1570193X1104140926165906 4. Clayden, J.; MacLellan, P. Beilstein J. Org. Chem. 2011, 7, 582–595. doi:10.3762/bjoc.7.68 4. Clayden, J.; MacLellan, P. Beilstein J. Org. Chem. 2011, 7, 582–595. doi:10.3762/bjoc.7.68 2296 Beilstein J. Org. Chem. 2016, 12, 2293–2297. 5. Diosdado, S.; Etxabe, J.; Izquierdo, J.; Landa, A.; Mielgo, A.;
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6526–6531. doi:10.1002/chem.201304877 6. Diosdado, S.; López, R.; Palomo, C. Chem. – Eur. J. 2014, 2 6. Diosdado, S.; López, R.; Palomo, C. Chem. 6526–6531. doi:10.1002/chem.201304877 6526–6531. doi:10.1002/chem.201304877 7. Chen, W.; Hartwig, J. F. J. Am. Chem. Soc. 2014, 136, 377–382. doi:10.1021/ja410650e 8. Badiola, E.; Fiser, B.; Gómez-Bengoa, E.; Mielgo, A.; Olaizola, I.; 8. Badiola, E.; Fiser, B.; Gómez-Bengoa, E.; Mielgo, A.; Olaizola, I.;
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7, 6060–6067. doi:10.1039/C6SC02039A 7, 6060–6067. doi:10.1039/C6SC02039A 11.Katritzky, A. R.; Yao, J.; Qi, M.; Chou, Y.; Sikora, D. J.; Davis, S 11.Katritzky, A. R.; Yao, J.; Qi, M.; Chou, Y.; Sikora, D. J.; Davis, S. Heterocycles 1998, 48, 2677–2691. doi:10.3987/REV-98-506 Heterocycles 1998, 48, 2677–2691. doi:10.3987/REV-98-506 12.Ballini, R.; Bosica, G.; Cioci, G.; Fiorini, D.; Petrini, M. Tetrahedron 12.Ballini, R.; Bosica, G.; Cioci, G.; Fiorini, D.; Petrini, M. Tetrahedron
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English
| null |
RISK MANAGEMENT PRACTICE IN CHINA'S PUBLIC-PRIVATE PARTNERSHIP PROJECTS
|
Journal of civil engineering and management
| 2,012
|
cc-by
| 7,488
|
JOURNAL OF CIVIL ENGINEERING AND MANAGEMENT
ISSN 1392-3730 print/ISSN 1822-3605 online
2012 Volume 18(5): 675–684
doi:10.3846/13923730.2012.723380 1. Introduction from the central government, and the rest would have to
be topped up by the local government, and/or the private
sector (NDRC 2009). Since most of the local govern-
ments were still subject to severe budgetary pressure,
there was a heavy reliance on the private sector invest-
ment. PPP financing modality, with the ability of attrac-
ting foreign and private capital in the development of
infrastructure, was therefore considered as an innovative
tool for financing major infrastructure projects (Yuan
et al. 2010; Cheung et al. 2010). As a result, the second
boom of private investment in infrastructure development
appeared (Ke et al. 2009). RISK MANAGEMENT PRACTICE IN CHINA’S PUBLIC-PRIVATE
PARTNERSHIP PROJECTS Yongjian Ke1, ShouQing Wang2, Albert P. C. Chan3
1Department of Construction Management, Tsinghua University, Beijing 100084, China;
Department of Building, National University of Singapore, 4 Architecture Drive, Singapore 117566
2Department of Construction Management, Tsinghua University, Beijing 100084, China
3Department of Building and Real Estate, The Hong Kong Polytechnic University, Hung Hom, Kowloon,
Hong Kong Special Administrative Region, China
E-mails: 1bdgky@nus.edu.sg (corresponding author); 2sqwang@tsinghua.edu.cn; 3bsachan@polyu.edu.hk
Received 18 Aug. 2010; accepted 30 May 2011 Abstract. Project risks were not properly managed in the many past Public-Private Partnership (PPP) projects in China. Although numerous research studies have been conducted on risk management in China’s PPP projects, the realization of
risk management in China’s construction industry especially in PPP projects with high risk exposure and complicated con-
tract structure has hitherto not been well studied. This paper therefore attempted to examine the current use of risk man-
agement in China’s PPP projects by an empirical survey. The results indicated that the use of risk management was inade-
quate; qualitative risk analysis methods were preferred to quantitative and semi-quantitative methods; risk management
usage in the execution was found to be much higher than in the planning, conceptual or termination stage; interviewees
were unfamiliar with most of the risk identification and assessment tools. All above could be partly due to the project na-
ture, but more fundamentally due to the local industrial culture. The absence of risk management culture was found to be
the dominant factor which limited the implementation of risk management in practice. Recommendations to alleviate the
difficulties of risk management were thereafter provided in this paper. Keywords: risk management, public-private partnership (PPP), China, infrastructure. Copyright © 2012 Vilnius Gediminas Technical University (VGTU) Press Technika
www.tandfonline.com/TCEM 1.1. Public-Private Partnership in China Public-Private Partnership (PPP) modality was adopted to
relieve the Chinese government’s budgetary pressure in
infrastructure construction and development, which was
first introduced by local governments in 1980s. Later
after 1996, several state-approved pilot Build-Operate-
Transfer (BOT) projects were awarded in order to intro-
duce BOT on a larger scale (Chen 2009). Since then, the
investment of private investors in infrastructure develop-
ment had improved greatly. However, at the end of the
decade to cope with the adverse influence of the financial
crisis in Asia, the central government invested huge
amounts of treasury bonds in infrastructure, and was de-
termined to clean up the unregulated or illegal projects,
which led to a termination of the first round of private
investment (Shen et al. 2005). Chinese government’s active attitude has been seen
to encourage and support private investors to participate
in the infrastructure construction and public service su-
pply. It is worth noting that “Several Opinions of the
State Council on Encouraging and Guiding the Healthy
Development of Private Investment” (so called “new 36
clauses”) issued in May 2010 further widen the field and
scope of private investment, which include railway, water
conservancy projects, petroleum gas, telecommunication,
land control, exploration and development of mineral
resources, policy-related housing, medical industry, edu-
cation, social welfare service, and as well as national
defense science and technology industry (State Council Due to the fast economic growth in China and espe-
cially Beijing’s success for the 2008 Olympic Games,
public facilities were in high demand to cope with sustai-
nable development in 2000s. The 4 trillion RMB stimulus
plan as announced by the Chinese government in 2008
embarked on a massive infrastructure spending program
to boost domestic demand (Chinese Government’s Offi-
cial Web Portal 2008). However, only 1.18 trillion came 675 Copyright © 2012 Vilnius Gediminas Technical University (VGTU) Press Technika
www.tandfonline.com/TCEM 676 Y. Ke et al. Risk management practice in China’s Public-Private Partnership projects 2010). This new regulation would no doubt further pro-
mote the PPP implementations in near future. Ezell et al. (2000). Research interests in risk management
have continued to develop in recent years. Researchers
have been adopting more complicated methods, such as
fuzzy set theory (Thomas et al. 1.1. Public-Private Partnership in China 2006), game theory
(Medda 2007), option pricing (Leung, Hui 2005), proac-
tive contracting (Tieva, Junnonen 2009), minimum reve-
nue guarantee and revenue cap agreements (Jun 2010),
etc., instead of qualitative analyses that were used in ear-
lier research work. 1.2. Risks in China’s PPP projects However, the PPP application in China has evolved in an
ad hoc and experimental manner and a mature Chinese
PPP framework has not been established yet (Chen,
Messner 2005). Therefore, only a few PPP projects in
China could perform successfully (Chan et al. 2010a). In
the authors’ other publications, a desktop literature re-
view and telephone interviews were conducted to collect
actual data from PPP projects in China (Ke et al. 2010,
2011). As a part of the findings, some principal risks
causing the failures of past cases were identified as
shown in Table 1. Therefore, a proper management
framework both theoretically and practically is important
for PPP implementations due to the large project scale,
long concession period, complexity and social sensitivity
usually associated (Zhang 2005; Ke et al. 2010; Chan
et al. 2010b). A comprehensive collection of literature regarding
PPP projects in China has been carried out as well. For
example, Zhang and Kumaraswamy (2001) provided an
overview and analysis of the BOT-based approaches for
infrastructure development in China. Ye and Tiong
(2000b) looked at the relationship between government
support and risk-return trade-off of the private partner in
China’s BOT power projects. The research team led by
Dr Robert Tiong in Singapore contributed a lot of efforts
on the risk and its management in China’s BOT projects. These included the evaluation and management of politi-
cal (Wang et al. 1999a, 2000a), foreign exchange and
revenue risks (Wang et al. 2000b, c), risk management
framework (Wang et al. 1999b), and also other studies on
BOT projects in China (Zhang et al. 1998). Sachs et al. (2007) conducted a questionnaire survey to analyze the
political risks and opportunities of PPP in China and
other selected Asian countries. Ke et al. (2010) contribu-
ted to provide a preferred risk allocation scheme for PPP
implementations in China. An impressive improvement
of the Chinese governmental behaviors in PPP was seen
in their results. Capitalizing on the Chinese government’s
increased PPP experience in the last two decades, they
have made a lot of efforts to improve the investment en-
vironment, including moving towards the adoption of
international contractual practices and working out an
equitable risk-sharing scheme (Wang, Ke 2008; Chen,
Doloi 2008). 1.3. Literature review The subject of risk in PPP projects has become a very
popular topic in recent years and is a much discussed
issue amongst all levels of industry and government. Many researchers have offered detailed PPP risk registers
and have assessed their relative importance, such as the
United Nations Development Organization (UNIDO)
(1996), Ozdoganm and Birgonul (2000), Zayed and
Chang (2002), Hardcastle and Boothroyd (2003), Thomas
et al. (2003), Ke et al. (2011), etc. Some of the frequently
adopted risk management analysis techniques reported in
international construction management journals included
Monte Carlo simulation (Ye, Tiong 2000a), Analytical
Hierarchy Process (AHP) (Hastak, Shaked 2000), and
event tree and expedience probability as demonstrated by Table 1. Principal risks encountered in past PPP projects of China Table 1. Principal risks encountered in past PPP projects of China
Risk Case
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Change in law
√
√
√
Approval and permit
√
Poor political decision-making
√
√
√
Public/Political opposition
√
Government’s reliability
√
√
√
√
√
√
√
√
√
Force majeure
√
√
Financial risk
√
Insufficient income
√
√
√
√
Competition (Exclusive right)
√
√
√
√
Supporting utilities risk
√
Market demand change
√
√
√
√
√
Tariff change
√
Corruption
√
Case 1: Jiangsu **** Sewage Treatment Plant; Case 2: Changchun Huijin Sewage Treatment Plant; Case 3: Shanghai Dachang
Water Plant; Case 4: Beijing No. 10 Water Plant; Case 5: Hunan **** Power Plant; Case 6: Tianjin Shuanggang Waste-to-
Energy Plant; Case 7: Qingdao Veolia Sewage Treatment Plant; Case 8: Hangzhou Bay Bridge; Case 9: Fujian Xinyuan Min-
jiang No. 4 Bridge; Case 10: Shandong Zhonghua Power Plant; Case 11: Guangdong Lianjiang Sino-French Water Plant; Case
12: Fujian Quanzhou Citong Bridge; Case 13: Wuhan Tangshunhu Sewage Treatment Plant; Case 14: Shanghai Yan’an
Road.(E) Tunnel; Case 15: Shenyang No. 9 Water Plant; Case 16: Beijing Jingtong Expressway. Case 1: Jiangsu **** Sewage Treatment Plant; Case 2: Changchun Huijin Sewage Treatment Plant; Case 3: Shanghai Dachang
Water Plant; Case 4: Beijing No. 10 Water Plant; Case 5: Hunan **** Power Plant; Case 6: Tianjin Shuanggang Waste-to-
Energy Plant; Case 7: Qingdao Veolia Sewage Treatment Plant; Case 8: Hangzhou Bay Bridge; Case 9: Fujian Xinyuan Min-
jiang No. 1.3. Literature review 4 Bridge; Case 10: Shandong Zhonghua Power Plant; Case 11: Guangdong Lianjiang Sino-French Water Plant; Case
12: Fujian Quanzhou Citong Bridge; Case 13: Wuhan Tangshunhu Sewage Treatment Plant; Case 14: Shanghai Yan’an
Road.(E) Tunnel; Case 15: Shenyang No. 9 Water Plant; Case 16: Beijing Jingtong Expressway. 677 Journal of Civil Engineering and Management, 2012, 18(5): 675–684 While a few studies on the actual use of risk mana-
gement in practice have been done, these primarily focu-
sed on developed construction industries such as Canada
and the United States (Hegazy, Moselhi 1995), the United
Kingdom (Akintoye, MacLeod 1997; Baker et al. 1999),
Australia (Uher, Toakley 1999; Lyons, Skitmore 2004),
etc. Although numerous researches have been conducted
on the subject of risk management as well as PPP imple-
mentations in China, the realization of risk management
in China’s construction industry especially in PPP pro-
jects has hitherto not been well studied. Given the lesson
that project risks were not properly managed in the past
PPP projects in China (Ke et al. 2009), it is therefore
necessary to examine the usage of risk management in
PPP projects. management practice in China’s PPP projects and their
expectations on the form and function of risk allocation
and management tools. Answers for sections 2 and 3
were solicited on a 5-point Likert scale, i.e. 1 = very low,
2 = low, 3 = moderate, 4 = high, and 5 = very high, while
the questions in the last section were open questions to
seek for the experience and understandings of experts in
this topic. p
By administering the questionnaire of Lyons and
Skitmore (2004) again but in a different country would be
of interest for comparison purposes in the future. The
research scope in this paper is limited to risk management
in PPP infrastructure projects instead of those in general
construction projects as studied by Lyons and Skitmore
(2004). Some characteristics distinguishing PPP projects
from traditional construction projects include the huge
investment, complicated contract structure, high risk,
long concession period (usually covering the design,
construction and operation), etc. Several questions were
thus added to reflect the new context of PPP projects, i.e. roles of the organization in the PPP project, organizatio-
nal use of risk allocation during contract negotiation,
rationale of risk allocation in PPP contracts, form and
functions of expected tools for risk allocation in PPP
projects, etc. 1.4. Aim and objectives of this paper Because of the unique economic, environmental, cultural
and political background in China, and the lack of ad-
vanced technology and management in the Chinese con-
struction industry (Zou et al. 2007), this paper therefore
aims to examine the current risk management practice in
PPP projects in China. Other objectives of this study
include to identify factors limiting the application of risk
management theories, and to identify measures to im-
prove the project risk management practice. A total of twenty managers from 20 different com-
panies agreed to be interviewed after much persuasion
and follow-up telephone calls. The twenty interviewees
are senior or middle level managers in their companies. Ten out of twenty interviewees have more than 10 years
working experience, eight with 5–10 years experience
and only two with less than 5 years. Three companies
have an annual turnover of less than 50 million RMB
(US$1 ≈ 6.78 RMB), three others have an annual turno-
ver of 50–100 million RMB, and the remaining fourteen
companies have an annual turnover of more than 100
million RMB. All these respondents have been involved
in one or more PPP projects in China. This confirms that
respondents participating in this survey have rich
experience in PPP financing and therefore their views
would be useful for further analysis. The data of
interview survey were presented in Table 2. 2. Research methodology Ke et al. Risk management practice in China’s Public-Private Partnership projects 678 End of Table 2
Items
Freq./Mean
SD
Factors preventing implementation of
risk management
Cost effectiveness
3.11
1.183
Lack of time
3.00
1.085
Lack of information
3.06
1.259
Difficulties in seeing the benefits
2.89
0.900
Human/organizational resistance
3.35
1.348
Lack of accepted industry
tools/techniques for analysis
3.39
1.335
Lack of experts familiar with the
tools/techniques
3.72
1.406 Continue of Table 2
Items
Freq./Mean
SD
Frequency of use of computers
Cost accounting
4.59
0.618
Databases
3.00
1.225
Risk management
2.24
0.970
Scheduling
3.76
0.970
Risk analysis method usage frequency
Qualitative
3.71
1.047
Semi-quantitative
2.47
1.179
Quantitative
2.53
1.179
Risk management element usage
frequency
Risk management planning
2.35
1.115
Risk identification
3.35
1.057
Risk assessment
3.24
1.147
Risk allocation
2.76
1.147
Risk response
3.07
1.100
Risk documentation
2.35
1.169
Risk management usage in project life
cycle phases
Conceptual
2.88
1.364
Planning
3.06
1.029
Execution
3.76
0.831
Termination
2.35
1.169
Risk identification tool usage
Documentation Reviews
3.35
1.169
Brainstorming
2.35
1.057
Delphi technique
1.53
0.717
Interviewing
2.35
1.222
SWOT analysis
2.94
1.197
Checklist analysis
2.06
1.088
Similar cases comparison
3.00
1.173
Cause-and-effect/Influence diagrams
2.18
1.015
System or process flow charts
2.29
1.105
Scenario assumptions Analysis
1.82
1.131
Frequency of recording risks in a risk
management database
2.31
1.195
Historical risk data usage frequency
3.00
1.265
Risk analysis technique usage
Sensitivity analysis
3.00
1.225
Expected monetary value
2.19
1.167
Decision tree analysis
2.53
0.943
Monte Carlo simulation
1.59
0.618
Risk probability and impact matrix
2.12
0.928
Risk adjusted discount rate
1.71
0.849
Risk premium
1.71
0.849
Risk categorization
2.94
1.345
Risk urgency assessment
3.00
1.118
Intuition/judgment/experience
3.65
0.862
Risk response technique usage
Contingencies
3.35
1.367
Contractual transfer
3.18
1.015
Insurance
2.29
1.160
Contingency percentage usage
0–5%
4
–
6–10%
9
–
11–15%
0
–
16–20%
1
–
>20%
1
– One limitation of this study is the small sample size. Therefore, the findings are not appropriate for comparati-
ve statistical analysis of responses from different types of
organizations (i.e. contractors, consultants, owners and
developers). It is possible that Chinese and Australian
managers might respond differently to questions of this
nature due to the cultural differences (e.g. respondents
may be too optimistic or modest to give an imprecise
score on the self-evaluation questions by the linguistic
scale). 2. Research methodology The comparative analysis between the survey
responses in this paper and Lyons and Skitmore (2004)
may be questionable to some extent. The cultural diffe-
rences would therefore be considered when analyzing the
differences of the survey results. 2. Research methodology In light of the lessons learnt from past PPP projects, this
study developed an empirical survey to collect feedback
from practitioners on the following aspects of risk man-
agement in China’s PPP projects. A series of structured
interviews were conducted during February and April
2009 targeting managers who had hands-on experience in
managing PPP projects in China. The questionnaire of Lyons and Skitmore (2004)
was selected as a basis for the structured interviews as
presented in this paper with their prior permission. The
usage of the following aspects of risk management in
China’s PPP projects would be examined: − Risk tolerance of individuals and companies; − Frequency of use of risk management techniques; Table 2. Survey results
Items
Freq./Mean
SD
Personal risk tolerance
Averse
3
–
Neutral
12
–
Taker
5
–
Personal knowledge of risk management
2.90
0.718
Level of training in risk management
1.89
0.737
Organizational risk tolerance
Averse
5
–
Neutral
14
–
Taker
1
–
Organizational use of risk management
in PPP projects
2.45
0.826
Organizational use of risk allocation in
contract negotiation
2.70
0.979
Rationality of risk allocation in PPP
contracts
2.88
0.806 Table 2. Survey results
Items
Freq./Mean
SD
Personal risk tolerance
Averse
3
–
Neutral
12
–
Taker
5
–
Personal knowledge of risk management
2.90
0.718
Level of training in risk management
1.89
0.737
Organizational risk tolerance
Averse
5
–
Neutral
14
–
Taker
1
–
Organizational use of risk management
in PPP projects
2.45
0.826
Organizational use of risk allocation in
contract negotiation
2.70
0.979
Rationality of risk allocation in PPP
contracts
2.88
0.806 − Risk management usage in each of the project life
cycle phases; − The recording and use of historical risk data; − Factors limiting the risk management practice. The questionnaire adopted in the interviews compri-
sed four sections. In the first section, background infor-
mation was sought, such as business category, annual
turnover, position in the company, years of experience. The second section investigated the respondents’ risk
tolerance, the risk management training respondents have
had and the benefits obtained. The third section focused
on the organizational experience with the application of
risk management, including the company’s risk tolerance,
the frequency of use of risk management techniques, the
recording and use of historical risk data and factors limi-
ting the implementation of risk management. The last
section looked for the opinions of respondents on the risk Y. 3.1. Individual risk tolerance and training The majority of interviewees considered themselves as
risk neutral. Interviewees considered their individual
experience/knowledge of risk management to be low to
moderate with a score of 2.90 as shown in Fig. 1 and
Table 2, which is different from the finding of “moderate
to high” in the Australian survey by Lyons and Skitmore
(2004). This observation may reflect that managers in
China’s PPP projects have less experience in risk man-
agement than those in the Australian construction indus-
try. It is also possible that due to the cultural differences
Chinese managers may be modest to undervalue their
experience/knowledge level. Moreover, the level of training in risk analysis and
management techniques was found to be very low with a
score of 1.89, which clearly indicated that there was a
lack of training in risk analysis and management
techniques by most of the interviewees. The maximum
score by the interviewees regarding the level of their
training in risk management was 3 (moderate), which yet
again reinforces the lack of risk management culture in Fig. 1. Individual risk management knowledge and training Fig. 1. Individual risk management knowledge and training 679 Journal of Civil Engineering and Management, 2012, 18(5): 675–684 China’s PPP projects. Ten respondents have indicated the
most beneficial risk management training that they had
received. These responses comprised three long-term
course studies and seven seminars, which were all about
project management and strategic management. hence understandable that computers were not frequently
used in risk management. But the absence of computer
application in current management in PPP projects in
China now may reflect the lack of the risk management
culture of these companies, as commercial risk manage-
ment software has experienced a great development in
recent years (Dikmen et al. 2004). 3.2. Organizational use of risk management Risk management element usage in PPP projects Fig. 4. Risk analysis method usage in PPP projects Fig. 2. Organizational use of risk allocation & management Fig. 4. Risk analysis method usage in PPP projects Fig. 2. Organizational use of risk allocation & management 3.2. Organizational use of risk management The usage frequency was greater for qualitative than
for semi-quantitative and quantitative methods (Fig. 4). Risk identification, assessment and response were the
most often used risk management elements than risk allo-
cation, management planning and documentation (Fig. 5). In addition as presented in Fig. 6, risk management usage
in the execution stage (score 3.76) was found to be much
higher than in the other three stages of the project life
cycle, i.e. planning (score 3.06), conceptual (score 2.88)
or termination (score 2.35). The late start of risk mana-
gement was identified in China’s PPP projects, which
may create rushing and the missing out of vital details
and hence reduce the efficacy of risk assessment and
response (Chinyio, Fergusson 2003). These observations
again reinforce the assertion that investors in PPP pro-
jects in China are in lack of risk management planning
and skills. This also has caused the failure of several past
PPP projects in China (Wang, Ke 2008) and hence why it
was considered as an obstacle to successful implementa-
tion of PPP projects in China (Chan et al. 2010a). Compared to the individual risk tolerance, more respond-
ents considered their companies as risk averse or neutral. In other words, organizations are more prudent towards
risk taking compared to individuals, which could explain
the following arguments to some extent. When investing
a PPP project, the companies undertook risk management
on a low frequency of 2.45 and risk allocation during the
contract negotiation on a low to moderate frequency of
2.70. The rationale of risk allocation in PPP contracts
were considered as low to moderate with a score of 2.88. However, according to Fig. 2, a lower agreement of the
respondents on the risk allocation usage in contract nego-
tiation could be observed. It means that although the
mean value was smaller than 3, there were still some
companies who already took into account risk allocation
in the contract negotiation. This is a positive sign because
risk allocation, the definition and division of responsibil-
ity associated with a possible future loss or gain, seeks to
assign responsibility for a wide variety of hypothetical
circumstances, which is commonly defined through the
contractual documents as a part of a risk management
strategy (Lam et al. 2007). Fig. 4. Risk analysis method usage in PPP projects
Fig. 5. Risk management element usage in PPP projects
Fig. 6. Y. Ke et al. Risk management practice in China’s Public-Private Partnership projects 680 Y. Ke et al. Risk management practice in China’s Public-Private Partnership projects Fig. 8. Risk analysis technique usage in PPP projects Table 2 and Fig. 7 indicated that most of the risk
identification tools were seldom used in past PPP pro-
jects. Seven out of ten tools in the list received a mean
score lower than 2.35. Documentation reviews, similar
cases comparison and SWOT analysis were the most
frequently used tools to identify risks. However, the usa-
ge frequency of these three tools was only a little greater
than moderate. Moreover, the use of risk management
databases to record project risks was found to be low to
moderate (score 2.31), along with the moderate usage of
such risk data on other projects (score 3.00). This may be
because the use of computers for risk management was
much lower than for other tasks as described above, and
interviewees had to record risks in other ways, e.g. by the
use of hand written risk registers, which obviously reduce
the possibility and willingness of the companies for risk
documentation. This situation was exacerbated when PPP
practice was relatively new as there was often no
experience or track record to follow. Fig. 8. Risk analysis technique usage in PPP projects Fig. 7. Risk identification tool usage in PPP projects Among the risk response techniques, it was found in
Fig. 9 that contingencies was the most frequently used
one (score 3.35) than contractual transfer (score 3.18) and
insurance (score 2.29). The most often adopted percenta-
ge range for the contingencies was 6–10%. However,
when asking how to determine this percentage,
interviewees all considered it as a common practice
without precise calculations. It could be remarked that the
efficacy of risk assessments cannot be ascertained in view
of the foregoing issue, as it is easy to say a 10% for con-
tingencies while in reality it may be more than 10%. In
this case, even though projects are completed or are run-
ning successfully, the client may not get the optimal va-
lue for money. It is noted that insurance was seldom used
in China’s PPP projects. This may be due to the fact that
PPP financing model is a relatively new financial modali-
ty and is unfamiliar to the insurers. Although PPP pro-
jects via BOT model were first seen in 1980s, it is still in
an immature and developing stage. Y. Ke et al. Risk management practice in China’s Public-Private Partnership projects For example, there is
no official PPP law in place yet and there has been a big
public accountability concern to the private investors. Therefore, insurers chose not to present their support in
these projects. Fig. 7. Risk identification tool usage in PPP projects Among the ten risk analysis techniques, intuition,
judgment and experience was the most frequently used
tools with a highest score of 3.65. The other frequently
used tools included risk urgency assessment, sensitivity
analysis and risk categorization. Sensitivity analysis was
the only quantitative technique frequently used, which
again reinforces that qualitative methods of risk asses-
sment were used more frequently than quantitative and
semi-qualitative methods. The interviewees scored very
low to the remaining quantitative tools such as expected
monetary value, decision tree analysis, Monte Carlo sim-
ulation, risk probability and impact matrix, risk adjusted
discount rate and risk premium. It could be also seen that
the maximum scores of many risk analysis techniques
were only 3 or 4 from Fig. 8, which means that not even
one company had been active to adopt these techniques in
any project. One of the reasons may be due to the fact
that risks were assessed on the basis of subjective judg-
ment and individual experience because of the dearth of
historical data documented. Fig. 9. Risk response technique usage in PPP projects
3.4. Factors limiting the implementation of risk Fig. 9. Risk response technique usage in PPP projects 3.4. Factors limiting the implementation of risk
management 3.3. Risk management usage in practice For the information technology, the use of computers was
found to be much lower for risk management than for
cost accounting, scheduling or databases (Fig. 3). This is
also in line with the findings of Lyons and Skitmore
(2004) in Australia. However, computer based risk man-
agement programs were not as popular as time manage-
ment programs at the time of their survey in 2002. It is Fig. 5. Risk management element usage in PPP projects Fig. 5. Risk management element usage in PPP projects Fig. 3. Computer usage in PPP projects
Fig. 5. Risk management element usage in PPP projects
Fig. 6. Risk management element usage in PPP projects Fig. 3. Computer usage in PPP projects
Fig. 5. Risk m
Fig. 6. Risk m Fig. 3. Computer usage in PPP projects Fig. 6. Risk management element usage in PPP projects 3.4. Factors limiting the implementation of risk
management The survey results as presented in Fig. 10 and Table 2
showed that the most dominant factor was “Lack of ex-
perts familiar with the tools/techniques”, which had the
highest mean score of 3.72. Factors “Lack of accepted
industry tools/techniques
for analysis” and “Hu-
man/organizational resistance” were ranked 2nd and 3rd. Journal of Civil Engineering and Management, 2012, 18(5): 675–684 681 Referring back to the individual knowledge and ex-
perience of risk management of the interviewees de-
scribed in section 3.1, it was found that most senior and
middle level managers in the companies admitted their
individual experience and knowledge of risk management
as below moderate. It is hence understandable why “Lack
of experts familiar with the tools/techniques” and “Hu-
man/organizational resistance” have been considered as
critical factors preventing the implementation of risk
management in practice. However, because the interview
results showed that the interviewees did not seem to be
familiar with some common risk identification or assess-
ment tools listed in the questionnaire, the authors would
argue that the factor “Lack of accepted industry
tools/techniques for analysis” may not be a critical rea-
son. The establishment of standard risk management
process in the company is another efficient measure. The
process may include how to use a PPP database, when to
start the risk analysis, who to take the responsibilities,
and what structured approach to be adopted. After run-
ning PPP projects for a while, participants would be able
to develop more reliable information on similar procure-
ment and also understand better what may constitute a
risk and how to manage it. It is therefore beneficial to set
up a PPP database to support risk analysis and manage-
ment. In a PPP project, which is obviously different from
traditional construction projects, the laxity of risk review
and late start of risk management of the private compa-
nies would become potential risks during the concession
period and may cause huge loss. More detailed risk ma-
nagement planning would surely help the participants
identify a myriad of risks that could easily go unnoticed
(Chinyio, Fergusson 2003). The interview survey showed
that the interviewees were not totally familiar with some
common risk identification and assessment tools. Further
training to executives on risk analysis as recommended
above is then essential. 3.4. Factors limiting the implementation of risk
management Moreover, adopting a more struc-
tured approach to risk assessment might be effective,
since it would introduce transparency to the process
(Chinyio, Fergusson 2003) and make it easier for the risk
documentation process. All in all, it could be concluded that these internal
factors (such as lack of experts familiar with the
tools/techniques and human/organizational resistance)
inside the companies were more important than the
external ones, such as lack of time, resources, informa-
tion, etc. It can be thereafter construed that the dominant
reason which limits the implementation of risk manage-
ment was the absence of risk management culture in these
companies. Fig. 10. Factors preventing risk management practices
4 Measures alleviating the difficulties of risk 5. Conclusions Although numerous researches have been carried out on
the subject of risk management in PPP projects, project
risks were still not well managed in many past PPP pro-
jects in China. This observation inspired the authors that
there might be a gap in between the risk management
theory study and the realization of these theories via vari-
ous tools in the management in China’s PPP projects. This paper thus attempts to examine the current PPP risk
management practice in China. A series of interviews
were used to canvass the opinions of managers who have
hands-on experience in PPP projects in China from Feb-
ruary to April 2009. As a brief summary, the results from
the survey included that: − The absence of risk management culture is the
dominant factor that limits the implementation of
risk management in practice. It stems from the re-
sult that internal factors like the lack of experts
who are familiar with the risk management tools
and human/organizational resistance were consid-
ered as more critical than those external factors; Fig. 10. Factors preventing risk management practices 4. Measures alleviating the difficulties of risk
management 4. Measures alleviating the difficulties of risk
management In light of the problems discussed above, solutions were
also sought on how to alleviate the difficulties of risk
management in practice. First of all, the training of risk
analysis and management is considered as one of the
notable preferred solutions, because it would theoretically
and practically facilitate a better understanding of risks
for the organizations and their employees. Given that the
risks pertaining to the public sectors’ behaviors were
always regarded as the principal ones affecting the suc-
cess of PPP projects in China (Sachs et al. 2007), the
need for motivating them to be more alert to the potential
risks is becoming more urgent. − In particular, these senior and middle level man-
agers considered their personal experience and
knowledge of risk management to be below mod-
erate; and the level of training in risk analysis and
management was also found to be very low; − The companies undertake risk management on a
low frequency especially risk allocation during
the contract negotiation when investing a PPP
project. The rationale for risk allocation in PPP
contracts was considered as low to moderate; − The usage frequency was greater for qualitative
methods of risk analysis than for quantitative and 682 Y. Ke et al. Risk management practice in China’s Public-Private Partnership projects Chan, A. P. C.; Lam, P. T. I.; Chan, D. W. M.; Cheung, E.;
Ke, Y. 2010b. Critical success factors for PPPs in infra-
structure developments: Chinese perspective, Journal of
Construction Engineering and Management ASCE
136(5): 484–494. http://dx.doi.org/10.1061/(ASCE)CO. 1943-7862.0000152 semi-qualitative methods. Documentation re-
views, similar cases comparison and SWOT anal-
ysis were the most frequently used tools to identi-
fy risks. Intuition, judgment and experience were
the most frequently used tools to assess risks; semi-qualitative methods. Documentation re-
views, similar cases comparison and SWOT anal-
ysis were the most frequently used tools to identi-
fy risks. Intuition, judgment and experience were
the most frequently used tools to assess risks; − Risk identification, assessment and response were
the most often used risk management elements
than risk allocation, management planning and
documentation. In addition, risk management us-
age in the execution phase was found to be much
higher than in the other three stages of the project
life cycle, i.e. planning, conceptual or termina-
tion; Chen, C.; Messner, J. I. 2005. An investigation of Chinese BOT
projects in water supply: a comparative perspective, Con-
struction Management and Economics 23(9): 913–925. Acknowledgements The work described in this paper was fully supported by a
joint grant from the National Natural Science Foundation
of China (Project No. 70731160634) and the Research
Grants Council of the Hong Kong Special Administrative
Region, China (RGC Project N_PolyU514/07). Sincere
thanks goes to Dr. Terry Lyons and Prof. Martin
Skitmore for permitting the research team to adapt their
survey questionnaire template. Special gratitude is also
extended to those industrial practitioners, who have kind-
ly participated in the interviews reported in this paper
from February to April 2009. Dikmen, I.; Birgonul, M. T.; Arikan, A. E. 2004. A critical
review of risk management support tools, in Proc. of the
20th Annual Association of Researchers in Construction
Management (ARCOM) Conference, 1–3 September,
2004, Heriot-Watt University, Edinburgh, UK, 1145–
1154. Ezell, B. C.; Farr, J. V.; Wiese, I. 2000. Infrastructure risk anal-
ysis of municipal water distribution system, Journal of In-
frastructure Systems ASCE 6(3): 118–122. http://dx.doi.org/10.1061/(ASCE)1076-0342(2000)6:
3(118) Hardcastle, C.; Boothroyd, K. 2003. Risks overview in public–
private partnership, in A. Akintoye, M. Beck, C. Hardcas-
tle. Public-Private Partnerships: Managing Risks and
Opportunities. Oxford: Blackwell Science Ltd., 31–57. 4. Measures alleviating the difficulties of risk
management http://dx.doi.org/10.1080/01446190500184121 Chen, C.; Doloi, H. 2008. BOT application in China: Driving
and impeding factors, International Journal of Project
Management 26(4): 388–398. http://dx.doi.org/10.1016/j.ijproman.2007.07.002 − Among the risk response techniques, it was found
that provision for contingencies was the most pre-
ferred technique than contractual transfer and in-
surance. The most often adopted percentage range
for the contingencies was 6–10%. Chen, C. 2009. Can the pilot BOT Project provide a template
for future projects? A case study of the Chengdu No. 6
Water Plant B Project, International Journal of Project
Management 27(6): 573–583. http://dx.doi.org/10.1016/j.ijproman.2008.10.006 Cheung, E.; Chan, A. P. C.; Kajewski, S. 2010. The public
sector’s perspective on procuring public works projects –
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Australia, Journal of Civil Engineering and Management
16(1): 19–32. http://dx.doi.org/10.3846/jcem.2010.02 All the findings above clearly prove that there is ap-
parently a significant gap in between the theory study and
the practice of risk management in China’s PPP projects. Recommendations to alleviate the difficulties of risk ma-
nagement were also provided in this paper, for example,
training of risk analysis and management especially for
the public sectors. The establishment of standard risk
management process in the company is another efficient
measure, which may include how to use a PPP database,
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lities, and what structured approach to be adopted. Chinese Government’s Official Web Portal. 2008. China’s 4
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f
y
( )
http://dx.doi.org/10.1061/(ASCE)1076-0342(2001)7: Yongjian KE. Currently a research fellow in the Department of Building at National University of Singapore, Singapore. This paper is based on a part of his PhD thesis defended at Department of Construction Management, Tsinghua Universi-
ty, China. His research interests include project management (especially risk management) in public construction projects
and Public-Private Partnership projects. ShouQing WANG. References Professor in the Department of Construction Management, and Deputy Director of the Institute of In-
ternational Engineering Project Management, Tsinghua University, China. He is also the Chairman of China National Col-
laboration Network for M.Eng. (PM) Education. His research interests include project/risk management in Public-Private
Partnership projects. Albert P. C. CHAN. Professor in the Department of Building and Real Estate, The Hong Kong Polytechnic University,
Hong Kong, China. He is also an Associate Dean of the Faculty of Construction and Environment. His research interests
include project management & project success, project finance & Public-Private Partnerships, construction procurement &
relationship contracting, construction industry development, construction safety.
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Editorial: Endocrine Forms of Hypertension: Clinical and Emerging Molecular Aspects
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Frontiers in endocrinology
| 2,019
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cc-by
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Endocrine Forms of Hypertension: Clinical and Emerging Molecular Aspects Secondary hypertension is responsible for about 5–20% of all hypertension cases depending
on the examined cohorts (1–3). In contrast with primary essential hypertension that can
only be treated, secondary hypertension often manifesting itself as resistant hypertension
includes potentially curable diseases (1–3). Research on endocrine diseases leading to secondary
hypertension is a progressive field, and better elucidating their pathophysiology and clinical
features, might pave the way for novel diagnostic modalities and treatments. The major
endocrine diseases as causes of hypertension include primary aldosteronism, hypercortisolism,
pheochromocytoma/paraganglioma,
thyroid
diseases,
primary
hyperparathyroidism,
and acromegaly. Peter Igaz 1,2† and Teresa Maria Seccia 3*† Peter Igaz 1,2† and Teresa Maria Seccia 3*† 1 Second Department of Internal Medicine, Faculty of Medicine, Semmelweis University, Budapest, Hungary, 2 MTA-SE
Molecular Medicine Research Group, Hungarian Academy of Sciences, Semmelweis University, Budapest, Hungary, 3 Clinica
dell’Ipertensione Arteriosa, Department of Medicine, University of Padua, Padua, Italy Keywords: hypertension, primary aldosteronism, thyroid, licorice, Cushing’s syndrome, microRNA EDITORIAL
published: 06 December 2019
doi: 10.3389/fendo.2019.00857 EDITORIAL published: 06 December 2019
doi: 10.3389/fendo.2019.00857 Edited and reviewed by:
Pierre De Meyts,
Université Catholique de
Louvain, Belgium In this Research Topic, we have compiled several articles on different issues of endocrine
secondary hypertension regarding both molecular and clinical features. Primary aldosteronism
is the most common endocrine cause of secondary hypertension affecting 5–13% of hypertensive
patients in different studies (4–6). Three articles are dedicated to its field in this Research Topic. q
Louvain, Belgium
*Correspondence:
Teresa Maria Seccia
teresamaria.seccia@unipd.it *Correspondence:
Teresa Maria Seccia
teresamaria.seccia@unipd.it *Correspondence:
Teresa Maria Seccia
teresamaria.seccia@unipd.it The study by Mohideen et al. was aimed at establishing the prevalence of KCNJ5 gene mutations
in APAs from a Malaysian cohort. The authors, by examining 54 APAs from a Malaysian cohort,
found that its prevalence was 31%, similarly as reported in Caucasians, however, with no prevalence
of mutants in females. KCNJ5 mutant and wild type APAs showed similar percentages of ZF- and
ZG- like cells, but KCNJ5 mutant APAs with a ZF-like profile tended to be associated with larger
APA. A higher expression of CYP11B2 was found in females, who also had adrenalectomy at a
younger age than males. An interesting hypothesis was provided by the Authors: since females had
often larger tumors, the phenotype previously associated with KCNJ5 mutant APAs could be the
phenotype of APAs from female patients, not the phenotype of all KCNJ5 mutant APAs. †These authors have contributed
equally to this work †These authors have contributed
equally to this work †These authors have contributed
equally to this work Specialty section:
This article was submitted to
Molecular and Structural
Endocrinology,
a section of the journal
Frontiers in Endocrinology
Received: 21 November 2019
Accepted: 22 November 2019
Published: 06 December 2019 In the original study by Decmann et al., circulating microRNAs belonging to the group of
non-coding RNA molecules (7, 8) are investigated as potential novel diagnostic tools for the
differentiation of the two major forms of primary aldosteronism i.e., bilateral adrenal hyperplasia
and unilateral adenoma. Three microRNAs were validated on a large cohort of samples to
be overexpressed in bilateral hyperplasia relative to the unilateral adenoma samples. Although
sensitivity and specificity values were not found to be high enough for clinical introduction
at present, this study presents a novel class of potential diagnostic markers for differentiating
subclasses of endocrine hypertension, but also underlines that primary aldosteronism could be
regarded as a spectrum disease. AUTHOR CONTRIBUTIONS Both authors listed have made a substantial, direct and
intellectual
contribution
to
the
work,
and
approved
it
for publication. Citation: Igaz P and Seccia TM (2019) Editorial:
Endocrine Forms of Hypertension:
Clinical and Emerging Molecular
Aspects. Front. Endocrinol. 10:857. doi: 10.3389/fendo.2019.00857 December 2019 | Volume 10 | Article 857 Frontiers in Endocrinology | www.frontiersin.org Editorial: Endocrine Forms of Hypertension Igaz and Seccia genetics
and
treatment
issues
of
pheochromocytoma
are presented. A
comprehensive
review
on
licorice-induced
pseudohyperaldosteronism
is
given
by
Sabbadin
et
al.,
who discussed its biochemical picture and the mechanisms
underlying the mineralocorticoid effect. Of interest, they
also discuss the potential therapeutic use of licorice related
to
its
anti-androgen
and
estrogen-like
activity,
mostly
exploited for treatment of polycystic ovary syndrome on
top
of
spironolactone,
and
the
anti-inflammatory
effects
of
licorice,
unveiling
unfamiliar
properties
of
an
old
medicinal plant. Thyroid diseases are very frequent, but they less often affect
blood pressure values. A comprehensive view of the effects of
thyroid hormones on the cardiovascular system is provided by
Berta et al., who also discuss the genetic background that may
favor cardiovascular damage. We hope that the reader will find this Research Topic
interesting, and the molecular and clinical issues covered by the
articles will be helpful in both research and clinical management
of secondary endocrine hypertension. The
pathophysiology
and
treatment
of
hypertension
in Cushing’s syndrome is discussed in the mini review
by Barbot et al.. Several relevant molecular mechanisms
are
presented,
and
most
notably
antihypertensive
and
also
cortisol-lowering
treatments
are
discussed. Clinically
relevant issues are also discussed in the mini review by
Canu et al. where the hypertension-related clinical picture, REFERENCES 6. Monticone
S,
D’Ascenzo
F,
Moretti
C,
Williams
TA,
Veglio
F,
Gaita
F,
et
al. Cardiovascular
events
and
target
organ
damage
in
primary
aldosteronism
compared
with
essential
hypertension:
a
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Mol Life Sci. (2014) 71:4081–7. doi: 10.1007/s00018-014-1682-4 2. Rossi
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Diabetes Obes. (2019) 26:155–9. doi: 10.1097/MED.0000000000000472 3. Wei FF, Zhang ZY, Huang QF, Staessen JA. Diagnosis and management of
resistant hypertension: state of the art. Nat Rev Nephrol. (2018) 14:428–
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resistant hypertension: state of the art. Nat Rev Nephrol. (2018) 14:428–
41. doi: 10.1038/s41581-018-0006-6 Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest. 4. Rossi
GP,
Bernini
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et
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48:2293–300. doi: 10.1016/j.jacc.2006.07.059 Copyright © 2019 Igaz and Seccia. This is an open-access article distributed under the
terms of the Creative Commons Attribution License (CC BY). The use, distribution
or reproduction in other forums is permitted, provided the original author(s) and
the copyright owner(s) are credited and that the original publication in this journal
is cited, in accordance with accepted academic practice. No use, distribution or
reproduction is permitted which does not comply with these terms. 5. Käyser SC, Dekkers T, Groenewoud HJ, Van Der Wilt GJ, Carel Bakx J, Van
Der Wel MC, et al. Study heterogeneity and estimation of prevalence of
primary aldosteronism: a systematic review and meta-regression analysis. J Clin
Endocrinol Metab. (2016) 101:2826–35. Frontiers in Endocrinology | www.frontiersin.org December 2019 | Volume 10 | Article 857 REFERENCES doi: 10.1210/jc.2016-1472 5. Käyser SC, Dekkers T, Groenewoud HJ, Van Der Wilt GJ, Carel Bakx J, Van
Der Wel MC, et al. Study heterogeneity and estimation of prevalence of
primary aldosteronism: a systematic review and meta-regression analysis. J Clin
Endocrinol Metab. (2016) 101:2826–35. doi: 10.1210/jc.2016-1472 December 2019 | Volume 10 | Article 857 Frontiers in Endocrinology | www.frontiersin.org Frontiers in Endocrinology | www.frontiersin.org 2
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Un modelo de periodización arqueológica: la zona de Elche
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Estos créditos pertenecen a la edición impresa de la obra.
Edición electrónica:
Espagrafic
ARQUEOLOGÍA
DEL PAÍS VALENCIANO:
Panorama y perspectivas
R. Ramos
Un modelo de periodización arqueológica:
La zona de Elche
Índice
Portada
Créditos
Un modelo de periodización arqueológica
La zona de Elche
R. Ramos.....................................................................
5
Bibliografía.............................................................
58
R. Ramos
Un modelo de periodización arqueológica
R. Ramos
Museo Arqueológico de Elche
Un modelo de periodización arqueológica:
La zona de Elche
a zona de Elche ofrece una periodización arqueológica
que permite observar, por las sucesiones estratigráficas de los yacimientos y porque la abundancia de ellos
aporta datos complementarios, una secuencia cultural casi
completa de la seriación de facies que se desarrollaron en
ella, si bien hay que indicar que para obtener esta documentación se han correlacionado aspectos parciales de diferentes yacimientos y que a pesar de ello no se ha obtenido una
secuencia total para la sucesión prehistórica, por lo que debemos quitar rigurosidad al título de esta ponencia, aunque
la sucesión sí es completa en lo referente a las fases protohistóricas y a la Edad Antigua.
L
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Este proceso se inició en Elche en unos momentos todavía
imprecisos de la primera mitad del III milenio a. J.C.. con los
primeros emplazamientos urbanos. El yacimiento que permite seguir la evolución de la población asentada en el territorio ilicitano es La Alcudia, donde se aprecia el origen y evolución de las sucesivas fases culturales que integran su pasado prehistórico, protohistórico e histórico.
La Alcudia, situado a dos kilómetros al Sur de la actual ciudad de Elche, constituyó un lugar de vida ininterrumpida desde el Eneolítico hasta el fin del período visigodo. Su emplazamiento ofrecía la doble ventaja de tener fácil acceso tanto
al río Vinalopó, que supuso en la antigüedad una vía de penetración natural hacia las tierras del interior puesto que sus
riberas marcaron un camino continuo de infiltración cultural,
como el puerto, fuente del comercio, situado en la actual
Santa Pola, que dominaba el antes llamado Seno Illicitano.
El solar ocupado por La Alcudia sobresale en altura unos
seis metros sobre las tierras de la llanura en que está situado, lo que le da un aspecto de montículo y de ahí su nombre.
Este montículo, un gran “tell”, es completamente artificial, es
fruto de la actividad del hombre, pues su formación se ha debido a la destrucción sucesiva de varios poblados y ciudades
allí erigidos, cuyos escombros han llegado a configurarlo.
ÍNDICE
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Un modelo de periodización arqueológica
El hecho de que las primitivas gentes de esta comarca, luego correspondientes a distintos estadios culturales de este
paraje en el pasado, eligieran este lugar para su asentamiento se debió a un factor geográfico. El paisaje de la zona
y su subsuelo nos han documentado sobre las condiciones
de habitabilidad y defensa que reunía, y que lo hacían apto
como emplazamiento, pues en aquellas épocas de nuestro
pasado el solar de Illici, las tierras hoy denominadas La
Alcudia, constituyeron un islote rodeado por las aguas de un
río, cuya fuente, Animeta, está virtualmente extinguida en la
actualidad y su cauce, en buena parte, terraplenado por labores agrícolas, que en este punto remansaba su caudal y
abrazaba a los poblados y después ciudades erigidos sobre
aquel lugar y convertidos así en auténticas fortalezas atendiendo al foso natural que el lecho de dichas aguas les confería y que daba a los núcleos de población allí ubicados el
requisito esencial de su emplazamiento. Estas magníficas
cualidades estratégicas, aunadas a la fertilidad de las tierras
circundantes, explica sobradamente la elección de su lugar
de asentamiento.
En aquel lugar que ahora se denomina La Alcudia se desarrolló la vida de nuestros antepasados durante casi cuatro
milenios. El testimonio de esta existencia se manifiesta en
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este yacimiento con la realidad de una superposición de niveles que responden a nueve estratos arqueológicos, que no
sólo marcan los modos de vida y los cambios sufridos por
sus habitantes en los determinados períodos que comprenden sino algo más: su evolución.
El primer asentamiento de pobladores en el solar de La
Alcudia hubo de corresponder al período Eneolítico, puesto
que los restos descubiertos han proporcionado buena cantidad de materiales de tipología definida. Existen suficientes
testimonios para que, aunque no hayamos encontrado documentación estratigráfica relacionada con las estructuras de
su poblado, podamos tratar de este período en este yacimiento: cuchillos de sílex y puntas de flecha bifaciales de pedúnculo y aletas y foliáceas, fragmentos campaniformes de
tipo campaniforme inciso, idolillos y colgantes de hueso, y
puntas de flecha de cobre de largo pedúnculo lo avalan.
Durante esta etapa la población del territorio ilicitano estuvo
diseminada en grupos o clanes asentados en aldeas o poblados en las cercanías del Vinalopó, plenamente vinculados
a estos habitantes de La Alcudia. Así, a muy corta distancia
de este yacimiento se han localizado los restos de una agrupación de cabañas alineadas formando calles en el paraje
denominado La Figuera Reona, que prácticamente enlaza
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Un modelo de periodización arqueológica
con el pequeño establecimiento de El Promontori donde se
evidencian estratigráficamente los primeros escalones del
proceso cultural en estas tierras, con manufacturas líticas y
cerámicas de clara tipología que responden a los primeros
conjuntos materiales asociables a aquellas primeras comunidades urbanas.
La Figuera Reona, en pleno III milenio a. J.C., debió constituir un poblado integrado por gentes dedicadas a prácticas
agrícolas que explotaban las fértiles tierras que les circundaban y que además recolectaban moluscos, criaban animales domésticos y cazaban los salvajes.
Este poblado, extendido por la ladera derecha del Vinalopó,
con un foco central en la actual salida del puente del ferrocarril, estuvo formado por cabañas circulares, cuyo diámetro
oscila entre 1’20 y 2 metros; de fondo rehundido en el suelo,
que con su excavación ofrecieron materiales cerámicos con
tipos frecuentes de base plana y generalmente provistos de
elementos de suspensión amamelonados. Su cocción, generalmente oxidante, es irregular y sus pastas son de dos tipos fundamentales: unas que contienen gruesos desengrasantes y frecuentemente superficies sin preparación; y otras
de buena calidad, con acabado bruñido o espatulado.
También son muy abundantes en este yacimiento los mateÍNDICE
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panorama y perspectivas
riales líticos que responden a una variada gama de puntas
de flecha bifaciales con predominio de los tipos foliáceos y
de pedúnculo y aletas, de retoque invasor total y similares a
las halladas en La Alcudia, al igual que ocurre con los materiales cerámicos descubiertos, así como a una variada serie
de hachas y azuelas.
Consecuentemente este poblado de La Figuera Reona fue
contemporáneo del emplazamiento inicial identificado por
sus materiales en La Alcudia y al estrato inferior de El
Promontori.
El yacimiento de El Promontori, al Norte de La Figuera
Reona y casi unido a ella, está situado en el cauce del
Vinalopó y constituye una pequeña meseta, recortada entre
el propio río y una suave barranca, cuya superficie se encuentra a unos veinticinco metros de altura sobre el actual
lecho de las aguas del río, si bien, atendiendo a la constante excavación de su curso. Puede suponerse que durante la
época de vida del yacimiento su superficie estuvo situada
entre unos dos y cuatro metros sobre dicho nivel de las
aguas.
ÍNDICE
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Un modelo de periodización arqueológica
1. Puntas de La Figuera Reona. (Col. Bañón).
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2.1. Materiales eneolíticos de La Alcudia.
2.2. Cerámicas de tipo campaniforme y otros útiles de El Promontori.
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La excavación efectuada en El Promontori ha mostrado la
existencia de una clara estratigrafía que precisa una evolución del núcleo de población allí emplazado desde una primera fase eneolítica, Eneolítico I, hasta el período de
Transición a la Edad del Bronce.
El primero de sus estratos, denominado C, corresponde al
Eneolítico I y supone la fase de enlace con el que se ha llamado Neolítico Final de Transición evidenciada por el hallazgo de un fragmento de cuello de un recipiente tipo botella,
por la presencia de varios fragmentos de cerámica “a la almagra” y por el hallazgo de un raspador frontal aquillado de
sílex, y está caracterizada por cerámicas que en general son
de buenas pastas, marrones y negras, con formas de cuencos, de vasos de paredes rectas y bases aplanadas y de vasos con suave perfil en S., y todas exclusivamente lisas, si
bien es evidente que estas formas preludian a aquellas posteriores que con su decoración llamaremos campaniformes.
Sobre este primer estrato se sitúa el denominado B, que responde a la segunda fase eneolítica, Eneolítico II, que se encuentra asociada a abundante cerámica de tipo campaniforme, cuya decoración es predominantemente incisa, con motivos de líneas, triángulos y reticulados, y con alternancias
de impresiones que configuran bandas pseudoexcisas lograÍNDICE
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Arqueología del País Valenciano:
panorama y perspectivas
das a punzón, con formas de cuencos y de vasos acampanados o de perfil en S. A estas cerámicas de tipo campaniforme las acompañan cerámicas lisas de pastas negras y
buena calidad, con superficies cuidadas, y cerámicas de
pastas marrones y amarillentas con formas de cuencos, de
vasijas de mamelones y de recipientes grandes de tipo ovoide muy similares a los pertenecientes al estrato anterior.
Estas cerámicas aparecen asociadas a conchas perforadas,
puntas de hueso y fragmentos de cuchillos de sílex.
El último estrato de este yacimiento, el estrato A, que identificamos con un período de Transición a la Edad del Bronce,
viene representado por la existencia de cerámicas lisas de
pastas amarillentas y calidades deficientes asociadas a escasas decoraciones incisas en recipientes cerámicos de
abundante desengrasante micáceo, algo muy distinto a las
calidades, tanto en pastas como en decoración, apreciadas
en los materiales del estrato B, aunque conservando reminiscencia de aquellos en sus motivos decorativos pero con
un aspecto, textura y composición diferentes. Con estas cerámicas aparecieron varias astillas y un cuchillo incompleto
de sílex, un fragmento de azuela de piedra pulida y un punzón metálico fusiforme de sección cuadrangular de 40 mm.
de longitud.
ÍNDICE
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Un modelo de periodización arqueológica
Consecuentemente la variedad de motivos decorativos cerámicos así como sus buenas técnicas y calidades responden
exclusivamente a los materiales del estrato B y sólo un reflejo de ellos perdura en el estrato A puesto que las cerámicas
con decoración a él asociadas no son más que decadentes
pervivencias tradicionales que simplemente recuerdan por
sus temas de decoración aquella producción anterior, porque
ni las calidades de las pastas de las vasijas ni la ahora tosca
técnica de decoración incisa ofrecen paralelos reales con los
auténticos tipos campaniformes de este yacimiento.
Los materiales cerámicos decorados pertenecientes al estrato A responden a cuencos modelados con pastas de baja
calidad, negruzca y rojiza en su interior o bien amarillenta terrosa y porosa, con abundante desengrasante micáceo, de
cocción deficiente y de superficies exteriores lavadas y nunca bruñidas. Las incisiones con que están realizados sus
motivos decorativos, frisos de líneas quebradas y zonas
triangulares apuntadas hacia la base logradas por series de
líneas convergentes, ofrecen la peculiaridad de su burda técnica que ocasiona surcos de marcado irregular con reborde
lateral originado por el desplazamiento del barro.
Por todo ello, basados en la estratigrafía del yacimiento y en
la realidad de los materiales hallados, exponemos la profunÍNDICE
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Arqueología del País Valenciano:
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da diferenciación existente entre los que podemos llamar auténticos tipos campaniformes correspondientes al Eneolítico
II, que cronológicamente podrían situarse dentro de la segunda mitad del III milenio a. J.C., y los tipos decadentes
ocasionados por un consecuente proceso evolutivo, reflejo
de una tradición ceramista, que en este yacimiento se muestran asociados al período de Transición a la Edad del Bronce,
período que pudo ocupar los dos primeros siglos del I milenio a. J.C.
El aspecto del yacimiento y la documentación obtenida tras
la realización de las campañas de excavación indican que
debió responder al emplazamiento de un núcleo de habitación muy posiblemente vinculado al poblado de La Figuera
Reona y en el que se realizaba un trabajo especializado.
Avala además esta consideración de lugar de habitación el
hecho de que sus cerámicas son de uso doméstico, tanto las
lisas como las de tipo y decoración campaniforme, puesto
que algunas de ellas mostraban indicios de utilización. Estas
piezas, en general, las mismas que en las tumbas integran
los ajuares, no debieron tener consiguientemente intencionalidad religiosa, sino que respondieron a las vasijas de calidad empleadas por una comunidad y debió ser precisamente de ellas de las que tal vez se escogían las piezas más
ricas para ser destinadas a constituir ajuares funerarios.
ÍNDICE
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Un modelo de periodización arqueológica
3.1. y 3.2. Aspectos parciales de la excavación practicada en El
Promontori.
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La secuencia estratigráfica observada en esta excavación de
Elche conlleva lógicamente una evolución de su población,
documentada por los materiales arqueológicos descubiertos
y por la inexistencia de niveles estériles, hecho fundamental
para sostener un criterio evolutivo en las gentes que utilizaron las cerámicas de tipo campaniforme. En consecuencia,
para precisar sobre la segunda fase eneolítica debemos
atender a la difusión de una modalidad en la decoración de
la cerámica puesto que este núcleo de habitación es esencialmente el que fue precampaniforme sin más innovación
en su segunda fase que la presencia de este nuevo tipo cerámico o lo que es lo mismo: la aplicación de decoración a
vasos y cuencos.
Por ello la “aparición de cerámicas campaniformes deben valorarse como el resultado de una moda ya de importaciones
o de producciones locales, problema que posiblemente resuelvan los análisis de pastas de los materiales cerámicos
aquí descubiertos comparados con las muestras extraídas
de la bolsada de arcilla existente junto al yacimiento. Por ello
esta presencia cerámica no responde a la llegada de una
nueva población, puesto que no existe ruptura en el resto del
complejo material integrante de su período cultural, hecho
avalado por el mantenimiento de los modos de vida y de las
ÍNDICE
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Un modelo de periodización arqueológica
formas cerámicas que evidencian una clara evolución de los
mismos ceramistas tanto en las formas lisas presentes en
sus tres estratos como en las que por su decoración pasarán
a ser campaniformes desde la aparición y desarrollo de sus
temáticas decorativas en su segundo estrato, y en las pervivencias de ellas en su último estrato. Consecuentemente el
mantenimiento general de pastas, técnicas y formas cerámicas acreditan la citada evolución.
Además, tras la excavación, observamos que sólo existe un
fondo de cabaña circular de 2’30 mts. de diámetro, de interior revestido de arcilla en la que se aprecia su aplicación sobre cañas y ramaje, y podemos suponer que en la zona arrasada por las extracciones de arena, anteriores al descubrimiento de este yacimiento, a lo sumo pudo haber otras dos,
lo que no permite tratar de un auténtico poblado sino más
bien de una comunidad familiar dedicada a una actividad
concreta.
Es muy significativo el hecho de no haber hallado ni una sola punta de flecha en nuestra excavación, lo que metodológicamente implica que sus gentes no desarrollaron la normal
actividad cazadora tan patente en La Figuera Reona y también es significativo el hecho de que en un área de poco más
de 100 m2 hayan aparecido más de 4.500 fragmentos ceráÍNDICE
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Arqueología del País Valenciano:
panorama y perspectivas
micos pertenecientes a la totalidad de sus fases de los que
498 pertenecen a tipos campaniformes. Este volumen de
material cerámico es indudablemente excesivo para el uso
de la comunidad a que pertenece, lo cual, basados para ello
en otros hallazgos que siguen la ruta de penetración hacia el
interior que marca el Vinalopó, nos sugiere la posibilidad de
que esta comunidad practicara la actividad alfarera y que este yacimiento hubiera constituido en su época de vida un rico alfar que no sólo abasteció de productos cerámicos a la
zona de Elche sino a lugares comunicados con ella pero
considerablemente alejados de este lugar de producción.
4.1. Restos de fortificaciones de la Edad del Bronce del Sector 5-F de La
Alcudia.
ÍNDICE
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Un modelo de periodización arqueológica
4.2. Restos de fortificaciones de épocas preibéricas en el Sector 4-B de
La Alcudia.
Todos estos testimonios manifiestan la existencia de unas
gentes que durante largo tiempo desarrollaron unas actividades, de muy variado tipo, que hicieron surgir en ellas el
concepto de riqueza, la posesión de bienes, pero a causa de
ello debieron iniciarse ya a principios del II milenio a. J.C.,
hostilidades ocasionadas por grupos ajenos a estas comunidades con las que comenzó un período de inestabilidad económica que hizo descender la productividad de los habitanÍNDICE
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Arqueología del País Valenciano:
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tes de esta zona y que condujo, hacia el 1800 a. J.C., a la
que se ha llamado Edad del Bronce. Las gentes de este territorio, ante la necesidad de protección para sus casas y
pertenencias, buscaron lugares que les permitieran fortificarse y algunos se vieron obligados a desplazar sus poblados a
lugares de fácil defensa natural. La Alcudia reunía tales requisitos pero no La Figuera Reona cuyos pobladores se trasladaron a puntos de difícil acceso, se encastillaron en parajes como El Castellar, la Serra del Búho y La Moleta, y además establecieron puestos vigías, auténticos fortines, como
el ya parcialmente excavado junto al Vinalopó, cerca de La
Moleta, y el localizado en la parte alta del Barranco de Los
Arcos, que permiten dominar los caminos de acceso a esta
zona.
Es por lo tanto éste un nuevo estadio cultural caracterizado por
la presencia de evidentes cambios sociales instrumentales.
En La Alcudia sus habitantes debieron mantener su establecimiento en el que, además de la existencia de su foso natural, construyeron muros defensivos para mejor protección de
su poblado, si bien quedaron manifiestamente empobrecidos
a causa del belicismo supuesto para esta Edad del Bronce
en este territorio.
ÍNDICE
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Este yacimiento ha ofrecido materiales significativos de la
etapa como dientes de hoz, hachas y azuelas de piedra pulida, molinos de mano barquiformes y manos de molino, y vasijas cerámicas con apéndices de mamelones.
El Castellar de Morera, yacimiento situado sobre una alta
plataforma inclinada y de difíciles accesos, en donde todavía
no hemos practicado prospección directa, y cuyos materiales proceden de afloraciones recogidas a principios de siglo,
debe de constituir uno de los recintos fortificados de esta
época siendo en él relativamente frecuentes los hallazgos de
dientes de hoz y raspadores de sílex, hachas y azuelas de
piedra pulida, fragmentos cerámicos modelados en los que
es abundante la presencia de mamelones aplicados y también se asocia a él el descubrimiento de una sepultura en fosa que con los restos óseos contenía un brazalete y unos
aretes de bronce.
El Puntal del Búho y los otros tres picos de la sierra de este
nombre contienen indicios de lugares de habitación y en el
primero de ellos fueron descubiertas varias sepulturas en
cista. Sus materiales consisten en ollas globulares con mamelones, tazas con asa, cuencos, fragmentos de vasos carenados, un posible pie de copa, una pulsera de bronce incompleta y un puñal de remaches. El conjunto podría resÍNDICE
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ponder a una facies argárica dados los hallazgos del poblado, enterramientos y materiales, pero son pocos los elementos de que disponemos para emitir opinión al respecto. Sin
embargo sí es evidente que el Vinalopó es la zona de confluencia entre el Bronce Valenciano y el Bronce Argárico por
lo que su atribución al segundo sería posible.
La Moleta es una eminencia de superficie plana que domina
dos barrancos y que se halla estratégicamente situada frente al estrecho de Manga, único camino para franquear por dicha zona el paso de las sierras del Búho y de Animeta.
Conserva restos de muros que debieron corresponder a fortificaciones y el reconocimiento de la superficie del terreno
ha aportado dientes de hoz y materiales cerámicos asociables a este período.
Al Oeste de La Moleta, en un escarpe rocoso volado sobre
el Vinalopó, Cara Moro, se encuentra el que hemos denominado “Fortín I” consistente en una fortificación de esta Edad
del Bronce sólidamente defendida con murallas, de hasta
cinco encintados en su zona de acceso. Es una construcción
arriñonada adaptada al terreno sobre el que se situó, con
una distribución interior que expresa la evidencia de su carácter defensivo: un pasillo estrecho que conduce a una habitación cortada por un muro que obliga a una entrada indiÍNDICE
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Un modelo de periodización arqueológica
vidualizada que da acceso a una segunda habitación que
contiene un hogar semicircular adosado y bancos laterales,
que da salida a una terraza de vigilancia también protegida
por muros. Los trabajos de excavación efectuados, además
del conocimiento todavía parcial de sus estructuras, ha proporcionado abundante material cerámico, caracterizado por
piezas carenadas, molinos de mano barquiformes, dientes
de hoz y punzones de hueso.
5.1. Fotografía aérea de El Fortín I.
ÍNDICE
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Arqueología del País Valenciano:
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5.2. Aspecto parcial de la zona excavada en El Fortín I.
En La Alcudia los últimos niveles de su estrato H son asociables a un largo período identificado con el Bronce Final y
representado por hallazgos de excavación consistentes en
cuencos con apéndices de mamelones y escudillas modeladas a mano, de pasta gris con desengrasantes misáceos,
fragmentos cerámicos modelados con decoración exterior
arañada y superficies lavadas, hachas de piedra pulida, raspadores de sílex y molinos de mano barquiformes así como
el descubrimiento ocasional de hachas planas de bronce.
ÍNDICE
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Un modelo de periodización arqueológica
Con esta denominación de Bronce Final aludimos a una fase caracterizada por la presencia de un principio de nuevas
corrientes culturales que incidirán de formas distintas en las
diferentes regiones peninsulares y que matizarán los diferentes substratos culturales existentes en ella. Así, en los últimos años del II milenio a. J.C., surgieron nuevos factores
que originaron la configuración de pueblos que o bien estaban ya inmersos en cauces protohistóricos o bien iban a desembocar en general en facies propias de la Edad del Hierro,
preludio de fases ajenas a la Prehistoria. Las distintas agrupaciones culturales de nuestra Península ofrecen matices diferenciadores originados tanto por las propias manifestaciones locales autóctonas como por las influencias debidas a
relaciones esencialmente de tipo comercial.
El Bronce Final en la zona ilicitana ofrece una producción cerámica que evidencia una continuidad con respecto a la fase
anterior y que está representada por formas comunes pertenecientes a grandes vasijas globulares o de paredes rectas
y fondo generalmente plano, y a cuencos y escudillas; mientras que su metalurgia de bronce ofrece hachas planas de
apéndices laterales, características de su período, destacando como conjunto el depósito de La Alcudia, integrado por
más de cincuenta piezas de las que hoy se conservan cinco
ÍNDICE
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en nuestro Museo Arqueológico y doce en el Arqueológico
Nacional.
En este yacimiento de La Alcudia, sobre la base del Bronce
local con sus típicos materiales cerámicos de abundante
desengrasante micáceo con formas de cuencos y vasos de
mamelones, se desarrolla una fase situada entre la segunda
mitad del siglo VI y la del V a. J.C., que responde a un claro
estrato arqueológico, estrato G, y que supone el período que
hemos denominado Preibérico y que, por su personalidad,
debemos empezar a denominar Ibérico Arcaico, que representa a una tradicional metalurgia del Bronce asociada a estructuras del Hierro I e inmersa en un mundo de colonizaciones que implica un auténtico proceso de aculturación con
modelos procedentes esencialmente del Mediterráneo
Oriental.
En La Alcudia este período Ibérico Arcaico está caracterizado por la presencia de materiales cerámicos torneados, posiblemente importaciones de diversos centros del
Mediterráneo (Fenicia, Chipre, Jonia, Siria ... ) realizadas a
través del comercio fenicio ya iniciado hacia fines de la fase
anterior y también procedentes de algunos puntos del Sur y
de la Meseta peninsulares, con decoraciones pintadas con
motivos de bandas, líneas y grupos de meandros, con tintas
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rojas, blancas, negras y marrones, con temas geométricos
en una sola tinta siena o con pinturas rosadas y terrosas, y
con la presencia de ánforas de tipología oriental y de ollas de
orejetas, observándose a lo largo del siglo VI a. J.C., la progresiva presencia de la cerámica de decoración monócroma,
preludio de lo propiamente ibérico, con motivos de bandas,
semicírculos y círculos concéntricos.
Parece evidente que los orígenes del pueblo ibero se encuentran en la primitiva población de las tierras en que, en su
momento, se desarrolló la que llamamos su cultura. A principios del siglo V a. J.C., ya existía la cultura ibérica plenamente formada, y los materiales hallados en La Alcudia precisan
el carácter evolutivo de su gestación. Consecuentemente hoy
podemos afirmar con respecto a ella que sus gentes y su
producción material son autóctonos, que en conjunto constituyen una cultura distinta a las demás y de gran personalidad, y que a lo largo de su vida podemos seguir un claro proceso evolutivo. Por todo ello opinamos con respecto a su origen que los iberos no vinieron a este suelo español de otras
tierras porque antes de aquellos momentos iniciales ya étnicamente habitaban aquí: también apreciamos que no fueron
influenciados directamente en su creación por otro pueblo
concreto, sino por un ambiente mediterráneo; y además preÍNDICE
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6.1. Vaso cerámico de El Fortín I.
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cisamos que forjaron su cultura con el contexto de una realidad: el mundo de su época.
En La Alcudia, tras los materiales cerámicos preibéricos o
ibérico-arcaicos integrantes de su estrato arqueológico correspondiente, en los que se aprecia la ya indicada paulatina
tendencia a las decoraciones monócromas, se manifiesta a
principios del siglo V a. J.C., la cultura ibérica con la configuración de una auténtica ciudad que pertenece al estrato F,
Ibérico I o Ibérico Antiguo, y que responde a unas claras normas de urbanismo: calles rectas que se cruzan en ángulo
recto, construcción popular con viviendas de habitaciones
rectangulares y edificios monumentales de sillería.
Esta ciudad encierra la época de la escultura ibérica que, por
tanto, cronológicamente se encuentra centrada entre los siglos V y III a. J.C. Afirmación que se fundamenta en el hecho
de que, a excepción de La Dama, hallada casualmente en el
escondrijo en que en su época se la ocultó, los demás fragmentos escultóricos descubiertos, expuestos en el Museo
Monográfico de La Alcudia, proceden de tareas sistemáticas
de excavación y se han encontrado formando parte del material de construcción de las edificaciones del estrato que cubre a éste al que pertenecen, bien hallados entre las piezas
del pavimento de una calle o bien constituyendo parte de
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6.2. Materiales ibérico-arcaicos de La Alcudia.
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muros, es decir integradas en una época en la que ya no se
producía ni se valoraba la obra escultórica anterior.
Además el estrato Ibérico Antiguo perteneciente a esta primera ciudad ibérica ofrece, asociadas a la producción de escultura y arquitectura monumental ibéricas, de cantería con
ensambladura de plomo, unos materiales cerámicos caracterizados por su decoración pintada de bandas, líneas, círculos, semicírculos y segmentos de círculo concéntricos, decoración esencialmente geométrica en la cual también están
presentes, aunque en pequeña proporción, ciertos temas vegetales simples de tradición mediterránea así como representaciones de zoomorfos realizados a tinta plana pero con
la peculiaridad, por lo general, de que estas representaciones se encuentran inscritas en temas geométricos, como el
ciervo intercalado en el vano libre de una zona de triángulos
como los cuadrúpedos, los peces o los soleiformes que, si
bien con mayor identidad, separan zonas de bandas y líneas, y que indudablemente recogen en estos motivos viejas
tradiciones emparentadas con representaciones pictóricas
de edades pasadas que informan de su autoctonía dentro
del complejo cultural en que se encuentran integradas. Estas
cerámicas ibéricas aparecen acompañadas de escasas producciones áticas de figuras rojas y de vasijas de cerámica
común.
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Los temas decorativos exclusivamente geométricos repiten
insistentemente los mismos motivos que siempre suelen estar concebidos y resueltos de la misma forma; más variedad
y soltura se encuentra en las decoraciones zoomorfas y vegetales, aunque su mayor espontaneidad afecta más a los
temas que a las soluciones técnicas. Sin embargo dentro de
esa uniformidad han de señalarse distintas tendencias que
no han de atribuirse únicamente a factores cronológicos sino
que hay que relacionarlas con la existencia de alfares diferentes y singularmente con la diversa capacidad artística de
los decoradores.
En el último tercio del siglo III a. J.C., la ciudad ibérica existente en La Alcudia fue totalmente demolida, realidad evidenciada por la estratigrafía del yacimiento. A partir de tales
momentos se reconstruye la ciudad, se configura en consecuencia otro estrato, y se inicia el II Período Ibérico, que hemos denominado Ibérico II, que comprende desde los últimos años del siglo III hasta mediados del I a. J.C.
Esta segunda etapa está caracterizada por la ausencia de
producción escultórica y por la nueva temática de la decoración cerámica, con representaciones de las fuerzas de la vida y de la muerte en actitudes violentas que contrastan con
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la serenidad con que pintaron a la Gran Diosa que normalmente preside las escenas.
A este Ibérico II corresponden los vasos decorados con rostros o con figuras humanas, aunque en ellas debemos distinguir por una parte la propia figura humana de las posibles
escenas narrativas y por otra parte las representaciones antropomorfas de tipo simbólico así como las decoraciones de
aves, carnívoros, caballos..., vasos cuya decoración es a veces un simbolismo religioso y que ocasionalmente contienen
plasmaciones de animales sagrados orientalizantes en personalísimas interpretaciones iberas.
La cerámica, como auténtica definidora de todo proceso cultural, es la que caracteriza a esta etapa que hemos llamado
ibérico II. Su personalidad, su barroquismo, su independencia, su desprecio por los cánones clásicos y su singular identificación evidenciada por sus representaciones simbólicas,
humanas, animales y vegetales constituye el más claro índice que puede precisarse para fijar un paso más en la secuencia cultural ibera.
Este tipo de cerámica es muy abundante y repite con frecuencia temas simbólicos, especialmente de aves, carnívoros y representaciones antropomorfas de interpretación ibeÍNDICE
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ra, además de las figuras humanas en escenas de variado tipo. En consecuencia su nota dominante la dan sus ricas decoraciones pintadas. La ejecución de su temática figurada
que ocupa la zona principal de los vasos está realizada a
mano libre, mientras que la de sus motivos geométricos es
fija y con el tradicional compás o peine, con manifestaciones
muy variadas, ya en semicírculos, segmentos y más rara vez
círculos completos, y unos y otros, casi siempre, en grupos
concéntricos que, agrupados en bandas, constituyen armazones de delimitación de zonas siendo en sí esta ornamentación geométrica un elemento secundario de decoración y
no los temas generalmente únicos y principales que caracterizaban la etapa anterior.
En La Alcudia, en este estrato, junto a la cerámica ibérica
decorada aparece, además de la indígena sin decorar, cerámica de Gnatia, calena, campaniense A y B, cerámica de
Megara y cerámica helenística de engobe blanco.
A principios de la segunda mitad del siglo I a. J.C., se produce una nueva remodelación en la ciudad ibérica existente
en La Alcudia tras su conversión en colonia romana. Se mantuvo en ella el urbanismo ibero y se inició un principio de “romanización” que en esta etapa, representada arqueológicamente por un nuevo estrato, sólo afectó realmente a cambios
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7.1. Composición de los fragmentos pertenecientes a una dama sedente.
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de mandos políticos y militares, pero no modificó sustancialmente las tradiciones indígenas que una vez más se manifiestan en su producción cerámica, que responde consiguientemente a un nuevo período en el proceso cultural ibero. Desde los momentos en que se inició esta nueva etapa
esta ciudad figura en la historia con el nombre de Colonia
Iulia Illici Augusta, es inmune y tiene derecho a acuñar moneda, en ella llegaron a concurrir dos legiones, sus ciudadanos obtuvieron el derecho itálico y su idioma oficial fue el latín, idioma que aquellos iberos no debían saber hablar puesto que así lo evidencia el hecho del hallazgo de inscripciones
realizadas en ibérico aunque con letras latinas: una manera
peculiar de cumplir la ley.
La cerámica indígena de este período comprendido entre
mediados del siglo I a. J.C., y mediados del siglo I de J.C., es
decir su cerámica ibérica, ofrece características concretas.
Supone una nueva fase que designamos como Ibérico III o
período Ibero-romano puesto que tanto en formas como en
temas decorativos es algo realmente distinto a las producciones de las dos fases anteriores, y precisamente la presencia de ciertas pervivencias decorativas manifiesta su
sentido de transición y evolución.
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Estas cerámicas pintadas iberorromanas ofrecen una temática diferente así como diferentes ejecuciones y soluciones
en la realización de sus motivos: las bandas de SSS que anteriormente se empleaban como motivo secundario de decoración pasan ahora a ser el tema principal y único que decora algunos vasos: surge un nuevo motivo de tallos y hojas
muy esquemático; aparecen nuevos tipos de hojas pintadas
7.2. Fragmento de un vaso de cerámica ibérica pintado con temas geométricos en el que en una zona libre de una banda de triángulos se representó un ciervo.
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a tinta plana; se realiza una modalidad técnica consistente
en realzar el dibujo por medio de líneas esgrafiadas que lo
siluetean o marcan sus ejes; predominan los finos reticulados con rellenos simétricos de espacios como tema principal
de decoración; van desapareciendo progresivamente las
bandas de semicírculos concéntricos y prácticamente ya no
se dibujan círculos y segmentos de círculo concéntricos.
Las características decorativas de la cerámica de este período Ibérico III, Iberorromano o tercer período de la producción cerámica ibérica, manifiestan la personalidad y la sencilla identidad de la etapa, y consecuentemente deben datarse entre mediados del siglo I a. J.C., y mediados del I de
J.C., es decir que deben situarse cronológicamente en la fase representada por este estrato de La Alcudia.
Estas cerámicas iberromanas se encuentran asociadas a
campanienses B y C, que en ocasiones se ofrecen con estampillas de letras latinas, y a cerámicas rojas con palmetas
impresas, por lo que indican que éste es el momento de la
evolución de la cerámica campaniense a la sigillata ya que
se emplean indistintamente las marcas de una y otra en cerámicas de barniz negro o rojo; también comienza a parecer
la sigillata aretina, con marcas rectangulares distribuidas sobre el fondo de los platos y posteriormente con una sola marÍNDICE
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ca central, y finalmente la sudgálica. Asimismo estos materiales se encuentran acompañados de las monedas de la ceca local, de Illici.
La estratigrafía existente en el yacimiento de La Alcudia de
Elche, apreciable en todos los cortes efectuados, incluso con
pavimentos duros e irrefutables en muchos de ellos, avala
esta clasificación por épocas de su cerámica que no puede
ser objeto de variación puesto que los materiales son componentes de los niveles arqueológicos asignados. Así pues,
las diferentes decoraciones cerámicas, por su asociación a
distintos y sucesivos estratos, responden a épocas distintas
y al mismo tiempo evidencian un manifiesto proceso evolutivo, por lo que los llamados estilos suponen conjuntos y etapas que cronológicamente determinan la sucesión cultural a
la que pertenecen las temáticas decorativas de la cerámica
ibérica.
Consecuentemente la cerámica ibérica debe clasificarse por
épocas y atender a que sus diferentes tipos, en función de
su temática decorativa, responden a sus sucesivas fases, si
bien en cada una de ellas deberá valorarse tanto la personalidad como la distinta capacidad de sus autores. Asimismo
será preciso distinguir entre estilos y escenas, puesto que en
cada etapa existen temáticas comunes desarrolladas con diÍNDICE
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ferentes estilos, lo que equivale a exponer una solución inversa a los esquemas tradicionales: dos vasos de una misma
época con una misma temática decorativa pueden responder
a dos autores que ejecutan su obra con estilo pictórico diferente, como se aprecia en los vasos de El Campesino y de El
Héroe, hallados en este yacimiento en su estrato correspondiente, que respectivamente ofrecen dos personajes pintados
con diferente modalidad técnica de dibujo y de pincel; igualmente con respecto a los llamados estilos narrativo y simbólico opinamos que no deben ser designados como tales sino
como escenas distintas, puesto que sobre el mismo vaso un
mismo decorador, en ocasiones, realiza ambos como puede
observarse en las dos escenas del Vaso del Campesino: el
personaje con su caballo y el ave. Pero ese personaje, así como otros, ¿no serán también representaciones simbólicas?
No obstante se podrá tratar de escenas narrativas y simbólicas porque tales escenas no implican estilo.
Por todo ello afirmamos que la cerámica ibérica responde
por temática a épocas y que además ofrece pervivencias
transicionales que evidencian su evolución; si bien debemos
matizar que la existencia de modalidades en ella es fruto de
la vida de distintos talleres en una misma ciudad y en diferentes ciudades pero dentro de un tipo decorativo que resÍNDICE
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ponde a una época determinada con predominio de determinados temas pero con soluciones decorativas similares,
de lo que se desprende la homogeneidad temática general
de cada una de sus etapas. La identificación de los talleres
supondrá una base fundamental en este estudio.
Todo lo expuesto en cuanto al establecimiento de nuevas bases para la clasificación de la cerámica ibérica está plenamente avalado por los materiales y por los cortes estratigráficos realizados en los sectores 3-F, 4-B, 4-C, 5-E, 6-F, 7-F,
10-A, 10-B, 10-D y 10-E que cubren la realidad estratigráfica
del yacimiento de La Alcudia y que manifiestan cómo el estrato ibérico queda cubierto por enlosados que constituyen el
nivel de pavimento del estrato ibérico II, cerrado a su vez por
una capa de cal y gravas, adobes e incluso mosaicos que
responden al nivel de pavimento del estrato ibérico III o contramano, a su vez cubierto por un grueso mortero de cal que
constituye el nivel de pavimento general del estrato romano.
A mediados del siglo I de J.C., se produjo una destrucción en
la ciudad de Illici que supuso su reconstrucción y, consecuentemente, la formación de un nuevo estrato en La
Alcudia. Se inicia por tanto este período en esta ciudad a
partir de un suceso extraordinario que debió acaecer sin que
podamos precisar sus causas concretas. Esta época, y conÍNDICE
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siguientemente la ciudad aquí edificada, fue ya plenamente
romana. A lo largo de ella se desarrolla un arte provincial evidenciado por la disposición de las viviendas, por los mosaicos y por las pinturas murales, así como por otros materiales que se exponen en la Sala IV del Museo Monográfico de
La Alcudia y por las construcciones de las termas y el alcantarillado. Pero no por ello desaparecieron los alfareros y
decoradores iberos cuya personalidad continuó reflejándose
en sus productos cerámicos.
8.1 y 8.2. Restos del horno calefactor de la villa romana del Parque de
Elche.
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8.1 y 8.2. Restos del horno calefactor de la villa romana del Parque de
Elche.
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En general los conjuntos de materiales de este estrato consisten en piezas de sigillata sudgálica así como hispánica,
clara y común, cerámicas de Acco y cerámicas vidriadas con
barniz verde, si bien este yacimiento, al igual que otros de vida anterior a lo romano, ofrece características propias en sus
conjuntos cerámicos, pues además de las variedades citadas prosigue, como hemos indicado, la tradición de la cerámica pintada, aunque en vasos romanos por lo general, especialmente olpes, cuya decoración suele ser sencilla, con
roleos, volutas y elementos vegetales, persistiendo también
las decoraciones de peces que, aunque bien ejecutados, tienen tipo diferente a los de épocas anteriores. Por todo ello a
este tipo cerámico lo hemos llamado romano de tradición
ibérica.
La fecha final de este estrato ha sido obtenida con carácter
absoluto por el hallazgo de la ocultación de un conjunto en
plata perteneciente a un equipo de tocador al que se sumaron varias piezas monetales cuya documentación informa
que esta ciudad romana de La Alcudia, Illici, fue destruida de
forma violenta a mediados del siglo III de J.C., durante el reinado de Galieno, por el ataque de los francos, puesto que la
coincidencia cronológica entre el nivel de destrucción en el
yacimiento y el suceso reseñado por las fuentes es evidente.
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Durante estos períodos romanos en torno a Illici fueron edificándose villas rústicas que posiblemente pertenecieron al
parcelario de sus centurias, pues la “deductio” de veteranos
registrada por las monedas para la creación de la Colonia
Iulia Illici Augusta trajo consigo la parcelación y reparto de la
tierra. El estudio realizado sobre fotografía aérea en este territorio muestra la existencia de la cuadrícula centurial. Por
consiguiente en el campo de Elche se ha localizado perfectamente la centuriación cuyos límites, cuando no se materializan en caminos o sendas, pueden observarse en diferencias del terrazgo o en cercas de diversa categoría.
Esta “centuriatio” romana se hizo en Elche en una superficie
de 11.340 Ha (225 centurias) con centro en la antigua Illici y
su pervivencia hasta nuestros días queda bien patente a
simple vista puesto que muchos caminos han fosilizado esta
parcelación. Además, esta “Centuriatio” del campo de Elche
es hoy fundamental para el estudio de la parcelación agraria
actual, del regadío, de la red de carreteras y caminos e incluso para el trazado urbano de la ciudad de Elche.
Así, el emplazamiento urbano de Illici marcó el centro de esta “centuriatio”, cuyo cardo máximo vino representado por un
eje de dirección N-S coincidente con la recta de la calle del
Filet de Fora que se prolonga por la carretera de Dolores y
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que luego enlaza con caminos; y cuyo decumano máximo
debió corresponder al todavía vivo camino de Vizcarra.
Establecida pues la realidad de la existencia de la centuriación de Illici, hemos de señalar el descubrimiento hasta hoy
de veinticinco de las villas pertenecientes a ella y pendientes
de excavación.
La información arqueológica obtenida en el yacimiento de La
Alcudia indica que sobre los restos de su ciudad arrasada a
mediados del siglo III de J.C., se levantó otra, que responde
a un nuevo estrato que cubre al anterior y que supone la ciudad reconstruida a partir de la incursión de los francos que,
con sus murallas restauradas, perduró hasta la llamada “invasión” de los bárbaros, o por expresarlo con mayor precisión, hasta el momento de las destrucciones ocasionadas
por las pasajeras devastaciones que causaron sus gentes a
principios del siglo V de J.C. Si bien, durante esta nueva etapa, cuando las estructuras lo permitían, se reutilizaron las viviendas de la época anterior que soportaron la destrucción y
permitieron un aprovechamiento parcial, con adaptaciones al
momento de crisis correspondiente, por lo que en tales casos el estrato romano del Bajo imperio no existe individualizado en los puntos concretos en que esto sucede y sus maÍNDICE
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teriales se muestran asociados a estructuras anteriores a su
época.
Las viviendas de esta ciudad, en su estrato correspondiente,
contienen habitaciones de dimensiones más reducidas que
las del estrato anterior. Sus materiales, especialmente los
cerámicos, vienen caracterizados por la presencia de símbolos cristianos, como así lo evidencian elementos decorativos como la cruz, los corderos, el pez, la ballena, la palma,
la espiga, la paloma, etc.; o como la efigie identificada por
iconografía de San Abdón; o como la escena bíblica del sacrificio de Isaac. Aunque la problemática actual que ello plantea es la de hasta qué punto estas decoraciones no son más
que eso y la de cuando suponen cristianización, problemática que se acentúa en este yacimiento por el hecho de haber
descubierto una ocultación de estos símbolos, lo que implica unos condicionantes que desconocemos.
A esta ciudad corresponde un monumento extraordinario, la
que se ha llamado Sinagoga-Basílica de Illici, construcción
pavimentada de un mosaico polícromo, emparentable a
otros de villas del mismo Elche y por tanto labrado por artífices de una misma escuela, que podemos atribuir a un taller
romano tardío y a una fecha sincrónica a la de su estrato arqueológico correspondiente.
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También a esta ciudad pertenece una necrópolis, emplazada dentro del núcleo urbano, integrada por cajas monolíticas
cubiertas con una gran losa tallada a dos vertientes y por fosas revestidas de tableros de piedra y cerradas de la misma
forma, conteniendo algunas de ellas ajuares funerarios compuestos de pendientes, anillos y collares, así como vasos de
vidrio.
Los materiales cerámicos de este estrato, de esta época del
Bajo Imperio, vienen caracterizados por la presencia de la
cerámica estampada, tanto de pasta roja como gris, con estampillas muy variadas, predominando las circunferencias,
palmetas, rombos y rosetas en múltiples combinaciones y,
con menor frecuencia, también las decoraciones animales y
humanas. Esta cerámica estampada, datada consecuentemente en el siglo IV de J.C., se encuentra asociada a la común, la gris, la sigillata clara y los olpes con decoración pintada de tradición ibérica.
Así pues durante toda la época romana, además de las variedades cerámicas indicadas, prosiguió la tradición de la cerámica pintada ibérica, modalidad cerámica a la que, como
ya hemos precisado, debemos designar como cerámica romana de tradición ibérica, que se mantuvo hasta principios
del siglo V de nuestra Era.
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R. Ramos
Un modelo de periodización arqueológica
Por todo ello, en función de los estudios estratigráficos de este yacimiento, ese término tan amplio de cerámica ibérica
queda identificado en el tiempo, en sus casi diez siglos de
producción pero en sus cuatro etapas, y sirve de patrón cronológico para, por asociación o de forma complementaria,
aplicar sus dataciones atendiendo a factores tipológicos a la
secuencia ibérica en general. Quedan así establecidos los
períodos ibéricos puesto que las sucesiones estratigráficas
9.1. Reconstrucción del impluvium de la casa romana del sector 3-F de
La Alcudia.
ÍNDICE
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Arqueología del País Valenciano:
panorama y perspectivas
del yacimiento de La Alcudia, caracterizadas por un diferente tipo decorativo de la cerámica en cada uno de sus estratos, vienen a ofrecer datos concretos que aportan documentación objetiva al problema del conocimiento de la cultura
ibérica.
La Alcudia es el yacimiento que permite observar los orígenes y el desarrollo total de esta cultura Ibérica. Allí se aprecia cómo surge el mundo ibérico a partir de la población indígena que asimila y crea: cómo tras una etapa de adaptaciones y mantenimiento de sus raíces se configura y personaliza en su primera fase con arquitectura, escultura y cerámica de decoración básicamente geométrica; como se extingue la producción escultórica y hace su aparición la nueva
decoración cerámica de tipo simbólico y representativo en el
segundo período ibérico: cómo se produce a mediados del
siglo I a. J.C., la dominación política y militar romana sin que
ello altere en sus líneas generales las bases de la cultura
ibérica y desarrollándose Un tercer período ibérico que también podemos denominar iberorromano: y cómo aparece la
que hemos llamado cerámica romana de tradición ibérica,
cuando ya se había producido la romanización, con pervivencia de los alfares ibéricos.
ÍNDICE
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R. Ramos
Un modelo de periodización arqueológica
Además, en la excavación de una de las casas de la segunda época romana, del estrato asociado al Bajo Imperio, escondido bajo un gran sillar existente en una de las esquinas
interiores de una habitación, descubrimos un tesorillo con claros indicios de que fue ocultado deliberadamente. Este hallazgo constituyó una base segura para fechar el fin de este
período. Sus piezas, todas de oro y con extraordinaria labor
de orfebrería, consisten en dos pares de pendientes, seis anillos, un lingote, dos sólidos áureos de Honorio, un semis de
Arcadio y varias ágatas con entalle. La presencia del lingote
y el hecho de que las joyas no estén terminadas, pues los cabujones de los pendientes y de casi todos los anillos están a
falta de colocar las piedras y cerrarlos, nos hace suponer que
pertenecían a un taller de joyería y que su creador estaba
confeccionándolas en el momento en que hubo de ocultarlas.
En consecuencia corresponden exactamente a la moda existente en aquella época y además, por el conjunto de monedas de este tesorillo, podemos deducir que la última fase de
habitabilidad de estas viviendas correspondió a principios del
siglo V, y ello aclara y explica que esta suma de alhajas fueran ocultadas con motivo de los ataques bárbaros.
Sobre el nivel de escombros de esta ciudad romana de Illici,
violentamente arrasada, se configuró un nuevo estrato que
ÍNDICE
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Arqueología del País Valenciano:
panorama y perspectivas
responde al período que de forma genérica podríamos llamar “visigodo”, aunque de hecho en él la ciudad de Illici, tras
los saqueos de los bárbaros, continuó su existencia tardorromana pasando más tarde a depender del poder bizantino,
dependencia de tipo más nominal que real, y después al
mundo hispanogodo. Durante esta etapa cambiaron los
mandos políticos de la ciudad, pero la vida de sus gentes
progresivamente empobrecidas tuvo pocas modificaciones
9.2. Aspecto parcial de las excavaciones en curso en el Sector 5-F de La
Alcudia.
ÍNDICE
54
R. Ramos
Un modelo de periodización arqueológica
puesto que la auténtica visigotización de la población de La
Alcudia sólo se realizó a partir de los comienzos del siglo VII
de J.C.
Durante este período también se reaprovecharon, ocasionando el fenómeno ya citado en el estrato anterior, las casas
que permanecieron en pie tras las “invasiones”, si bien considerada la ciudad estratigráficamente alcanza hasta la superficie del terreno. Las edificaciones propias de su nivel arqueológico, asociadas a materiales cerámicos de su época,
ofrecen algunos restos pobrísimos de tipo constructivo a base de canto rodado y piedras cogidas con barro o cal.
En esta época se reutiliza el edificio destinado a la basílica a
la que se incorpora el cancel, alcanzando Illici la categoría
eclesiástica de Obispado.
El tipo general de las cerámicas de esta etapa es basto, de
pastas con grueso desengrasante o con impurezas, y de torneado o modelado así como cocción deficientes. Este proceso regresivo culmina en los materiales cerámicos del siglo
VII y de la primera mitad del VIII de J.C., así como en los
años inmediatos de pertenencia a un régimen de autonomía
local bajo el protectorado musulmán, que expresan con toda
exactitud la crisis económica y cultural que en estos moÍNDICE
55
Arqueología del País Valenciano:
panorama y perspectivas
mentos vivió esta ciudad, puesto que responden a piezas cerámicas confeccionadas a mano, sin torno, que tecnológicamente son en todo similares a las que este mismo yacimiento ofrece como pertenecientes a la Edad del Bronce.
Todos estos datos, comprobados a lo largo de cuarenta y
nueve campañas de excavaciones demostrables en cualquiera de los cortes estratigráficos abiertos, no sólo permiten
precisar el conocimiento del proceso evolutivo ibérico en este yacimiento sino que, complementado con los hallazgos
materiales, esencialmente escultura y cerámica en sus cuatro tipos, de otros yacimientos logra establecer las bases de
la cultura ibérica y de su consecuente secuencia como manifestación de unas gentes durante el transcurso de los cinco siglos anteriores a J.C., y de las pervivencias de su personalidad en las épocas romanas.
El último período de vida de La Alcudia, de Illici, supone ya
la relación de sus habitantes con las gentes de los nuevos
establecimientos musulmanes, que implicó su comunicación,
su dependencia y su agotamiento, pues hacia el año 713 de
C., los pobladores de estas tierras se vieron obligados a pactar con los conquistadores musulmanes, hecho que en principio no ofreció alteraciones en los modos de vida ya que realmente sólo supuso un reconocimiento y el que la comuniÍNDICE
56
R. Ramos
Un modelo de periodización arqueológica
dad no islámica de este territorio pasara a estar representada por Teodomiro, pues la oligarquía visigoda mantuvo sus
privilegios y su culto aunque quedó sujeta al pago de impuestos que los no musulmanes debían al Califa. Pero entre
los años 743 y 744 de C., grupos de sirios se instalaron en
esta zona y si bien no se establecieron en la ciudad cristiana sí lo hicieron en unas fincas de su campo situadas unos
dos kilómetros al norte de ella que posteriormente dieron lugar a la nueva ciudad: Elche.
Este establecimiento musulmán evidenció a la población hispano-romana la existencia de un modo de vida distinto al suyo que permitía el ascenso social, por lo que paulatinamente, a lo largo del siglo IX, algunos ilicitanos se convirtieron al
Islam, no por convencimiento religioso sino por conveniencia, puesto que la exención de impuestos sólo alcanzaba a
los musulmanes y porque al islamizarse pasaban a ser ciudadanos con todos los derechos de aquella comunidad, lo
que no habían conseguido en los siglos anteriores siendo
súbditos de la dominación visigoda.
Así pues, a partir del establecimiento islámico en lo que hoy
es Elche, las gentes de La Alcudia iniciaron un progresivo
traslado a la ciudad nueva hasta que Illici quedó totalmente
abandonada y no fue habitada de nuevo, ni siquiera cultivaÍNDICE
57
Arqueología del País Valenciano:
panorama y perspectivas
da como tierra de labor, hasta el siglo XIX en el que allí se
construyo una vivienda para las gentes que iniciaron actividades agrícolas en ella y que en la actualidad, ampliada, ha
dado lugar a la instalación de su Museo Monográfico.
Este ensayo de periodización, con sus indicados vacíos prehistóricos, ofrece una secuencia que hilvana y marca la evolución de los períodos ibéricos, precisando las características y los matices propios de cada fase, y muestra la sucesión de las etapas romanas, tardorromanas e hispanogoda
con el aval estratigráfico y la tipología cerámica correspondientes.
Bibliografía
ALBERTINI, E., 1906 y 1907: Fouilles d’Elche. Bull. Hisp.,
Burdeos.
BAÑÓN ANTÓN, J. 1949: Hallazgos arqueológicos en Elche.
IV C.A.S.E. Elche, 1948. Cartagena.
BELTRÁN VILLAGRASA, P. 1945: Las primeras monedas latinas de Illici. J.M. Arq- Cartagena.
HERNÁNDEZ PÉREZ, M. 1982: P. Ibarra Ruiz y La Figuera
Reona. Festa d’Elig/82. Elche.
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58
R. Ramos
Un modelo de periodización arqueológica
IBARRA MANZONI, A. 1879: Illici, su situación y antigüedades. Alicante.
IBARRA RUIZ, P. 1926: Elche, materiales para su historia.
Cuenca.
LIBROS DE ACTAS DEL ARCHIVO MUNICIPAL, DE ELCHE.
MATA CARRIAZO, J. 1954: La Edad del Bronce. H.ª E.M.P.,
I. Madrid.
MAYANS Y SISCAR, A. 1771: Illici, hoy la villa de Elche.
Valencia.
PARIS, P. 1903: Essai sur l’art et l’industrie de l’Espagne primitive. Paris.
– 1907: Promenades archeologiques en Espagne. Elche.
Bull. Hisp. Burdeos.
RAMOS FERNÁNDEZ, R. 1974: De Heliké a Illici.
Such-Serra. Alicante.
– 1975: La ciudad romana de Illici. I.E.A., II, 7. Alicante.
– 1977 (2.ª ed. 1981): Arqueología. Métodos y técnicas.
Bellaterra. Barcelona.
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Arqueología del País Valenciano:
panorama y perspectivas
– 1976: Excavaciones en La Alcudia de Elche. E.A.E., 91.
Madrid.
– 1980: Lecciones de Arqueología. U.N.E.D. Elche.
– 1982: Arqueología prehistórica de la península Ibérica.
Ed. Picher. Elche.
– 1983: La Alcudia de Elche. C.A.A.M. Elche.
– 1965: Las invasiones de los francos en España. A.U.M.,
XXIII, 3-4. Murcia.
– 1966: Memoria de las excavaciones practicadas en La
Alcudia de Elche en el año 1964. (Col. A. Ramos) Not.
Arq. Hisp., VIII y IX, 1-3. Madrid.
– 1969: Amuletos de tipo púnico descubiertos en la Alcudia
(Elche). I.E.A., 2. Alicante.
– 1969: Inscripciones ibéricas de La Alcudia de Elche.
A.P.L., XII. Valencia.
– 1974: Tipología de los pondus de La Alcudia de Elche en
sus distintas épocas. M.A.. XXV A.C.A., t. II. Barcelona.
– 1975: Aureliano Ibarra y la investigación arqueológica. IV
A.C.E. 74. I.E.A.. Alicante.
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R. Ramos
Un modelo de periodización arqueológica
– 1976: La antigüedad de Elche. Festa d’Elig/76. Elche.
– 1977: Las villas de la centuriación de Illici. Symposion de
Ciudades Augusteas, II. Zaragoza.
– 1976: Excavaciones al Este del Parque Infantil de Tráfico
en Elche. (CI. A. Ramos) Not. Arq. Hisp. Arqueología IV.
Madrid.
– 1977: La Alcudia de Elche. Valencia A., n.º 508. Valencia.
– 1977: Estratigrafía de La Alcudia de Elche. ITEM. 1.
Alicante.
– 1978: Elche. Materiales para su Historia. Festa d’Elig/78.
Elche.
– 1978: La ciudad musulmana de Elche. M-C, 1 . Elche.
– 1979: Vaso de tipo “megárico” del Portus Illicitanus.
ITEM, 3. Alicante.
– 1979: La Dama de Elche. Historia 16, n.º 45. Madrid.
– 1980: La Alcudia y su Museo Monográfico. Historia 16,
n.º 46. Madrid.
– 1979: El poblamiento ilicitano. Poblad.. 1. Elche.
ÍNDICE
61
Arqueología del País Valenciano:
panorama y perspectivas
– 1980: Actividad arqueológica de los Museos de Elche.
Festa d’Elig/80. Elche.
– 1980: Vasos cerámicos de tipo campaniforme en Elche.
Poblad., 2. Elche.
– 1980: Las cerámicas campaniformes de Elche. Historia
16, n.º 53. Madrid.
– 1981: Nuevas aportaciones para el conocimiento del
Eneolítico. I.E.A., 32. Alicante.
– 1981: Illici Romana. Excavaciones en La Alcudia de
Elche. Historia 16, n.º 68. Madrid.
– 1981: Aspectos culturales de La Alcudia de Elche:
Ensayo de interpretación arqueológica. ITEM, 5.
Alicante.
– 1981: Pervivencias del mundo islámico en Elche. M-C, 4.
Elche.
– 1982: Precisiones para la clasificación de la cerámica
ibérica. LVCENTVM. 1. Alicante.
– 1982: Actividad arqueológica de los Museos de Elche.
Festa d’Elig/82. Elche.
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62
R. Ramos
Un modelo de periodización arqueológica
– 1982: Illici y los Francos. Poblad., 4. Elche.
– 1983: La Alcudia de Elche. Rev. de Arqueología, n.º 24.
Madrid.
– 1983: Elche. B.I.H.F.M., 3. Murcia.
– 1983: La cerámica medieval en Elche. M-C. 6. Elche.
– 1983: Precisiones evolutivas sobre cerámicas de tipo
campaniforme. XVI C.N.A. Murcia-Cartagena. Zaragoza.
– 1983: Estratigrafía del sector 5-F de La Alcudia.
LVCENTVM. 2. Alicante.
– Historia general sobre la investigación del fenómeno ibérico. La Cultura Ibérica. Gandía-La Safor. II C. H.ª y C.V.
(e.p.).
– Memoria de las Excavaciones de La Alcudia. Campanas
1975-76-77-78 y 79 (Subdirección Gral. de
Arqueología).
– Memoria de las Excavaciones en El Promontori. I
Campaña, 1979. (Subdirección Gral. de Arqueología).
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Arqueología del País Valenciano:
panorama y perspectivas
– Memoria de las Excavaciones en El Promontori.
Campañas
1980-81. (Subdirección
Gral. de
Arqueología).
– Aportaciones estratigráficas para el conocimiento de lo
campaniforme en Elche. Homenaje a M. Almagro (e.p.).
RAMOS FOLQUES, A. 1933: Nuevos descubrimientos en
Illice. A.E.A. y A, 26. Madrid.
– 1941: Nuevas excavaciones en La Alcudia de Elche.
C.E., S.E. de A.E. y P, I. Madrid.
– 1943: Hallazgos cerámicos en Elche y algunas consideraciones sobre el origen de ciertos temas. A.E. Arq., 52.
Madrid
– 1943: Museo Arqueológico Municipal de Elche. M.M.A.P.,
IV. Madrid.
– 1944: La Dama de Elche. Nuevas aportaciones a su estudio. A.E. Ar., 56, Madrid.
– 1945: La Dama de Elche. Madrid.
– 1947: Museo Municipal de Elche. M.M.A.P., VIII. Madrid.
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64
R. Ramos
Un modelo de periodización arqueológica
– 1947: Problemas de cerámica. II C.A.S.-E. Esp.,
(Albacete 1946). Albacete.
– 1948: La Dama de Elche. Datos para su cronología. El
problema del nivel arqueológico de su hallazgo. III
C.A.S-E. Esp. (Murcia, 1947). Cartagena.
– 1949: Un tesorillo bizantino en La Alcudia. IV C.A.S.-E.
Esp. (Elche, 1948). Cartagena.
– 1950: La Alcudia de Elche, antes y durante la dominación
púnica. I C.N.A. y V C.A.S.-E. (Almería, 1949),
Cartagena.
– 1950: Hallazgos escultóricos en La Alcudia de Elche.
A.E. Arq., XXIII, n.º 81. Madrid.
– 1951: Influencia del arte griego, etrusco y púnico sobre el
ibérico. VI C.A.S.-E. Esp. (Alcoy, 1950). Cartagena.
– 1952: Una vajilla de cerámica ibérica en La Alcudia.
A.P.L., III. Valencia.
– 1952: La escultura ibérica y las excavaciones de Albertini
en La Alcudia. A.E. Arq.. XXV, n.º 85. Madrid.
– 1952: Perfiles de la cerámica de La Alcudia. II C.N.A.
(Madrid, 1951). Zaragoza.
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65
Arqueología del País Valenciano:
panorama y perspectivas
– 1953: Museo Municipal de Elche. Memoria correspondiente a los años 1949-50. M.M.A.P., XI-XII. Madrid.
– 1953: Mapa arqueológico del término municipal de
Elche. A.E. Ar.: Madrid.
– 1953: Molde romano hallado en La Alcudia de Elche.
ZEPHYRUS, IV. Salamanca.
– 1953: Hallazgos monetarios en Elche. Num. Hisp., VIII 15 y 16.
– 1953: Vestigios cartagineses en La Alcudia de Elche. I
Cong. Arq. Marruecos Español. Tetuán.
– 1953: Campañas de excavaciones en La Alcudia durante los años 1940 a 1948. Not. Arq. Hisp., II, L-III. Madrid.
– 1954: Fragmento de cerámica pintada de La Alcudia reproduciendo una figura clásica. III C.A.N. (Galicia, 1953).
Zaragoza.
– 1955: Sobre escultura y cerámica ilicitanas. Estud.
Ibéricos, III. Valencia.
– 1956: Campañas de excavaciones en La Alcudia de
Elche durante los años 1949 a 1952. Not. Arq. Hisp., III
y IV, 1-3. Madrid.
ÍNDICE
66
R. Ramos
Un modelo de periodización arqueológica
– 1956: Cerámicas que acompañan a la cerámica pintada
de Elche en La Alcudia. IV Cong. Int. C.P.P. (Madrid,
1954). Zaragoza.
– 1957: Elche y su arqueología. Universidad Católica de
Sao Paulo, XIII. Sao Paulo.
– 1958: Las invasiones germánicas en La Alcudia (Elche).
I Cong. Esp. Est. Clásicos (Madrid, 1956). Madrid.
– 1958: Cerámica estampada de La Alcudia de Elche.
S.E.A. y A. de la Universidad de Valladolid. Valladolid.
– 1958: Peine cartaginés de La Alcudia. ZEPHYRUS, IX.
Salamanca.
– 1959: La escultura ibérica de Elche (Alicante). V Congr.
Int. Arq. (Hamburgo, 1958). Hamburgo,
– 1960: Las invasiones germánicas en la provincia de
Alicante (siglos III y V de Jesucristo). I.E.A., XVII.
Alicante.
– 1960: Esquema de la Historia de Elche. E.A.E. Elche.
– 1960: Hallazgos monetarios de Elche. Numario Hispánico,
VIII.
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67
Arqueología del País Valenciano:
panorama y perspectivas
– 1960: Un mosaico helenístico en La Alcudia. Festa
d’Elig/60. Elche.
– 1959: Cerámicas de Azaila y Elche. V C.A.N. Zaragoza.
– 1961: Los jinetes con lanza en la cerámica pintada de La
Alcudia. VI C.A.N. (Oviedo, 1959). Zaragoza.
– 1961: Estado actual de las excavaciones en La Alcudia
de Elche. VII C.A.N. Barcelona.
– 1962: Memoria de las excavaciones efectuadas en La
Alcudia de Elche en 1961. Exc. Arq. en España, 8.
Madrid.
– 1962: Cerámica presigillata de La Alcudia de Elche. VII
C.A.N. (Barcelona, 1960). Zaragoza.
– 1962: Memorias de Excavaciones. Campañas realizadas
durante los años 1956 y 1961. Not. Arq. Hisp., V. Madrid.
– 1962: Cerámicas ibéricas, antiguas, del Sudeste español. S.H. y A. Albacete.
– 1962: Cerámicas esmaltadas de La Alcudia. Homenaje a
C. de Mergelina. Murcia.
ÍNDICE
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R. Ramos
Un modelo de periodización arqueológica
– 1963: Unos pozos manantiales de época romana en La
Alcudia de Elche. A.E. Arq., XXXVI, 107 y 108. Madrid.
– 1964: Esculturas ibéricas de Elche. II Congr. Esp. Est.
Clásicos (Madrid, 1961). Madrid.
– 1964: Los peces en la cerámica pintada de La Alcudia de
Elche. VIII C.N.A. (Sevilla-Málaga. 1963). Zaragoza.
– 1964: Numismática y Arqueología de Elche. Estudios de
Numismática Romana. I.P. y A. Barcelona.
– 1965: La Dama de Elche. Ed. Peñíscola. Barcelona.
– 1962: Elche y la Dama de Elche. Latin-Phila, 3-4. Lima.
– 1966: Fragmento de escultura ibérica de Elche. A.P.L.,
XI. Valencia.
– 1966: La cerámica ibérica de La Alcudia de Elche. VI
Cong. Int. C. P. y P., V-VII (Roma, 1962). Roma.
– 1966: Un kernos y otros vasos de La Alcudia de Elche. IX
C.N.A. (Valladolid, 1965). Zaragoza, 1966.
– 1966: Memoria de las excavaciones practicadas en La
Alcudia de Elche en el año 1964. Not. Arq. Hisp., VIII y
IX. Madrid.
ÍNDICE
69
Arqueología del País Valenciano:
panorama y perspectivas
– 1967: Estratigrafía de La Alcudia de Elche. SAITABI, XVI.
Valencia.
– 1969: Torito ibérico hallado en La Alcudia de Elche. X
C.N.A. (Mahón, 1967). Zaragoza.
– 1969: Cerámica del Cabezo Lucero. A.E. Arq., 42.
119-120. Madrid.
– 1970: La Alcudia de Elche. Oro Verde, 10-77. Madrid.
– 1970: Excavaciones en La Alcudia. S.I.P., 39. Valencia.
– 1970: Religiones y cultos antiguos en Elche. I.E.A., II-3,
Alicante.
– 1970: Campanitas encontradas en La Alcudia de Elche.
XI C.N.A. (Mérida, 1969). Zaragoza.
– 1970: Evolución de la cerámica campaniense a la sigillata en La Alcudia de Elche. R.C.R.F., XI-XII. Munich.
– 1970: Historia de Elche. Elche.
– 1972: El nivel ibero-púnico de La Alcudia de Elche.
Rivista di Studi Liguri, XXXIV, 1-3 (Om. a F. Benoit, II).
Bordighera.
– 1972: Esquema de la historia de Elche. E.A.E. Elche.
ÍNDICE
70
R. Ramos
Un modelo de periodización arqueológica
– 1972: Un cancel visigodo en La Alcudia de Elche. PYRENAE, 8. Barcelona.
– 1973: Cerámicas de La Alcudia de Elche. XII C.N.A.
(Jaén, 1971). Zaragoza.
– 1973: Guía de La Alcudia y de su Museo. Elche.
– 1973: La industria, el comercio y la agricultura en Elche.
Elche.
– 1974: La Dama de Elche. Elche.
– 1974: Morteros de La Alcudia de Elche. Miscelánea
Arqueológica, II. XXV Aniversario de los Cursos Int. de
Preh.ª y Arq.ª en Ampurias. Barcelona.
– 1975: En la época ibero-púnica o Ibérico II, en La Alcudia
de Elche, ritos religiosos, XIII C.N.A. (Huelva, 1973).
Zaragoza.
– 1975: Un mosaico helenístico en La Alcudia de Elche.
A.P.L. XIV. Valencia.
– 1976: Excavaciones en La Alcudia de Elche. 1968 a
1973. Excavaciones Arqueológicas en España, 91.
Madrid.
ÍNDICE
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Arqueología del País Valenciano:
panorama y perspectivas
– 1976: Excavaciones al Este del Parque Infantil de Tráfico
de Elche. Not. Ar. Hisp., Arqueología 4. Madrid.
– 1977: Tablas y dados. XIV C.N.A. (Vitoria, 1975).
Zaragoza.
– 1979: Cerámica ibérica de La Alcudia de Elche configuras animales a tinta plana. XV C.N.A. (Lugo, 1977).
Zaragoza.
– (e.p.): El Eneolítico y la Edad del Bronce en la Comarca
de Elche. S.I.P. Valencia.
ROMÁN LAJARÍN, J.L. 1978: Materiales arqueológicos del
“Puntal del Búho” (Elche, Alicante). I.E.A., 24. Alicante.
– 1980: Los yacimientos de la Edad del Bronce de la “Serra
del Búho”. Festa d’Elig/80. Elche.
SANZ, C. 1.621 (mns.): Recopilación en que se da cuenta de
las cosas ansi antiguas como modernas de la inclita villa de Elche. (Editado en 1954 en Elche por Librería
Atenea bajo el título “Excelencias de la Villa de Elche”.)
TARRADELL, M. 1962: El País Valenciano del Neolítico a la
Iberización. Valencia.
– 1965: Prehistoria y Antiguitat. Historia del País Valenciá,
I. Barcelona.
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Technology-Facilitated Stalking and Unwanted Sexual Messages/Images in a College Campus Community: The Role of Negative Peer Support
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Technology-Facilitated Stalking and Unwanted Sexual Messages/
Technology-Facilitated Stalking and Unwanted Sexual Messages/
Images in a College Campus Community: The Role of Negative
Images in a College Campus Community: The Role of Negative
Peer Support
Peer Support Follow this and additional works at: https://researchrepository.wvu.edu/faculty_publication This article is available at The Research Repository @ WVU: https://researchrepository.wvu.edu/faculty_publications/
2399 Authors
Authors
Walter D. DeKeseredy, Martin D. Schwartz, Bridget Harris, Delanie Woodlock, James Nolan, and Amanda
Hall-Sanchez This article is available at The Research Repository @ WVU: https://researchrepository.wvu.edu/faculty_publications/
2399 Digital Commons Citation
Digital Commons Citation DeKeseredy, Walter D.; Schwartz, Martin D.; Harris, Bridget; Woodlock, Delanie; Nolan, James; and Hall-
Sanchez, Amanda, "Technology-Facilitated Stalking and Unwanted Sexual Messages/Images in a College
Campus Community: The Role of Negative Peer Support" (2019). Faculty & Staff Scholarship. 2399. https://researchrepository.wvu.edu/faculty_publications/2399 This Article is brought to you for free and open access by The Research Repository @ WVU. It has been accepted
for inclusion in Faculty & Staff Scholarship by an authorized administrator of The Research Repository @ WVU. For
more information, please contact researchrepository@mail.wvu.edu. Authors
Authors
Walter D. DeKeseredy, Martin D. Schwartz, Bridget Harris, Delanie Woodlock, James Nolan, and Amanda
Hall-Sanchez Keywords y
technology-facilitated stalking, unwanted sexual messages/images, negative peer support, intimate partner violence, sexual assault y
technology-facilitated stalking, unwanted sexual messages/images, negative peer support, intimate partner violence, sexual assault Research on physical and sexual assaults on North American
college women has mushroomed since the mid-1980s. We
now know that at least 25% of female undergraduates have
experienced one or more types of sexual assault during their
college career and that estimates of intimate partner nonsex-
ual violence against college women (e.g., slapping and
punching) range from 10% to 50% (Lindquist & Krebs,
2017; Powers & Kaukinen, 2017). One problem in the litera-
ture is that while there are a variety of forms of sexual
assaults, labels used by researchers have caused confusion. Many readers presume that sexual assaults have to involve
attempted or completed sexual acts. Some of these attacks,
however, may be those that involve power dynamics and
control over women more than overtly physical acts (Geeson,
2018). These behaviors, although widespread and damaging,
have not been the subject of extensive research. For example,
there are fewer studies of the stalking of college women. The
rare ones that do exist provide a dramatic range of preva-
lence rates (from 13% to 30%) and different samples and
variations in operationalizing stalking make it difficult to
make useful comparisons (Nobles & Fox, 2017). learning, it is now possible to stalk a person through newer
technology. This could include an unwanted presence on
social media, the use of now easily available spycraft tech-
niques such as hidden microphones or cameras, or hidden
GPS devices to facilitate learning a person’s location. Similarly sparse are studies of the dissemination of
unwanted sexual messages and images among college stu-
dents (Crisafi, Mullins, & Jasinski, 2016; Navarro, 2016;
Woodlock, 2017). However, it is estimated that there are
more than 3,000 sites devoted just to the narrower topic of
“revenge pornography” or sexual images posted by men
without the permission of the women portrayed (Lamphere
& Pikciunas, 2016). The best study, one of the general popu-
lation rather than college students, is Powell and Henry’s
(2016) national Australian survey. Nearly 3,000 people aged 1West Virginia University, Morgantown, USA
2The George Washington University, Washington, DC, USA
3Queensland University of Technology, Brisbane, Australia
4University of New England, Armidale, New South Wales, Australia
5Fairmont State University, WV, USA Corresponding Author:
Walter S. Authors
Authors 8231 SGOXXX10.1177/2158244019828231SAGE OpenDeKeseredy et al. 231 SGOXXX10.1177/2158244019828231SAGE OpenDeKeseredy et al. Article Article https://doi.org/10.1177/215824401982823
SAGE Open
January-March 2019: 1–12
© The Author(s) 2019
DOI: 10.1177/2158244019828231
journals.sagepub.com/home/sgo Technology-Facilitated Stalking and
Unwanted Sexual Messages/Images in a
College Campus Community: The Role of
Negative Peer Support Walter S. DeKeseredy1, Martin D. Schwartz2, Bridget Harris3,
Delanie Woodlock4, James Nolan1, and Amanda Hall-Sanchez5 Walter S. DeKeseredy1, Martin D. Schwartz2, Bridget Harris3,
Delanie Woodlock4, James Nolan1, and Amanda Hall-Sanchez5 Creative Commons CC BY: This article is distributed under the terms of the Creative Commons Attribution 4.0 License
(http://www.creativecommons.org/licenses/by/4.0/) which permits any use, reproduction and distribution of
the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages
(https://us.sagepub.com/en-us/nam/open-access-at-sage). Abstract Researchers have accumulated much social scientific knowledge about the scope, distribution, causes, and outcomes of
the physical and sexual abuse of female students in North American institutions of higher learning. However, surveys of
technology-facilitated stalking and the dissemination of unwanted sexual messages/images in college campus communities
are in short supply. The few that have been conducted do not identify key sociological risk factors associated with these
two electronic forms of victimization. This paper, then, has two objectives: (1) to examine the influence of two types of
negative peer support and (2) to determine if being the target of technology-facilitated stalking and receiving unwanted sexual
messages/images are associated with female students’ intimate partner violence and sexual assault experiences. The results
confirm that the two variants of negative peer support examined in this study are significant predictors of digital victimization
and that such abuse is strongly associated with intimate partner violence and sexual assault. 1West Virginia University, Morgantown, USA
2The George Washington University, Washington, DC, USA
3Queensland University of Technology, Brisbane, Australia
4University of New England, Armidale, New South Wales, Australia
5Fairmont State University, WV, USA Understanding Technology-Facilitated
Stalking and Unwanted Sexual
Messages/Images: The Relevance of
Negative Peer Support Researchers seeking to explain (face-to-face, offline) physi-
cal and sexual assaults on female college students have sug-
gested many causes or facilitating factors. One frequently
identified in survey research is negative peer support
(DeKeseredy, Hall-Sanchez, & Nolan, 2018; Hart, 2009). These are messages that mainly come from patriarchal male
friends but also occasionally from female friends. There are
various definitions of this concept, but here we offer a modi-
fied version of DeKeseredy’s (1988a) definition of male peer
support: attachments to peers and the resources they provide
that encourage and justify violence against college women. The theory based on this definition suggests that men often
seek advice from their peers about their problems relating to
women, whether they are in a relationship or possibly would
like to be in one, or just are dealing with friends. These peers
may offer positive and useful advice, but unfortunately some
men are instead given advice that encourages them to engage
in sexual, verbal, or physical abuse. Schwartz and
DeKeseredy (1997) assert that abusive patriarchal men situ-
ated in a patriarchal rape-supportive culture develop and
maintain friendships with friends who hold similar beliefs
and values. These attachments then help these men to develop
and then reinforce beliefs and values that promote the abuse
of women and in particular those women who represent a
threat to male patriarchal authority. These attachments are
particularly important to the reinforcement of values that
promote and reward abusive behavior toward women. There is an empirical literature exploring technology-
facilitated violence, but to date it has mostly focused on
“electronic dating violence” among high school and college
groups in the Global North (Borrajo, Gámez-Guadix, Pereda,
& Calvete, 2015; Dick et al., 2014; Hinduja & Patchin, 2011;
Lucero, Weisz, Smith-Darden, & Lucero, 2014; Reed,
Tolman, & Ward, 2016; Reed, Tolman, Ward, & Safyer,
2016; Temple et al., 2016; Watkins, Maldonado, & DiLillo,
2018; Wolford-Clevenger et al., 2016; Zweig, Dank, Yahner,
& Lachman, 2013). Another body of research centers on
“social media surveillance” (J. Fox & Warber, 2013; Lyndon,
Bonds-Raacke, & Cratty, 2011; Muise, Christofides, &
Desmarais, 2014; Utz & Beukeboom, 2011). These studies
mainly consist of lab-based psychological experiments with
college students that largely rely on decontextualized ques-
tion items that do not investigate the context, meaning, or
motives of behavior. Keywords In this vein, Powell and Henry
(2016) report that although women and men report experi-
encing similar overall prevalence of technology-facilitated
sexual violence victimization, the nature and impact of those
experiences differ in particular gendered ways that reflect
broader patterns in both gender relations and “offline” sexual
harassment. Women are more likely to have stronger emo-
tional reactions to this form of victimization, and more likely
to suffer negative consequences, especially from those on-
line sites that include the woman’s telephone number and
email address (DeKeseredy, Dragiewicz, & Schwartz, 2017). Keywords DeKeseredy, Anna Dean Carlson Endowed Chair of Social
Sciences, Director of the Research Center on Violence, and Professor of
Sociology, West Virginia University, Morgantown, WV 26506, USA. Email: Walter.dekeseredy@mail.wvu.edu Studies of technology-facilitated stalking on college cam-
puses are certainly not plentiful. This is problematic because
while nonelectronic forms of stalking (e.g., following a per-
son) still exist and are extensive at institutions of higher 2 SAGE Open SAGE Open 18 to 54 were surveyed and one in 10 respondents reported
that someone had posted online or sent to others nude or
seminude pictures of them without their permission and
9.6% reported that someone threatened to post such images
or to send them to others. Note, too, that in a review of the
extant empirical literature on the subject, Henry and Powell
(2018) found very little qualitative or quantitative informa-
tion about adults, such as college students, although there is
a growing body of research about children and younger ado-
lescents. What is more, they found almost no reliable data on
the nature, scope or impact of technology-facilitated sexual
violence. A significant void in this arena is quantitative
research on what associations might exist between victimiza-
tion by electronic means of college women and their accounts
of offline sexual assault and intimate physical violence. Special attention is devoted to exploring the relationship
between two types of negative peer support (attachments to
abusive peers and pro-abuse informational support) and the
above two forms of digital abuse. Since this article only
focuses on female survivors of digital and offline abuse, it is
not to be expected that the level of pro-abuse informational
support would be comparable to the kinds of messages that
male peers give to males (DeKeseredy & Schwartz, 2013),
but this does not mean that women do not receive these mes-
sages, or that the messages are not associated with their vic-
timization. This, of course, is an empirical question that we
will investigate. The same applies to the other component of
negative peer support: the survivor’s association with abu-
sive peers. Overall, the limited work that has been done on these
harms overlooks contextual elements, including the gen-
dered nature of these problems and relationships between
survivors and offenders. Understanding Technology-Facilitated
Stalking and Unwanted Sexual
Messages/Images: The Relevance of
Negative Peer Support Moreover, although we know that many
such victimizations involve past or present relationships
between the victim and the perpetrator, most studies do not
draw links to intimate partner violence. There are also practical aspects to these friends, in that
they also provide resources that involve specific verbal and
emotional support. This includes a vocabulary of motive that
defines some women as legitimate objects of abuse and sex-
ual assault. DeKeseredy and Schwartz (2013) outline a vari-
ety of contexts where male peer support allows some men to
feel normal and justified when committing violence against
current and former intimate partners. Using data gathered by a large-scale survey conducted at
a doctoral institution located in a South Atlantic part of the
United States, the main purpose of this article is to help fill
some of these research gaps. An important goal is to inves-
tigate the extent to which technology-facilitated stalking
and unwanted sexual messages/images are associated with
the face-to-face physical and sexual victimization of women. DeKeseredy et al. 3 These friends particularly suggest the legitimacy of such
abuse as a solution to the “problem” of women who deny
male supremacy through such actions as “talking back” or
failing to provide sex on demand. In addition to encourage-
ment, these men are sometimes offered advice on specific
techniques to handle these women through abuse. DeKeseredy and Schwartz (2013, 2015) have documented in
a wide range of studies conducted over 30 years that male
peer support is a powerful predictor of male physical and
sexual victimization of college women. Unfortunately,
almost all of the empirical research on this to date has been
heteronormative (covering male violence against females)
and only looks at male attachments to male peers, rather than
any support from female members of peer groups that are
either meant or taken to legitimate male violent acts. The support for expanding our study to mixed-sex college
peer groups, or more specifically including women as potential
sources of negative peer support, is a very small body of survey
work. There is certainly extensive anecdotal evidence and a
growing recognition in the literature that some women can be
hostilely sexist toward other women (Glick & Fiske, 1996;
Sibley, Overall, & Duckitt, 2007). Understanding Technology-Facilitated
Stalking and Unwanted Sexual
Messages/Images: The Relevance of
Negative Peer Support In more recent study, we
found that mixed-sex negative peer support was related to
offline stalking and sexual assault victimization within the les-
bian, gay, bisexual, transgender, and queer/questioning
(LGBTQ) community on a college campus (DeKeseredy et al.,
2017), while DeKeseredy et al. (2018) found evidence that
mixed-sex negative peer support contributed to female college
students’ offline sexual victimization. Gwartney-Gibbs and
Stockard (1989) found that “the sexual aggression of males
within a mixed-sex peer group appears to be an important
determinant of the probability that females within the group
will be sexually victimized” (p. 198). Similarly, Schwartz and
Pitts (1995) found that college women who are sexually
assaulted are more likely to have male friends who get women
drunk or high to have sex with them. Another arena where male peer support is associated with
violence against women is separation and divorce
(DeKeseredy, Dragiewicz, & Schwartz, 2017). For example,
many members of patriarchal peer groups view the victimiza-
tion of women, such as through beatings or sexual assault, as
a legitimate and effective means of responding to “damaged”
patriarchal masculinity and reaffirming a man’s right to con-
trol his female partner (Messerschmidt, 1993; Ray, 2011). Not only do these men privately and publicly state that these
forms of abuse are legitimate means of patriarchal authority
and domination, they also serve as role models because many
of them physically, sexually, and psychologically harm their
own intimate partners (DeKeseredy & Schwartz, 2013). A major goal, then, of this study is to determine whether
the mixed-sex negative peer support discovered by earlier
studies exists and influences online victimization in a recent
sample of college women. The small number of studies in
this field that attempt to test these theoretical speculations
was an important reason for this research. Another reason for focusing on negative peer support is
that theory testing in the field of technology-facilitated abuse
is limited to evaluating the utility of gender-blind criminologi-
cal perspectives, such as routine activity theory (Cohen &
Felson, 1979) and the general theory of crime (Gottfredson &
Hirschi, 1990). The powerful association between gender and
women’s risk of being harmed by cyberstalking and revenge
pornography cannot be satisfactorily accounted for via the use
of these and other “male stream” theories. Such criminological
perspectives were not specifically designed to address the gen-
dered nature of these crimes. Understanding Technology-Facilitated
Stalking and Unwanted Sexual
Messages/Images: The Relevance of
Negative Peer Support Thus, we would argue, of the
very limited theoretical work done so far, negative peer sup-
port theory seems the most promising. Since DeKeseredy and
Schwartz’s (2016) theory is shaped by feminist ways of know-
ing and masculinities studies, measures informed by these dis-
courses were utilized in this study. Moreover, as DeKeseredy
and Schwartz (2016) and Dragiewicz (2011) remind us, if pro-
abuse peer support has been found to be associated with vari-
ous types of face-to-face male-to-female victimization, there
is ample reason to investigate and propose that it is similarly
related to technology-facilitated types of woman abuse. Peer support motivates men to “lash out against the
women . . . they no longer can control” (Bourgois, 1995, p. 214). This can be especially clear when looking at lesbian
coming out experiences, which often include violence com-
mitted by ex-boyfriends or husbands. Bisexual women, the
Centers for Disease Control and Prevention (CDC) found,
are more likely to experience rape, physical violence, and
stalking from an intimate partner compared with heterosex-
ual women or lesbians, with males as almost always the
offenders (Messinger, 2017; Walters, Chan, & Breiding,
2013; Walters & Lippy, 2016). Stalking is another area where negative peer support may
be important. There is some support for the claim that non-
electronic types of stalking among college students is learned
or reinforced through social interactions with peers
(DeKeseredy, Hall-Sanchez, Nolan, & Schwartz, 2017; K. Fox, Nobles, & Akers, 2011). However, more evidence is
needed to determine whether negative peer support is as
influential online as it is offline. Still, while recognizing that most negative peer support
comes from men, the concept was expanded here to include
such problematic support from any source. Specifically, it is
important to include in any explanation of college life a
broader view of patriarchal practices and discourses. Indeed,
women in some friendship networks may contribute, legiti-
mate, and support their male friends’ belief that their hurtful
behaviors and sexist attitudes are regularized parts of campus
life (Gwartney-Gibbs & Stockard, 1989). Sample and Data Collection For the most part, as
described in Table 1, the sample is representative of the
entire student campus community. More women than men,
however, participated (n = 3,269). Since women are among
the highest risk of groups to experience many of the harms
addressed in the CQLS, especially sexual assault, it is to be
expected that the CQLS elicited a higher percentage of
females than that of the school’s general population, as well
as a lower percentage of men than that of the wider male
student community. Note that for the purpose of this article,
the results reported below are limited to women’s responses. Regardless of whether they elected to continue, all par-
ticipants were provided with information on free profes-
sional support from counseling services. Every page of the
survey that contained sensitive questions had a link to on-
campus resources, including one at the end of the instru-
ment. Located below the list of resources at the end of the
survey was the option for students to enter their email
addresses in a draw for a $50.00 VISA gift card. To further
preserve students’ confidentiality, spreadsheets containing
participants’ responses are securely stored by Qualtrics and
are only accessed by the research team. Recruiting participants involved a campus-wide effort
and entailed using multiple methods, including posters, fly-
ers, direct email communication, and in-class announce-
ments. Incentives were also used to recruit respondents. All
types of publicity informed students of the prospect of being
randomly chosen to get one of 20 $50.00 VISA gift cards
(also made explicit in the instrument). Lotteries are widely
used in Web surveys and are repeatedly found to be more
effective than other enticements (Couper & Bosnjak, 2010;
Pedersen & Nielsen, 2016). Following the first email invitation, three reminders were
sent out (1 a week) for a total of 4 weeks of data collection. Couper and Bosnjak (2010) contend that “much of the non-
response occurs at the early stages before we have a chance
to convince them of the importance of the study” (p. 539). This was not the case with the CQLS. Actually, nearly 2,500
students completed the survey within 5 days of the first email
invitation. Email invitations to participate in the CQLS were sent to
30,470 students, with the first of 4 weekly requests issued on
March 28, 2016. Sample and Data Collection Our data are derived from the Campus Quality of Life Survey
(CQLS), which was administered online in Spring 2016 to
30,470 students enrolled at the previously mentioned U.S. SAGE Open SAGE Open 4 Table 1. Demographic Characteristics of the Main Campus Population and the CQLS Sample. Status
Population (N = 28,488)
Sample (n = 5,718)
Female sample (n = 3,269)
Undergraduate
77.3
78.9
78.5
Professional
4.6
5.1
5.1
Graduate
18.2
15.9
16.5
Sex
Female
48.6
57.2
100
Male
51.4
37.1
n/a
Other
Not recorded
1.1
n/a
Race/ethnicity
Black/African American
5.1
4.4
4.0
White
88.4
83.8
85.8
Asian
2.0
3.3
2.8
Hawaiian/Pacific Islander
0.1
0.2
0.1
Native American
0.2
0.4
0.3
Hispanica
3.8
3.1
2.9
Other (including multi race)
4.2
4.7
4.1
Age
Average age
23.3
22.1
21.9
Note. Survey respondents from South Asia and Middle East (2.7%) are listed as “Other” along with multi race. CQLS = Campus Quality of Life Survey. aThe ethnic category “Hispanic” was considered separate from race in the population column and so the total exceeds 100%. Institutional race data are
limited to U.S. citizens which account for 92.1% of students. Data retrieved at https://institutionalresearch.wvu.edu/files/d/a2c20b50-78d9-4,603-8,653-
76d93b378d08/wvu_enrollment_trends_fall-2,017.pdf Table 1. Demographic Characteristics of the Main Campus Population and the CQLS Sample. Note. Survey respondents from South Asia and Middle East (2.7%) are listed as “Other” along with multi race. CQLS = Campus Quality of Life Survey. aThe ethnic category “Hispanic” was considered separate from race in the population column and so the total exceeds 100%. Institutional race data are
limited to U.S. citizens which account for 92.1% of students. Data retrieved at https://institutionalresearch.wvu.edu/files/d/a2c20b50-78d9-4,603-8,653-
76d93b378d08/wvu_enrollment_trends_fall-2,017.pdf Participants were asked to confirm that they were at least
18 years old and a current student. They were additionally
informed that any information they provided would be ano-
nymized. As well, it was made explicit that student confiden-
tiality is a priority and that any information they shared
would not be identified. Moreover, they were informed that
the research team cannot access their IP addresses or link the
survey to their names, student IDs, or email addresses. Furthermore, in line with research protocol, students were
told that participation in this study is strictly voluntary, ques-
tions can be skipped, and that they could stop at any time. college. Nearly 20% of the total student population (n =
5,718) completed the questionnaire. Measures Experiences Survey (Cronbach’s α = .80). The following
introduction was used and the response categories are “yes”
and “no”: Technology-facilitated stalking. Often referred to as cyberstalk-
ing, technology-facilitated stalking is defined in this study as
“the utilization of information and communication technolo-
gies to harass and/or stalk another person” (Navarro, 2016, p. 135). It was operationalized using two items included in a
broader measure of stalking included in the Centers for Dis-
ease Control and Prevention’s National Intimate Partner and
Sexual Violence Survey (NISVS; Black et al., 2011). Listed
below, they were introduced with a preamble included in the
Administrator-Researcher Campus Climate Collaborative
(ARC3; 2015) Survey’s introduction to stalking victimiza-
tion items. Participants were asked to report how many times
they experienced these behaviors and the response categories
(also derived from the ARC3 Survey) are “None,” “1-2,”
“3-5,” “6-8,” and “More than 8”: The following questions concern unwanted sexual experiences
that you may have had since you enrolled at XXX. We know that
these are personal questions, so we don’t want your name or
other identifying information. Your answers are completely
confidential. We hope that this helps you feel comfortable
answering each question honestly. Intimate physical violence. The eight items in Table 3 are
derived from the C.A.T.S. Survey and the revised Conflict
Tactics Scale (Straus, Hamby, Boney-McCoy, & Sugarman,
1996) (Cronbach’s α = .83). These were introduced with the
preamble below and the response categories are “Never (0
times,” “Once (1 time),” “Sometimes (2-5 times),” “Often
(6+ times),” and “Choose not answer”: ••
Watched or followed you from a distance, or spied on
you with a listening device, camera, or GPS [global
positioning system]? We are particularly interested in learning about your intimate or
romantic relationships. Since you started at XXX, how many
times has someone you were dating or a spouse/partner done the
following physical thing to you that were NOT done in a
joking or playful manner? ••
Sent you unwanted electronic messages such as texts,
voice messages, emails, or through social media apps? Unwanted sexual messages/images. One item included in the
University of Kentucky’s Research Center on Violence’s
(Center for Research on Violence Against Women, 2014)
Campus Attitudes Toward Safety (C.A.T.S.) Survey was
used to operationalize this variable. Sample and Data Collection In each one was a link to the questionnaire
that was administered using Qualtrics software. After clicking
the link to the survey in the email invitation and then clicking
a button to participate, participants were taken to a screen
including a consent form. Students who stated that they did not
want to participate were deleted from the email reminder list. DeKeseredy et al. 5 Table 2. Female Sexual Assault Victimization. Type of sexual assault
% yes
n
% no
n
Someone fondled, kissed, or rubbed up against the private areas of my
body (lips, breast/chest, crotch or butt) or removed some of my clothes
without my consent (but did not attempt sexual penetration)
29
860
71
2,009
Someone had oral sex with me or made me have oral sex with them
without my consent
7
193
93
2,765
Someone put their penis, fingers, or other objects into my vagina without
my consent
10
301
90
2,652
Someone put their penis, fingers, or other objects into my butt without
my consent
4
121
96
2,831
Even though it didn’t happen, someone TRIED to have oral, anal, or
vaginal sex with me without my consent
16
465
84
2,489 Experiences Survey (Cronbach’s α = .80). The following
introduction was used and the response categories are “yes”
and “no”: Type of informational support study uncovered evidence of mixed-sex negative peer sup-
port that predicted female college students’ sexual victimiza-
tion. Patriarchal practices and discourses occur in mixed-sex
college peer groups and women in some such cohorts may
influence their male friends to believe that their abusive
behaviors and chauvinist attitudes are standardized features
of campus life (Gwartney-Gibbs & Stockard, 1989). As well,
women can be hostilely sexist toward other women (Glick &
Fiske, 1996; Sibley et al., 2007). What is more, Schwartz and
Pitts (1995) found that undergraduate survivors of sexual
assault are more likely to have male peers who get women
intoxicated or high to have sex with them. Another key
objective, then, of this study is to establish whether the likeli-
hood found by earlier studies of mixed-sex pro-abuse peer
support exists within the area of online victimization. date is used, please think of anyone with whom you have or have
had a romantic or sexual relationship—short or long term. Please click the bubble which best represents your answer. To the best of your knowledge, did any of your friends tell you
that . . . Attachments to abusive peers. A slightly revised rendition of
one of DeKeseredy and Schwartz’s (1998) indices was used
to operationalize this form of negative peer support. The
response categories were none, 1 or 2, 3 to 5, 6 to 10, more
than 10, and don’t know (Cronbach’s α = .81). To the best of your knowledge, how many of your friends ••
have ever made physically forceful attempts at sexual
activity with dates which were disagreeable and offen-
sive enough that the dates responded in an offended
manner such as crying, fighting, screaming or
pleading? Table 4. Pro-Abuse Informational Support. Table 4. Pro-Abuse Informational Support. Table 4. Pro-Abuse Informational Support. Type of informational support
% yes
n
% no
n
You should respond to your dates’ challenges to your authority by using physical force,
such as hitting or slapping? 3
85
97
2,938
It is alright for someone to hit a date in certain situations
7
208
93
2,813
Your dates should have sex with you whenever you want
4
117
96
2,906
When you spend money on a date, the person should have sex with you in return
5
157
95
2,866
You should respond to your dates’ challenges to your authority by insulting them or
putting them down
3
92
97
2,927
You should respond to your dates’ sexual rejections by using physical force to have sex
1
26
99
2,991
It is alright to physically force a person to have sex under certain conditions
2
44
98
2,976 Measures It is part of a five-item
sexual harassment measure and was introduced as follows:
“Since you started at XXX, how often has someone (Not
someone you are dating or a spouse/partner) done any of
the following to you?” The item we used is “Sent sexual
messages or pictures that you did not want (including porn).”
The response categories are “Never (0 times),” “Once (1
time),” “Sometimes (2-5 times),” “Often (6+ times),” and
“Choose not to answer.” Peers’ pro-abuse informational support. This type of negative
peer support refers to guidance and advice that influences
men to sexually, physically, and psychologically abuse their
dating partners (DeKeseredy & Schwartz, 1998). To measure
this concept, an index was created by adding male and female
respondents’ scores on seven slightly modified items devel-
oped by DeKeseredy (1988b) and presented in Table 4
(Cronbach’s α = .80). The preamble below includes a state-
ment found in the ARC3 (2015) Survey’s introduction to
peer norms measures, and respondents were asked to answer
either “yes” or “no”: The next questions are about the information your current
friends may have given you concerning how to deal with
problems in intimate or romantic relationships. When the word Sexual assault. The five items in Table 2 are adapted from
some of those in Koss et al.’s (2007) Revised Sexual SAGE Open SAGE Open 6 Table 3. Intimate Physical Violence. Type of violence
Number one or more times
%
Shoved, shook, pinched or scratched you, or pulled your hair? 396
13.0
Slapped you. 186
6.1
Threw something at you that could hurt you. 234
7.7
Bent your fingers or twisted your arm. 187
6.1
Hit, punched, kicked, or bit you. 178
5.8
Dragged you by your hair, threw you down stairs or out of a car, or threw you
around. 75
2.5
Burned you, choked you, or tried to strangle or suffocate you. 96
3.1
Used, or threatened to use, a weapon against you
83
2.7 Table 5. Negative Peer Support and Technology-Facilitated Stalking. Unwanted sexual
messages/images
Technology-
facilitated stalking
Attachment to
abusive peers
Peers’ pro-abuse
informational support
Sexual
assault
Intimate physical
violence
Unwanted sexual messages/
images
1
.368**
.187**
.179**
.343**
.249**
Technology-facilitated stalking
.368**
1
.249**
.174**
.316**
.268**
Attachment to abusive peers
.187**
.249**
1
.217**
.261**
.234**
Peers’ pro-abuse informational
support
.179**
.174**
.217**
1
.187**
.157**
Sexual assault
.343**
.316**
.261**
.187**
1
.235**
Intimate physical violence
.249**
.268**
.234**
.157**
.235**
1
**P = <.01. Table 6. Negative Peer Support and Unwanted Sexual Messages/Images. Table 7. Bivariate Correlations. Unwanted sexual
messages/images
Technology-
facilitated stalking
Attachment to
abusive peers
Peers’ pro-abuse
informational support
Sexual
assault
Intimate physical
violence
Unwanted sexual messages/
images
1
.368**
.187**
.179**
.343**
.249**
Technology-facilitated stalking
.368**
1
.249**
.174**
.316**
.268**
Attachment to abusive peers
.187**
.249**
1
.217**
.261**
.234**
Peers’ pro-abuse informational
support
.179**
.174**
.217**
1
.187**
.157**
Sexual assault
.343**
.316**
.261**
.187**
1
.235**
Intimate physical violence
.249**
.268**
.234**
.157**
.235**
1
**P = <.01. 2002; Fremouw, Westrup, & Pennypacker, 1997; Nobles &
Fox, 2017). Fourteen percent (n = 426) of our sample were
watched or followed from a distance, or spied on with listen-
ing device, camera, or GPS. Twenty-eight percent (n = 855)
were sent unwanted messages such as texts, voice messages,
emails, or through social media apps. 2002; Fremouw, Westrup, & Pennypacker, 1997; Nobles &
Fox, 2017). Fourteen percent (n = 426) of our sample were
watched or followed from a distance, or spied on with listen-
ing device, camera, or GPS. Twenty-eight percent (n = 855)
were sent unwanted messages such as texts, voice messages,
emails, or through social media apps. support being more than twice as likely to report being vic-
timized than those who did not report negative peer associa-
tions. Thus, as is the case with face-to-face types of female
college student victimization, negative peer support is a
influential determinant of online victimization, which pro-
vides some substantiation of DeKeseredy and Schwartz’s
(2016) theory. Twenty-one percent (n = 633) received unwanted sexual
messages/images, including pornography. Again, comparing
this figure with those uncovered by other studies is difficult
because of methodological differences, but it is consistent
with those found by studies of similar harms using female
samples in the same age group (Powell & Henry, 2017). Table 5. Negative Peer Support and Technology-Facilitated Stalking. Table 5. Negative Peer Support and Technology-Facilitated Stalking. B
SE
Wald
df
Significance
Exp (B)
95% CI
LL
UL
Pro-abuse informational support
0.736
.116
40.141
1
.000
2.087
1.662
2.620
Abusive peers
1.018
.087
136.643
1
.000
2.768
2.332
3.286
Constant
–1.363
.069
389.554
1
.000
0.256
Note. CI = confidence interval; LL = lower limit; UL = upper limit. Table 6. Negative Peer Support and Unwanted Sexual Messages/Images. B
SE
Wald
df
Significance
Exp (B)
95% CI
LL
UL
Pro-abuse informational support
0.865
.122
50.548
1
.000
2.374
1.871
3.013
Abusive peers
0.845
.105
65.023
1
.000
2.328
1.896
2.859
Constant
–1.993
.085
544.869
1
.000
0.136
Note. CI = confidence interval; LL = lower limit; UL = upper limit. Table 7. Bivariate Correlations. Unwanted sexual
messages/images
Technology-
facilitated stalking
Attachment to
abusive peers
Peers’ pro-abuse
informational support
Sexual
assault
Intimate physical
violence
Unwanted sexual messages/
images
1
.368**
.187**
.179**
.343**
.249**
Technology-facilitated stalking
.368**
1
.249**
.174**
.316**
.268**
Attachment to abusive peers
.187**
.249**
1
.217**
.261**
.234**
Peers’ pro-abuse informational
support
.179**
.174**
.217**
1
.187**
.157**
Sexual assault
.343**
.316**
.261**
.187**
1
.235**
Intimate physical violence
.249**
.268**
.234**
.157**
.235**
1
**P = <.01. B
SE
Wald
df
Significance
Exp (B)
95% CI
LL
UL
Pro-abuse informational support
0.736
.116
40.141
1
.000
2.087
1.662
2.620
Abusive peers
1.018
.087
136.643
1
.000
2.768
2.332
3.286
Constant
–1.363
.069
389.554
1
.000
0.256
Note. CI = confidence interval; LL = lower limit; UL = upper limit. Note. CI = confidence interval; LL = lower limit; UL = upper limit. Table 6. Negative Peer Support and Unwanted Sexual Messages/Images. B
SE
Wald
df
Significance
Exp (B)
95% CI
LL
UL
Pro-abuse informational support
0.865
.122
50.548
1
.000
2.374
1.871
3.013
Abusive peers
0.845
.105
65.023
1
.000
2.328
1.896
2.859
Constant
–1.993
.085
544.869
1
.000
0.136
Note. CI = confidence interval; LL = lower limit; UL = upper limit. Table 7. Bivariate Correlations. Results In total, 34% (n = 1,041) of the female respondents reported
being targets of technology-facilitated stalking. Several other
studies of U.S. college students uncovered cyberstalking
prevalence rates ranging from 1% to 30%, but sound com-
parisons cannot be made because of different means of oper-
ationalizing technology-facilitated stalking and different
samples (Navarro, 2016; Nobles & Fox, 2017). Nevertheless,
our rate is higher than most estimates of female college stu-
dent stalking victimization of any sort, which range from
13% to 30% (Cantor et al., 2015; Fisher, Cullen, & Turner, ••
have ever used physical force, such as hitting or beat-
ing, to resolve conflicts with their dates? ••
insult their dates, swear at them, and/or withhold
affection? It is unclear how many women in the study received nega-
tive peer support from only men, only women, or a combina-
tion of both. Yet, DeKeseredy et al.’s (2018) analysis of
CQLS data and Gwartney-Gibbs and Stockard’s (1989) 7 DeKeseredy et al. Table 5. Negative Peer Support and Technology-Facilitated Stalking. Table 5. Negative Peer Support and Technology-Facilitated Stalking. Table 5. Negative Peer Support and Technology-Facilitated Stalking. Table 5. Negative Peer Support and Technology-Facilitated Stalking. B
SE
Wald
df
Significance
Exp (B)
95% CI
LL
UL
Pro-abuse informational support
0.376
.136
7.607
1
.006
1.456
1.115
1.902
Abusive peers
0.985
.125
62.361
1
.000
2.678
2.097
3.419
Technology-facilitated stalking
0.944
.114
68.842
1
.000
2.570
2.056
3.212
Unwanted sexual messages/images
0.779
.114
42.950
1
.000
2.180
1.727
2.752
On or off campus
1= on campus,
0 = off campus
−0.214
.130
2.710
1
.100
0.808
0.626
1.042
Age
−0.014
.015
0.838
1
.360
0.986
0.958
1.016
Race (1 = White, 0 = non-White)
−0.028
.163
0.030
1
.862
0.972
0.707
1.337
Constant
−2.458
.393
39.105
1
.000
0.086
Note. CI = confidence interval; LL = lower limit; UL = upper limit. Table 9. Informational Support, Abusive Peers, Stalking, Unwanted Sexual Messages/Images, and Intimate Physical Violence. 95% CI Table 9. Informational Support, Abusive Peers, Stalking, Unwanted Sexual Messages/Images, and Intimate Physical Violence. Table 9. Informational Support, Abusive Peers, Stalking, Unwanted Sexual Messages/Images, and Intimate Ph the conceptualization of technology-mediated harm against
women remains significantly underdeveloped” (p. 7). Guided
by male peer support theory, the study presented here addresses
this concern. Likewise, it is the first survey to examine whether
negative peer group dynamics are connected to cybercrimes
against women. Certainly, a contribution of this work is pro-
viding statistical evidence to support the claim that “peer sup-
port for sexual violence against women emerges as a
particularly challenging and troubling feature of sexual vio-
lence in the digital age” (Powell & Henry, 2017, p. 5). Also,
this project provides more empirical support for previous stud-
ies, such as Woodlock’s (2017), that found technology-
facilitated stalking and abuse in the contexts of face-to-face
sexual violence and intimate physical violence. Table 9 also includes a multivariate logistic regression
model. It shows that respondents who reported receiving pro-
abuse informational support were 50% more likely to state
having been targeted by intimate physical violence. Similarly,
participants who reported attachments to abusive peers were
2.7 times more likely to report such violence. Furthermore,
respondents who reported being the target of technology-facil-
itated stalking were 2.6 times more likely to report intimate
physical violence than women who did not report stalking. Finally, Table 9 shows women who reported unwanted sexual
messages/images were more than 2 times more likely to report
inmate physical violence than respondents who were not sent
these things. Table 5. Negative Peer Support and Technology-Facilitated Stalking. Moreover, Table 5 shows that women who reported receiv-
ing pro-abuse informational support were two times more
likely to report technology-facilitated stalking than female
respondents who did not receive such support. Respondents
with attachments to abusive peers were nearly 3 times as
likely to report this form of stalking than those who did not
have these associations. Table 6 shows that negative peer
support, too, is a determinant of receiving unwanted sexual
messages/images, with women reporting both types of this Slightly more than 18% (18.2%, n = 551) reported
experiencing one or more of the forms of intimate partner
violence itemized in Table 3, and 34% of the women
reported being harmed by at least one type of sexual assault
presented in Table 2. Not only, as described in Table 7, is
negative peer support a key predictor of sexual assault (as
well as intimate physical violence), but, as Table 8 shows,
respondents who report being the victim of technology-
facilitated stalking were 2.3 times more likely to report a
sexual assault than women who were not victimized. As
well, Table 8 shows that participants who reported receiv-
ing unwanted sexual messages/images were 3.4 times
more likely to state being sexually assaulted than women
who did not receive them. SAGE Open SAGE Open 8 Table 8. Informational Support, Abusive Peers, Stalking, Unwanted Sexual Messages/Images, and Sexual Assault. B
SE
Wald
df
Significance
Exp (B)
95% CI
LL
UL
Pro-abuse informational support
0.448
.129
12.019
1
.001
1.566
1.215
2.018
Abusive peers
0.797
.097
67.756
1
.000
2.218
1.835
2.682
Technology-facilitated stalking
0.871
.097
80.520
1
.000
2.390
1.976
2.892
Unwanted sexual messages/images
1.245
.111
126.912
1
.000
3.475
2.798
4.315
On or off campus
1 = on campus,
0 = off campus
0.139
.108
1.659
1
.198
1.149
0.930
1.419
Age
−0.048
.014
11.706
1
.001
0.953
0.928
0.980
Race (1 = White, 0 = non-White)
−0.120
.137
0.765
1
.382
0.887
0.678
1.161
Constant
−0.739
.353
4.393
1
.036
0.478
Note. CI = confidence interval; LL = lower limit; UL = upper limit. nal Support, Abusive Peers, Stalking, Unwanted Sexual Messages/Images, and Intimate Physical Violence. Table 9. Informational Support, Abusive Peers, Stalking, Unwanted Sexual Messages/Images, and Intimate Physical Violence. B
SE
Wald
df
Significance
Exp (B)
95% CI
LL
UL Table 9. Informational Support, Abusive Peers, Stalking, Unwanted Sexual Messages/Images, and Intimate Physical Violence. Table 5. Negative Peer Support and Technology-Facilitated Stalking. Several potentially confounding variables were
added to Table 9, including race, age, and whether the student
lived on campus or in off-campus housing, but none had a sig-
nificant effect on the table statistics. Several limitations need to be addressed in future research
in the fields covered in our research. First, the technology-
facilitated stalking and unwanted sexual messages/images
measures are rather limited in scope. For example, the mes-
sages/images item does not tell us how many women only
received pictures, only received messages, or how many
received a combination of both. This distinction does not Discussion As Henry and Powell (2015) remind us, “as a result of the
gender-blindness within studies of virtual or cyber criminality, DeKeseredy et al. 9 Nonetheless, preliminary evidence provided by the CQLS
strongly suggests that this relationship is an emerging prob-
lem, one that could possibly get worse in the future. appear because the CQLS was not specially designed to
solely examine electronic means of woman abuse and associ-
ated risk factors. Indeed, this topic has only recently received
social scientific attention, as the uptake of technology and
perpetration emerges and escalates. What we do know for sure from the data uncovered by
this study and similar ones (e.g., Woodlock, 2017) is that
“technology is altering, intensifying and facilitating gen-
dered violence” (Vitis & Segrave, 2017, p. 5). Thus, in addi-
tion to doing more empirical and theoretical work on
technology-facilitated forms of abuse, it is necessary to
develop new prevention and intervention strategies. The
growing number of experts in the field are collectively
emphasizing the importance of avoiding simplistic solutions
and the value of a multipronged approach involving legal
reforms, education and awareness programs, survivor sup-
port services, perpetrator reeducation, and corporate efforts
to combat digitized means of gender violence (Clevenger,
2016; DeKeseredy, Dragiewicz, & Schwartz, 2017; Hall &
Hearn, 2018; Powell & Henry, 2017). There are, for sure,
other initiatives that could be discussed here. Yet, regardless
of what solutions are proposed in the future, it is always nec-
essary to keep in mind this point raised by Klein (2012):
“Ending abuse is not only about specialized services deliv-
ered by trained professionals. It is perhaps more importantly
about ‘humdrum’ cultural change in which everyone does
things a little differently every day” (p. 127). Indeed, we all
have a role to play in making virtual spaces safer spaces
(Hall & Hearn, 2018). The next step, then, is to develop a framework that more
fully explores the ways in which negative peer support influ-
ences, facilitates, and enables electronic means of abusing
college women and how these harms are related to and inter-
sect with sexual violence and intimate physical violence. One useful resource is Powell and Henry’s (2016) technol-
ogy-facilitated sexual violence victimization survey, which
measures a much broader range of online sexual, gender, and
sexuality-based harassment. Discussion As well, as far as we know,
there is a dearth of qualitative data on the issues covered here
and rich in-depth interviews and ethnographic research are
likely to reveal some issues that are difficult, if not impossi-
ble, to measure using survey technology. Ideally, though, a
multimethod study, like that done by (Woodlock, 2017), is
what researchers should strive for when examining the utili-
zation of technology in intimate violence and stalking. One more limitation should again be addressed. Since we
used gender-neutral negative peer support measures, the
number of male and the number of female friends of partici-
pants who provided informational support and who engaged
in abusive behaviors cannot be identified. Consequently, the
number of women who were in pro-abuse mixed-sex peer
groups cannot be discerned, but many of them were likely in
such cohorts (DeKeseredy et al., 2018). Even so, future stud-
ies should ask respondents to report the sex or gender iden-
tity of peers who gave them informational support and who
abuse dates. Funding The author(s) received no financial support for the research, author-
ship, and/or publication of this article. Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect
to the research, authorship, and/or publication of this article. Authors’ Note This is a revised version of a paper presented at the February 2018
annual meetings of the Academy of Criminal Justice Sciences. Some men victimize women but do not communicate
with abusive friends on a face-to-face basis (DeKeseredy &
Olsson, 2011; DeKeseredy & Schwartz, 2013). Of course,
there still may be other environments/places in which their
peers influence them to abuse women. For instance, there are
patriarchal online communities with members who never
meet in person but often exchange written, audio, and visual
communication with their peers (DeKeseredy & Corsianos,
2016; Dragiewicz, 2011; Kimmel, 2008; Salter, 2017). Hence, another essential step for researchers is determining
whether peer support for technology-facilitated woman
abuse is mainly offline, online, or a combination of both con-
texts. Related to this new direction is the need to glean data
from potential male perpetrators and to inquire about how
their online and offline male peers influence them to abuse
women. Acknowledgment The authors would like to thank William F. Flack Jr. and Adam
Pritchard for their assistance. References Administrator-Researcher Campus Climate Collaborative. (2015). ARC3 survey of campus climate regarding sexual misconduct. Atlanta: Georgia State University. Peer support for various types of violence against
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aggression in relationships scale: A new multidimensional
measure of technology-based intimate partner aggression. Assessment, 25, 608-626. doi:10.1177/1073191116665696 Wolford-Clevenger, C., Zapor, H., Brasfield, H., Febres, J.,
Elmquist, J., Brem, M., . . . Stuart, G. L. (2016). An exami-
nation of the Partner Cyber Abuse Questionnaire in a college
student sample. Psychology of Violence, 6, 156-162. Powell, A., & Henry, N. (2016). Technology-facilitated sexual
violence victimization: Results from an online survey of
Australian adults. Journal of Interpersonal Violence. Advance
online publication. doi:10.1177/0886260516672055 Woodlock, D. (2017). The abuse of technology in domestic vio-
lence and stalking. Violence Against Women, 23, 584-602. Powell, A., & Henry, N. (2017). References Sexual violence in a digital age. London, England: Palgrave Macmillan. Zweig, J. M., Dank, M., Yahner, J., & Lachman, P. (2013). The
rate of cyber dating abuse among teens and how it relates to
other forms of teen dating violence. Journal of Youth and
Adolescence, 42, 1063-1077. Powers, R. A., & Kaukinen, C. (2017). Intimate partner violence
on college campuses. In C. Kaukinen, M. H. Miller, & R. A. Powers (Eds.), Addressing violence against women on college 12 SAGE Open and family violence, technology-facilitated violence, justice admin-
istration, and legal advocacy. and family violence, technology-facilitated violence, justice admin-
istration, and legal advocacy. Author Biographies Walter S. DeKeseredy is the Anna Deane Carlson Endowed Chair
of Social Sciences, director of the Research Center on Violence,
and profess of Sociology at West Virginia University. He has pub-
lished 24 books, 95 refereed journal articles and 80 scholarly book
chapters on issues such as woman abuse, rural criminology, and
criminological theory. Delanie Woodlock is a community researcher and Adjunct Lecturer
at University of New England. Her research interests include vio-
lence against women, domestic violence, and the medicalization of
women’s health. James Nolan is professor of Sociology at West Virginia University. His research and publications focus heavily on violence against women
on campus, neighborhood dynamics, police procedures, crime mea-
surement, hate crimes, and equity and inclusion in higher education. Martin D. Schwartz is professional lecturer at George Washington
University, professor emeritus at Ohio University, and the author,
co-author or editor of 16 books and more than 150 refereed articles,
chapters, essays, and reports. Amanda Hall-Sanchez is an assistant professor of Criminal Justice
at Fairmont State University. Her research and publications focus
heavily on violence against women on campus and separation/
divorce violence against women in rural communities. Bridget Harris is lecturer and member of the Crime and Justice
Research Center in the School of Justice at Queensland University
of Technology. She conducts research on access to justice, domestic
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Protecting genes from RNA silencing by destroying aberrant transcripts
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Science China. Life Sciences/Science China. Life sciences
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Protecting genes from RNA silencing by destroying aberrant
transcripts ZHANG Heng1* & ZHU JianKang1,2 Received May 6, 2015; accepted May 8, 2015; published online May 19, 2015 Citation:
Zhang H, Zhu JK. Protecting genes from RNA silencing by destroying aberrant transcripts. Sci China Life Sci, 2015, 58: 613–615,
doi: 10.1007/s11427-015-4871-y foreign nucleic acids, whether they are from viruses, trans-
posons or transgenes. In Arabidopsis, small RNAs are usu-
ally 2124 nucleotides in length. Different classes of small
RNAs (sRNA) all require a double stranded RNA (dsRNA)
precursor that is diced into small fragments by the RNase
III-class DICER enzymes. The DICER protein functions
like a ruler to determine the size of small RNA products. For example, siRNA generated by DCL2 are usually 22-nt
long and siRNA by DCL4 are 21-nt long. When the guide
strand of double stranded sRNAs are loaded into
ARGONAUTE (AGO) proteins and form the RNA-induced
silencing complex (RISC), the RISC complex binds to
complementary transcripts, resulting in transcript cleavage
or translational inhibition. The cleaved transcript can also
be converted into dsRNAs by the RNA-dependent RNA
polymerases (RDRs) and produce so-called secondary small
interfering RNAs, which increases siRNA levels and en-
sures silencing. Gene expression is intensively regulated at the transcript
level. It is the rate of transcription and the rate of degrada-
tion that together determine the steady-state level of a spe-
cific transcript. A whole-genome study indicates that the
half-life of Arabidopsis transcripts varies from 0.2 to >24 h,
indicating large variations in the turnover rate of specific
transcripts, which is related to their biological functions [1]. While RNA polymerase II is responsible for mRNA pro-
duction in all eukaryotic organisms, a large repertoire of
ribonucleases are involved in mRNA decay [2]. Different
components in the RNA degradation pathway usually target
specific sets of transcripts. The specificity can be at least
partly accounted for by the presence of RNA motifs within
the 3′ UTR regions and RNA binding proteins that recog-
nize them. In Arabidopsis, mRNAs targeted for degradation
are first deadenylated and subjected to digestion by at least
two major classes of exonucleases [3]. The exosome per-
forms 3′5′ degradation, whereas the XRN4 exonuclease
acts on further decapped transcripts and digests them from
the 5′ end. The dsRNA precursor can be generated from different
sources. MicroRNA (miRNA) genes produce transcripts
that form hairpin-loop structures and are processed into
mature miRNAs by DCL1. *Corresponding author (email: zhangheng@sibs.ac.cn) © The Author(s) 2015. This article is published with open access at link.springer.com • INSIGHT • June 2015 Vol.58 No.6: 613–615
doi: 10.1007/s11427-015-4871-y SCIENCE CHINA
Life Sciences SCIENCE CHINA
Life Sciences June 2015 Vol.58 No.6: 613–615
doi: 10.1007/s11427-015-4871-y life.scichina.com link.springer.com Protecting genes from RNA silencing by destroying aberrant
transcripts In other cases, aberrant tran-
scripts are sensed by the cell and converted into dsRNAs by
RDRs. For example, TRANS-ACTING SIRNA (TAS) genes
are targets of miRNAs. The cleaved transcripts by the Both animals and plants utilize small RNAs for posttran-
scriptional gene silencing (PTGS) and/or transcriptional
gene silencing (TGS). Evolutionarily, small RNA- mediated
gene silencing is believed to function in defense against life.scichina.com link.springer.com Zhang H, et al. Sci China Life Sci June (2015) Vol.58 No.6 614 miRNA-containing RISC complex are uncapped in the 5′
end and processed into 2122 nt siRNAs by the combined
action of RDR6 and DCL2/4. The resulting ta-siRNAs then
regulate other genes in trans. down-regulated genes in the ein5-1 ski2-3 mutant, whose
expression is presumably repressed by the ct-siRNAs. However, the up-regulated genes are also enriched for
ct-siRNAs, indicating that either the siRNA does not have a
silencing function or another antagonizing mechanism ex-
ists to protect the transcript from being destructed. An interesting question is how the cell protects its own
protein-coding transcripts from being targeted by the RNA
silencing machinery. A recent study by Zhang et al. indi-
cates that removal of aberrant transcripts by the RNA decay
pathway is the key [4]. Considering the definition of ct-siRNAs, we may expect
to identify more genes that produce them under different
mutant backgrounds or environmental stresses. In the ein5
single mutant, though morphologically normal, accumula-
tion of gene-derived siRNAs was reported for over one
hundred loci [6]. The mRNA decapping enzymes, which
function before XRN4 decay, also have a role in preventing
PTGS of transgenes [7]. More importantly, other conditions
that lead to accumulation of aberrant transcripts may also
trigger the production of ct-siRNAs. Mutations defective in
genes involved in RNA splicing and mRNA 3’ end for-
mation also enhanced PTGS at specific loci [8]. Recently it
was proposed that splicing is likely an important feature by
which the cell senses foreign/aberrant transcripts. The study
in human pathogenic yeast Cryptococcus neoformans shows
that transposons tend to have sub-optimal splicing sites that
may be sensed by spliceosomes and enhance siRNA bio-
genesis [9]. Indeed Arabidopsis intron-less genes have
higher exon siRNA density, and introducing an intron into a
GFP transgene suppresses spurious transgene silencing in
plants [10]. Thus cells probably utilized every feature of
normal mRNAs to recognize aberrant/foreign ones. 5
Gazzani S, Lawrenson T, Woodward C, Headon D, Sablowski R. A
link between mRNA turnover and RNA interference in Arabidopsis.
Science, 2004, 306: 10461048 4
Zhang XY, Zhu Y, Liu XD, Hong XY, Xu Y, Zhu P, Shen Y, Wu
HH, Ji YS, Wen X, Zhang C, Zhao Q, Wang YC, Lu J, Guo HW.
Suppression of endogenous gene silencing by bidirectional
cytoplasmic RNA decay in Arabidopsis. Science, 2015, 348:
120123 Protecting genes from RNA silencing by destroying aberrant
transcripts A prop-
er function of the RNA decay and RNA processing path-
ways is important to protect normal transcripts from being
targeted for gene silencing. The authors started by screening for mutants that sup-
press the ethylene hyper-response phenotype of EIN3 over-
expression lines. The result is the identification of several
genes functioning in RNA degradation, including the 5′3′
exonuclease XRN4/EIN5 and subunits of the SKI complex
that has RNA helicase activity and functions in feeding
transcripts into the exosome for degradation. EIN5 was
shown to suppress PTGS of specific transgenes and it was
proposed that defects in 5′3′ RNA degradation lead to ac-
cumulation of aberrant transgene transcripts, which are tar-
geted by the RNA silencing machinery [5]. Interestingly,
whereas the ein5 or the ski2 single mutant shows relatively
normal phenotypes, the ein5-1 ski2-2 double mutant is em-
bryonic lethal, indicating that EIN5 and SKI2 redundantly
target some endogenous transcripts. By using the weak al-
lele ski2-3, the ein5-1 ski2-3 mutant is viable but shows
severe developmental defects. Surprisingly, the mutation in
RDR6 almost completely restores normal phenotypes in
ein5-1 ski2-3 mutants. Further genetic analyses indicated
that the mutant phenotype of ein5 ski2 also depends on oth-
er known components of PTGS, including AGO1, SGS3
and DCL2/4. Thus disruption of both the 5′ and 3′ RNA
decay function is devastating to plants. This might be be-
cause some highly expressed genes tend to generate aber-
rant RNAs such as abnormally processed RNAs, which are
ordinarily destroyed by the RNA decay pathways. However,
the aberrant RNAs would over-accumulate in the ein5 ski2
RNA decay mutants, and the accumulated aberrant RNAs
may be channeled to the RDR6 protein, which initiates the
RNA interfering process, leading to silencing of the tran-
scripts. Whole-genome mRNA and small RNA profiling pro-
vides direct support for the above model. Transcriptome
analyses identified 596 differentially expressed genes in
ein5 ski2 and >80% of them were down-regulated. Very
similar transcriptome profiles were observed between rdr6
and ein5 ski2 rdr6, both of which show almost opposite
pattern to that of the ein5 ski2 mutant. In addition, more
than 400 protein-coding genes that produce RDR6-
dependent sRNAs in the ein5 ski2 mutant were identified
and named coding transcript-derived siRNAs (ct-siRNAs). As expected the ct-siRNA loci is over-represented in the 6
Gregory BD, O'Malley RC, Lister R, Urich MA, Tonti-Filippini J,
Chen H, Millar AH, Ecker JR. 6
Gregory BD, O'Malley RC, Lister R, Urich MA, Tonti-Filippini J,
Chen H, Millar AH, Ecker JR. A link between RNA metabolism and
silencing affecting Arabidopsis development. Dev Cell, 2008, 14:
854866 1
Narsai R, Howell KA, Millar AH, O'Toole N, Small I, Whelan J.
Genome-wide analysis of mRNA decay rates and their determinants
in Arabidopsis thaliana. Plant Cell, 2007, 19: 34183436
2
Stoecklin G, Muhlemann O. RNA decay mechanisms: specificity
through diversity. Biochim Biophys Acta, 2013, 1829: 487490
3
Belostotsky DA, Sieburth LE. Kill the messenger: mRNA decay and
plant development. Curr Opin Plant Biol, 2009, 12: 96102
4
Zhang XY, Zhu Y, Liu XD, Hong XY, Xu Y, Zhu P, Shen Y, Wu
HH, Ji YS, Wen X, Zhang C, Zhao Q, Wang YC, Lu J, Guo HW.
Suppression of endogenous gene silencing by bidirectional
cytoplasmic RNA decay in Arabidopsis. Science, 2015, 348:
120123
5
Gazzani S, Lawrenson T, Woodward C, Headon D, Sablowski R. A
link between mRNA turnover and RNA interference in Arabidopsis.
Science, 2004, 306: 10461048
6
Gregory BD, O'Malley RC, Lister R, Urich MA, Tonti-Filippini J,
Chen H, Millar AH, Ecker JR. A link between RNA metabolism and
silencing affecting Arabidopsis development. Dev Cell, 2008, 14:
854866
7
Thran M, Link K, Sonnewald U. The Arabidopsis DCP2 gene is 1
Narsai R, Howell KA, Millar AH, O'Toole N, Small I, Whelan J.
Genome-wide analysis of mRNA decay rates and their determinants
in Arabidopsis thaliana. Plant Cell, 2007, 19: 34183436
2
Stoecklin G, Muhlemann O. RNA decay mechanisms: specificity
through diversity. Biochim Biophys Acta, 2013, 1829: 487490
3
Belostotsky DA, Sieburth LE. Kill the messenger: mRNA decay and
plant development. Curr Opin Plant Biol, 2009, 12: 96102
4
Zhang XY, Zhu Y, Liu XD, Hong XY, Xu Y, Zhu P, Shen Y, Wu
HH, Ji YS, Wen X, Zhang C, Zhao Q, Wang YC, Lu J, Guo HW.
Suppression of endogenous gene silencing by bidirectional
cytoplasmic RNA decay in Arabidopsis. Science, 2015, 348:
120123 7
Thran M, Link K, Sonnewald U. The Arabidopsis DCP2 gene is
required for proper mRNA turnover and prevents transgene silencing 8
Herr AJ, Molnar A, Jones A, Baulcombe DC. Defective RNA
processing enhances RNA silencing and influences flowering of
Arabidopsis. Proc Natl Acad Sci USA, 2006, 103: 1499415001 in Arabidopsis. Plant J, 2012, 72: 368377 9
Dumesic PA, Natarajan P, Chen CB, Drinnenberg IA, Schiller BJ, Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction
in any medium, provided the original author(s) and source are credited. in Arabidopsis. Plant J, 2012, 72: 368377
8
Herr AJ, Molnar A, Jones A, Baulcombe DC. Defective RNA
processing enhances RNA silencing and influences flowering of
Arabidopsis. Proc Natl Acad Sci USA, 2006, 103: 1499415001
9
Dumesic PA, Natarajan P, Chen CB, Drinnenberg IA, Schiller BJ,
Thompson J, Moresco JJ, Yates JR, Bartel DP, Madhani HD. Stalled
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2013, 152: 957968
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Christie M, Croft LJ, Carroll BJ. Intron splicing suppresses RNA
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Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction
in any medium provided the original author(s) and source are credited Protecting genes from RNA silencing by destroying aberrant
transcripts A link between RNA metabolism and
silencing affecting Arabidopsis development. Dev Cell, 2008, 14:
854866 7
Thran M, Link K, Sonnewald U. The Arabidopsis DCP2 gene is
required for proper mRNA turnover and prevents transgene silencing 615 Zhang H, et al. Sci China Life Sci June (2015) Vol.58 No.6 Zhang H, et al. Sci China Life Sci June (2015) Vol.58 No.6 Zhang H, et al. Sci China Life Sci June (2015) Vol.58 No.6 8
Herr AJ, Molnar A, Jones A, Baulcombe DC. Defective RNA
processing enhances RNA silencing and influences flowering of
Arabidopsis. Proc Natl Acad Sci USA, 2006, 103: 1499415001 9
Dumesic PA, Natarajan P, Chen CB, Drinnenberg IA, Schiller BJ, Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction
in any medium, provided the original author(s) and source are credited.
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The clinical efficacy of novel vacuum suction ureteroscopic lithotripsy in the treatment of upper ureteral calculi
|
Research Square (Research Square)
| 2,021
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cc-by
| 3,894
|
LvWen zhang LvWen zhang
Sheng Jing Hospital
Yan Song
(
alexander-song@163.com
)
Sheng Jing Hospital
Xiang Fei
Sheng Jing Hospital Sheng Jing Hospital The clinical efficacy of novel vacuum suction ureteroscopic lithotripsy in
the treatment of upper ureteral calculi LvWen zhang
Sheng Jing Hospital
Yan Song
(
alexander-song@163.com
)
Sheng Jing Hospital
Xiang Fei
Sheng Jing Hospital Research Article eywords: Upper ureteral calculi, Ureteroscopic lithotripsy, Vacuum suction ureteroscopy, Clinical efficacy DOI: https://doi.org/10.21203/rs.3.rs-280213/v1 License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Page 1/7 Abstract This study investigated the clinical efficacy of a novel vacuum suction ureteroscopic approach in the treatment of upper ureteral calculi. A total of
160 patients with impacted upper ureteral calculi were included in this study. 50 patients underwent rigid ureteroscopic lithotripsy, 54 patients
underwent flexible ureteroscopic lithotripsy, and 56 patients underwent vacuum suction ureteroscopic lithotripsy. The operative time, length of
hospitalization, stone-free rate, complication rate and total treatment cost were compared among the three groups. Subgroup analysis was
performed based on the stone diameter over and below 1.5 cm. Compared with the other two groups, the vacuum suction ureteroscopy group had
higher stone-free rate at 3–5 days (90.0% vs. 61.9% vs. 55.6%, P < 0.05) and 1 month (96.4% vs. 77.7% vs. 74.0%, P < 0.05) postoperatively. In
subgroup analysis, the stone-free rate of the vacuum suction ureteroscopy group was significantly higher when the stone diameter was > 1.5 cm at 1
month postoperatively ( P < 0.05); however, there were no differences in postoperative complications.( P > 0.05). In conclusion, the novel vacuum
suction ureteroscopic lithotripsy has significantly improved the stone-free rate especially in complicated cases; however the complication and cost
was not increased. Introduction Ureteral stones are common and the obstruction caused by stones can adversely affect the quality of life of patients [1].There are many options for
treatment, such as extracorporeal shock wave lithotripsy (ESWL), percutaneous nephrolithotomy (PCNL), and retrograde intrarenal surgery (RIRS) [2]. Currently, RIRS is preferred by many clinicians, especially in the management of upper and middle ureteral stones [3]. In general, the absence of residual stones or the presence of stone fragments < 4 mm are clinical indicators of stone-free status. However, even small
residual stone fragments can still cause symptoms, such as low back pain, hematuria, and infection. Furthermore, the time required for the complete
elimination of stone fragments is variable, so achieving stone-free status in a theoretical sense is of great importance [4]. Ureteroscopic lithotripsy
with self-made negative pressure device that can improve the stone-free rate has been reported[5]. Recently, we have performed some ureteroscopic lithotripsy cases with a suction device that can effectively eliminate the stone fragments and
reduce the risk of infection. In this study, the clinical efficacy of this new device was evaluated. Results alculi were included in this study. Their demographic data and clinical characteristics are shown in Table 1. 160 patients with upper ureteral calculi were included in this study. Their demographic data and clinical characteristics are shown in Table 1. atients with upper ureteral calculi were included in this study. Their demographic data and clinical characte nts with upper ureteral calculi were included in this study. Their demographic data and clinical characteristics are shown in Table 1. Table 1
Demographic characteristics data of patients according to patients ‘group
Parameter
Vacuumsuctionureteroscopy
group
Flexible ureteroscopy
group
Rigid
ureteroscopy group
F/χ2
P
n
56
54
50
Age
53.8 ± 12.1
55.2 ± 10.2
54.3 ± 11.6
0.202
0.817
Gender
Male/Female
34/22
37/17
25/25
3.728
0.155
BMI
25.77 ± 3.37
26.91 ± 3.49
26.76 ± 3.40
1.804
0.168
Creatinine
80.06 ± 29.38
85.29 ± 24.09
79.92 ± 31.18
0.628
0.535
Stone duration (month)
2.90 ± 3.44
3.05 ± 3.13
3.61 ± 4.14
0.567
0.568
Stone hardness (1000 HU)
708.59
± 343.70
684.22
± 376.30
665.04
± 309.61
0.213
0.809
Stone diameter (mm)
13.9 + 4.7
12.7 + 5.5
12.48 + 4.9
2.769
0.606
Stone location
Left/right
28/28
25/29
23/27
0.217
0.897
Preoperative infection
Yes/No
5/40
4/37
4/38
0.727
0.695
SWL history
Yes/No
15/41
17/37
12/38
0.751
0.687 Page 2/7 The operative time of the rigid ureteroscopy group was significantly shorter than that of the flexible ureteroscopy group and vacuum suction
ureteroscopy group (P < 0.05). However, there were no differences in postoperative complications (P > 0.05).The total treatment cost in vacuum
suction ureteroscopy group was much lower than that in both rigid ureteroscopy group and flexible ureteroscopy group(P < 0.05). The operative time of the rigid ureteroscopy group was significantly shorter than that of the flexible ureteroscopy group and vacuum suction
ureteroscopy group (P < 0.05). However, there were no differences in postoperative complications (P > 0.05).The total treatment cost in vacuum
suction ureteroscopy group was much lower than that in both rigid ureteroscopy group and flexible ureteroscopy group(P < 0.05). In this study, the sheath placement was first failed in 3 patients, and the patients were treated successfully by the vacuum suction ureteroscopy two
week after D-J stent placement; however no ureteral stricture or trauma was found in this study during the follow up period (3 months). Results The stone-
free rate was significantly higher in the vacuum suction ureteroscopy group than that in the other two groups at 3–5 days and 1 month
postoperatively (P < 0.05) According to the modified Clavin grading system, there were no complications above grade IV in the three groups. There
were only two cases with grade I complications (infection and fever) in the vacuum suction ureteroscopy group; four cases with grade I
complications (three cases of infection and fever and one case of renal colic that was treated with an analgesic) in the rigid ureteroscopy group; and
three cases with grade I complications (infection and fever) in the flexible ureteroscopy group. (Table 2). Table 2 Table 2
Intraoperative and Postoperative data according to patients’ group
Parameter
Vacuum suction ureteroscopy
group
Flexible ureteroscopy group
Rigid ureteroscopy group
F/χ2
P
n
56
54
50
Operative time
46.4 ± 17.9a
57.3 ± 28.5b
39.1 ± 13.6c
10.06
0.01
Treatment cost(US. Dollar)
2622.6
± 794.7
3274.4
± 903.3
2883.6
± 1030.7
4.56
0.04
Complication rate (n (%))
2(3.6)
3(5.6)
4(8.0)
3.48
0.18
*Stone-free rate at 3–5
Days (n (%))
18/20(90.0)a
13/21(61.9)b
10/18(55.6)b
8.49
0.00
Stone-free rate at 1 month
(n (%))
54(96.4)a
42(77.7)b
37(74.0)b
5.48
0.01
Note: Different a, b and c indicate multiple comparisons among groups that are significantly different; * indicates the stone-free rate of patients
was re-examined by CT postoperatively. Intraoperative and Postoperative data according to Note: Different a, b and c indicate multiple comparisons among groups that are significantly different; * indicates the stone-free rate of patients
was re-examined by CT postoperatively. In stratified analysis according to the maximum stone diameter (< 1.5 cm and > 1.5 cm), there was no significant difference in the stone-free rate
among the three groups at 1 month postoperatively when the stone diameter was < 1.5 cm (P > 0.05). When the stone diameter was > 1.5 cm, the
stone-free rate of the vacuum suction ureteroscopy group was significantly higher than that of the other two groups at 1 month postoperatively (P <
0.05) (Table 3). In stratified analysis according to the maximum stone diameter (< 1.5 cm and > 1.5 cm), there was no significant difference in the stone-free rate
among the three groups at 1 month postoperatively when the stone diameter was < 1.5 cm (P > 0.05). Discussion The surgical treatment for upper ureteral stones is ureteroscopic lithotripsy and percutaneous nephrolithotomy. Percutaneous nephrolithotomy has a
very high stone-free rate, but its complications, including pain, bleeding, and even loss of kidney are not rare [7]. Therefore, ureteroscopic lithotripsy is
still regarded as the first-line of treatment by some clinicians [8–10]. Generally, ureteroscopic lithotripsy is safe and effective [8–11]. However, retrograde stone or stone fragments migration is an important factor
affecting the success of intraoperative lithotripsy in both rigid and flexible lithotripsy. The use of devices such as stone cone can indeed decrease the
risk of stone fragment retrograde retropulsion, but will undoubtedly increase the treatment cost. However, the stone-free rate of most patients
immediately after surgery is not high; a long time is needed for the stone residues to pass by themselves. There are two methods for the
intraoperative management of calculi by ureteroscopy. The first method (dusting) is to break the calculi into small pieces as tiny as possible, which
are slowly discharged postoperatively; the other method is to remove the stone fragments as best as possible by using the stone basket after the
stones are fragmented. The former prolongs the lithotripsy time and the latter may inevitably increase the damage to the ureter through the repeated
removal of the stone fragments. Although the ureteral access sheath (UAS) may alleviate this damage [12], it is not always satisfactory. Therefore,
further studies are needed to improve the initial stone-free rate of ureteroscopic lithotripsy [13]. The novel vacuum suction ureteroscopic approach combines the advantages of ureteroscopy and percutaneous nephrolithotomy. The principle of
continuous vacuum suction is basically the same as that of ultrasonic lithotripsy. During the whole process, stones can be continuously drawn and
flushed out. However, sometimes stone fragments may get stuck in the sheath while suctioning. If it did happen, it was always safe to break the
fragments by laser in the sheath because the metallic sheath can prevent ureteral injury effectively. By adjusting the vacuum suction device to control the force of suction, the stones are driven and pressed to the tip of the sheath by the vacuum
suction force, greatly improving the lithotripsy efficacy and reducing the operative time. Vacuum suction greatly decreased the risk of infection. Results When the stone diameter was > 1.5 cm, the
stone-free rate of the vacuum suction ureteroscopy group was significantly higher than that of the other two groups at 1 month postoperatively (P <
0.05) (Table 3). Table 3
Comparison of perioperative parameters of stone size stratification, stone-free rate, and postoperative complications
Variables
Stone ≤ 1.5cm
P
Stone > 1.5 cm
P
Sub-Group
Vacuumsuctionureteroscopy
group
Flexible
ureteroscopy
group
Rigid
ureteroscopy
group
Vacuum
suction
ureteroscopy
group
Flexible
ureteroscopy
group
Rigid
ureteroscopy
group
number
32
30
34
24
24
16
Operation
time
41.7 ± 10.8a
51.5 ± 24.2b
34.4 ± 9.7c
0.001
46.6 ± 11.2
59.1 ± 11.7
45.6 ± 18.6
0.475
Treatment
costs
2438.4
± 749.3
2944.3
± 880
2585.3
± 773.3
0.035
2796.3
± 947.4
3395.1
± 715.0
2875.1
± 852.4
0.047
Complication
(n(%))
1(3.1)
2(6.67)
2(5.9)
0.680
1(4.1)
1(4.1)
2(12.5)
0.234
Stone-free
rate 1month
(n(%))
32(100.0)
27(91.7)
29(85.2)
0.257
22(91.6) a
15(62.5) b
8(50.0) b
0.001
Note: Different a, b and c indicate multiple comparisons among groups that were significantly different. Table 3 Page 3/7 Discussion There
is plenty of space between the lithotripsy endoscope(6F) and the working sheath (13F); vacuum suction speeded up the circulation of irrigating water,
which helps to ensures the clear view of surgical field and helps to take away the heat generated by the holmium laser [14], thereby reducing thermal
injury. The cost of vacuum suction ureteroscopic lithotripsy was significant lower than that of the other two groups. Devices used to prevent the stone retro-
migration or help to clean the stone fragments such as stone cone or stone basket were not used in this group. The working sheath was reusable,
which further limited the cost of the procedure. If the stone retro-migration happened, the operation can be easily converted to flexible ureteroscopic
lithotripsy by just changing the scope without changing the working sheath. The stone-free rate of the vacuum suction ureteroscopy group was also much higher than that of the other two groups. In subgroup analysis, we
found that the stone-free rate became more significant when the stone diameter was > 1.5 cm, indicating that vacuum suction ureteroscopic
lithotripsy would be better in the treatment of larger or complicated stones. In terms of postoperative complications, there was no difference among
the three groups. It is undeniable that this novel vacuum suction ureteroscopy approach has some disadvantages. Firstly, the semi-flexible sheath is made of metallic
material, which is possible to cause damage to the ureter, requiring the surgeon to be very familiar with the anatomical structure and to be as gentle
as possible, especially in physiological bends. Sheath placement failure may occur in patients with ureteral stricture. For patients who fail to place
the sheaths, placing the D-J stent for 2 weeks is suggested. Secondly, although vacuum suction devices can significantly reduce the renal pelvic
pressure [15], there is a lack of effective devices to monitor the real-time changes of the suction force and renal pelvic pressure [16]. Therefore, it is
necessary for the surgeon to be experienced and to arrive at an effective judgment of the lithotripsy procedure. In conclusion, vacuum suction ureteroscopy has significantly improved the stone-free rate compared with traditional rigid and flexible approaches. When dealing with large and complicated upper ureteral stones, vacuum suction ureteroscopic lithotripsy is a better option. Surgical procedures The new vacuum suction ureteroscope (Figure 1) consisted of a standard ureteroscope (9.8F), a lithotripsy endoscope (6F), a standard semi-rigid
ureteroscopic access sheath (13F) and a vacuum suction device. In the vacuum suction ureteroscopy group, patients received general anesthesia and were placed in the lithotomy position. First, the sheath was
connected to the standard ureterscope and placed near the stones under the guidance of direct vision by the standard ureterscope, and then the
standard ureterscope was withdrawn. The vacuum suction device was connected to the end of the ureteroscopic sheath, the lithotripter (6F) was
fixed to the sheath, and a 200-μm holmium laser fiber was inserted into ureterscope for lithotripsy. After lithotripsy, a 6F D-J tube was indwelled. (Video 1). For patients who failed to place the sheaths, D-J stent were placed first and the patients were treated two week after D-J stent placement. In the rigid ureteroscopy group, patients received general anesthesia and were placed in the lithotomy position. An 8/9.8F ureteroscope was inserted
into the ureter under the guidance of a guide wire. After confirming the location, the stones were fragmented with holmium laser; Stone Cone
wasused in some cases to stop the stone from retrograde-migration and a 6F D-J stent was indwelled after the completion of operation. In the flexible ureteroscopy group, patients received general anesthesia, flexible ureteroscopic access sheath (13F) was placed near the stone, and a
holmium laser fiber was used to break the stones with the help of flexible ureteroscopy. Stone basket was used to remove the fragments. A 6F D-J
stent was indwelled routinely. The operative time was defined as the time from the start to the end of anesthesia. Patients were re-examined by CT at 3–5 days postoperatively, and
those patients who were not stone free were re-examined by CT to judge the residual stone status at 1 month postoperatively. The double-J stents
were usually removed 2 weeks after the operation. The stone-free rate was defined as the absence of residual stones upon CT examination. Complications were assessed using a modified Clavin grading system. All the patients were followed up for 3 months after the operationto
determine whether or not they had a post-surgical stricture. Due to the retrospective nature of the study, the need for informed consent was waived; additionally, the study design was approved by the
ShengJing hospital ethics review board. Statistical methods Measurement data were described as mean ± standard deviation (SD), and counting data were expressed as frequency (number). Data were
analyzed by SPSS 23.0 software. According to the normality and homogeneity of variance of measurement data, one-way ANOVA was used for
comparisons between groups, and the LSD-t test was used for multiple comparisons between groups. In addition, the RxC contingency table chi-
square test was used to compare distribution differences of the composition ratio between groups, and the chi-square segmentation method was
used for multiple comparisons between groups. P-values <0.05 were considered statistically significant. Surgical procedures Authors had no access to information that could identify individual participants during or after data
collection. All clinical investigations were conducted according to the principles expressed in the Declaration of Helsinki. Study Design The data of 160 patients with upper ureteral stones who were eligible for treatment at our hospital from January 2018 to January 2020 were
retrospectively collected in this study. Fifty patients underwent rigid ureteroscopic lithotripsy, 54 patients underwent flexible ureteroscopic lithotripsy,
and 56 patients underwent vacuum suction ureteroscopic lithotripsy. All patients underwent pre-operative CT, urine analysis, urine culture, routine blood analysis, coagulation function tests, and creatinine level. Preoperative prophylactic antibiotic therapy was given to all patients, and sensitive antibiotic was given to patients with positive urine cultures. Stents were preoperatively placed only in patients with obstructing stones and urinary sepsis. The inclusion criteria were as follows: patients with unilateral upper ureteral calculi who were not recommended for ESWL or patients refused to take
ESWL or PCNL. According to Chinese Urology Guidelines, the upper ureter is defined as the imaging segment, which extends from the pelviureteric Page 4/7 Page 4/7 junction to the upper margin of the sacroiliac joint (or the lower margin of the fourth lumbar vertebra). The exclusion criteria were as follows: patients
with a solitary kidney; those who needed concurrent treatment for kidney stones or bilateral ureteral stones; and those who were lost to follow-up. junction to the upper margin of the sacroiliac joint (or the lower margin of the fourth lumbar vertebra). The exclusion criteria were as follows: patients
with a solitary kidney; those who needed concurrent treatment for kidney stones or bilateral ureteral stones; and those who were lost to follow-up. The study design was approved by the ShengJing hospital ethics review board (NO.2018PS266K). The study was not a clinical trial, the data was
retrospectively collected from the hospital database. Due to the retrospective nature of the study, the need for informed consent was waived by
China Medical University. The study protocol was done following relevant guidelines. Authors' contributions: Authors' contributions: Page 5/7
LV Wen Zhang: Data collection, manuscript writing
Yan Song: Study design, supervision
Xiang Fei: Data analysis, manuscript writing
Declarations
Funding: No funding
Conflicts of interest:The authors declare that there are no conflicts of interest. Ethics approval: the study design was approved by the ShengJing hospital ethics review board. Declarations Funding: No funding Conflicts of interest:The authors declare that there are no conflicts of interest. Ethics approval: the study design was approved by the ShengJing hospital ethics review board. Page 5/7 Page 5/7 Consent to participate: Due to the retrospective nature of the study, the need for informed consent was waived Consent for publication: The study was approved by the ShengJing hospital ethics committees for Consent for publication Consent for publication: The study was approved by the ShengJing hospital ethics committees for Consent for publication Availability of data and material: Supporting data can be accessed via the hospital database by contacting the corresponding author upon request. References Availability of data and material: Supporting data can be accessed via the hospital database by contacting the corresponding author upon request. References References 1. Zeng, G. H. et al. Prevalenceof kidney stones in China: an ultrasonography based cross-sectional study. 2. Preminger, G. M. et al. 2007 Guideline for the management of ureteral calculi. Eur. Urol.52, 1610-31(2007 2. Preminger, G. M. et al. 2007 Guideline for the management of ureteral calculi. Eur. Urol.52, 1610-31(2007). 2. Preminger, G. M. et al. 2007 Guideline for the management of ureteral calculi. Eur. Urol.52, 1610-31(2007). 3. Hyams, E. S. et al. A prospective, multi-institutional study of flexible ureteroscopy for proximal ureteral stones smaller than 2 cm. J. Urol. 193,
165–169 (2015). 3. Hyams, E. S. et al. A prospective, multi-institutional study of flexible ureteroscopy for proximal ureteral stones smaller than 2 cm. J. Urol. 193,
165–169 (2015). 4. Lu, P. et al. Clinical efficacy and safety of flexible ureteroscopic lithotripsy using 365 µm holmium laser for nephrolithiasis: a prospective,
randomized, controlled trial. World. J. Urol. 38, 481–487 (2020). 4. Lu, P. et al. Clinical efficacy and safety of flexible ureteroscopic lithotripsy using 365 µm holmium laser for nephrolithiasis: a prospective,
randomized, controlled trial. World. J. Urol. 38, 481–487 (2020). 5. Wu, Z. et al. Negative-Pressure Ureteroscopic Holmium-YAG Laser Lithotripsy for Ureteral Stones. Urol. Int. 104, 752–757 (2020). 6. Deng, T. et al. Systematic review and cumulative analysis of the managements for proximal impacted ureteral stones. World. J. Urol. 37, 1687–
1701 (2019). 7. Wollin, D. A. et al. Percutaneous nephrolithotomy: complications and how to deal with them. Urolithiasis 8. Kallidonis, P. M. Invasive Surgical Ureterolithotomy Versus Ureteroscopic Lithotripsy for Large Ureteric Stones: A Systematic Review and Meta-
analysis of the Literature. Eur. Urol. Focus. 3, 554–566 (2017). 8. Kallidonis, P. M. Invasive Surgical Ureterolithotomy Versus Ureteroscopic Lithotripsy for Large Ureteric Stones: A Systematic Review and Meta-
analysis of the Literature. Eur. Urol. Focus. 3, 554–566 (2017). 9. Lai, S. et al. Optimal management of large proximal ureteral stones (> 10 mm): A systematic review and meta-analysis of 12 randomized
controlled trials. Int. J. Surg. 80, 205–217 (2020). 9. Lai, S. et al. Optimal management of large proximal ureteral stones (> 10 mm): A systematic review and meta-analysis of 12 randomized
controlled trials. Int. J. Surg. 80, 205–217 (2020). 10. Deng, T. al.Antibiotic prophylaxis in ureteroscopic lithotripsy: a systematic review and meta-analysis of comparative studies. BJU. Int. 122, 29–
39 (2018). et 10. Deng, T. al.Antibiotic prophylaxis in ureteroscopic lithotripsy: a systematic review and meta-analysis of comparative studies. BJU. References Int. 122, 29–
39 (2018). et 11. Elsheemy, M. S. et al. Holmium:YAG laser ureteroscopic lithotripsy for ureteric calculi in children: predictive factors for complications and
success. World. J. Urol. 32, 985–990 (2014). 12. Chen, Y. et al. Comparison of Safety and Efficacy in Preventing Postoperative Infectious Complications of a 14/16F Ureteral Access Sheath with
a 12/14F Ureteral Access Sheath in Flexible Ureteroscopic Lithotripsy. J. Endourol. 32, 923–927 (2018). 3. Rodríguez, D. et al. Minimally invasive surgical treatment for kidney stone disease. Adv. Chronic. Kidney. 13. Rodríguez, D. et al. Minimally invasive surgical treatment for kidney stone disease. Adv. Chronic. Kidney. Dis.22, 266 – 72(2015). 4. Liang, H. et al. Thermal effect of holmium laser during ureteroscopic lithotripsy.BMC. Urol.20,69(2020). 15. Jakobsen, J. S. et al. Endoluminal isoproterenol reduces renal pelvic pressure during semirigid ureterorenoscopy: a porcine model. BJU. Int. 105,
121–124 (2010). 16. Jung, H. et al. Endoluminal isoproterenol irrigation decreases renal pelvic pressure during flexible ureterorenoscopy: a clinical randomized,
controlled study. Eur. Urol. 54, 1404–1413 (2008). 16. Jung, H. et al. Endoluminal isoproterenol irrigation decreases renal pelvic pressure during flexible ureterorenoscopy: a clinical randomized,
controlled study. Eur. Urol. 54, 1404–1413 (2008). Figures Figures Page 6/7 Figure 1 The new vacuum suction ureteroscope (Figure 1) consisted of a standard ureteroscope (9.8F), a lithotripsy endoscope (6F), a standard semi-rigid
ureteroscopic access sheath (13F) and a vacuum suction device. A: Vacuum pipe. B: Vacuum suction control. C: Irrigation pipe. D: ureteroscopic
access sheath. E: lithotripsy endoscope. F: light. G: Camera. Thevacuumsuctionureteroscopiclithotripsy.mp4 Page 6/7 Figure 1
The new vacuum suction ureteroscope (Figure 1) consisted of a standard ureteroscope (9.8F), a lithotripsy endoscope (6F), a standard semi-rigid
ureteroscopic access sheath (13F) and a vacuum suction device. A: Vacuum pipe. B: Vacuum suction control. C: Irrigation pipe. D: ureteroscopic
access sheath. E: lithotripsy endoscope. F: light. G: Camera. Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Thevacuumsuctionureteroscopiclithotripsy.mp4 Page 7/7
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Análise e indexação de imagens na rede Flickr
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Em Questão
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Análise e indexação de imagens na rede Flickr Rafael Alves de Oliveira
Doutorando; Universidade Federal da Paraíba;
rraffael@gmail.com Luciane Paula Vital
Doutoranda; Universidade Federal de Santa Catarina;
lucianepv@yahoo.com.br Resumo: O artigo apresenta um breve panorama sobre a disseminação e
democratização da internet colaborativa, e de como esse processo popularizou o
uso da imagem em suporte digital. Faz uma breve análise sobre a rede social
Flickr, identificando seus objetivos, suas ferramentas, e alguns dos estudos
relativos à sua usabilidade no âmbito da Ciência da Informação. Delimita a
abordagem para as imagens armazenadas na rede, propondo um estudo
qualitativo em torno da indexação livre realizada por seus usuários. Aplica a
metodologiaestabelecida em estudo anterior como a mais apropriada para
indexação de imagens fotográficas em ambiente web, comparando os resultados
obtidos com a indexação livre realizada pelos usuários. Palavras-chave: Imagem. Indexação de imagens. Flickr. Redes sociais. Indexação social. Indexação social. E-ISSN 1808-5245 E-ISSN 1808-5245 Q
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doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 Questão, Porto Alegre, v. 21, n. 2, p. 7 30, mai/ago. 2015
http://dx.doi.org/10.19132/1808-5245212.7-30
| 7 Q
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doi: http://dx.doi.org/10.19132/1808-5245212.7-30 1 Introdução A década de 2001 a 2010 foi testemunha do surgimento de um novo conceito de
uso da internet. A possibilidade de publicação e tratamento livre de informações
por parte de seus usuários tornou-se a característica fundamental deste novo
modelo, que, embora não seja um consenso, os estudiosos nomearam de Web
2.0. Em alguma medida, pode-se dizer que a criação das redes sociais foi uma
consequência natural desse fato, e que a aceitação dessas por parte de uma
grande parcela dos usuários da internet é um reflexo de como este modelo se
estabeleceu, e tende a se aprimorar. Essas novas ferramentas disseminaram o conceito e democratizaram o
uso de um suporte informacional bastante peculiar: a imagem, que migrou dos | 7 Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital
E-ISSN 1808-5245 Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital álbuns de fotografias e das molduras de parede para o suporte digital, podendo
agora ser acessada a qualquer instante, de qualquer lugar do planeta. [...] com as novas tecnologias de comunicação e informação uma
nova fase inicia-se, e nesta, as imagens são disponibilizadas
livremente pelos usuários na internet. Essa liberdade de publicação,
naturalmente, impeliu os métodos de tratamento e recuperação para
essa mesma ideia. Agora o usuário não só pode publicar as imagens
como
também
categorizá-las. Esse
boom
de
imagens
disponibilizadas iniciou-se com o surgimento das redes sociais. A
sociedade aderiu a esta nova forma de interagir, e as redes
cresceram e popularizaram-se significativamente ao longo de
poucos anos. À medida que as ferramentas destas redes se
desenvolviam com o avanço dos seus servidores, as redes podiam
armazenar cada vez mais informações. A publicação livre de
imagens foi apenas um passo natural nesse desenvolvimento. (OLIVEIRA, 2011, p. 2). Partindo desse pressuposto, fica evidente que uma problemática tende a
surgir com esse avanço, e diz respeito especificamente à maneira como estas
imagens serão tratadas e recuperadas. Na literatura científica de Ciência da
Informação ainda não existe um acordo sobre como as imagens devem ser
tratadas, pois diversos fatores implicam nessa análise, incluindo o tipo de
imagem (pinturas, fotografias, etc.) e o suporte no qual se encontra. Entretanto,
alguns modelos bem construídos e eficientes podem ser encontrados, como o de
Manini (2002) e Rodrigues (2007). Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 Em Questão, Porto Alegre, v. 21, n. 2, p. 7 30, mai/ago. 2015
doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Q
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doi: http://dx.doi.org/10.19132/1808-5245212.7-30 1 Introdução No trabalho intitulado Metodologias para indexação de imagens
fotográficas em ambiente web (OLIVEIRA, 2011) são apresentados e analisados
os elementos propostos e o comportamento de três das metodologias para
indexação de imagens mais citadas na literatura científica da área da Ciência da
Informação, visando identificar aquela que poderia ser considerada mais
adequada à aplicação em imagens fotográficas coletadas em ambiente web. Este
estudoanalisa, dentre outras, a metodologia proposta por Rodrigues (2007), na
qual o autor revela que uma imagem não apenas mostra, mas também representa
algo, que pode não necessariamente ter uma relação direta aos objetos
apresentados. Devido a isso, entende-se que uma imagem terá dois níveis ou
sentidos principais: o denotativo, que se refere “[...] àquilo que a imagem
representa com ‘certa precisão’, no seu sentido real” (RODRIGUES, 2007,
p.69), e o conotativo,referente àquilo “[...] que a imagem pode ‘interpretar’ em | 8 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital
E-ISSN 1808-5245 Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 um determinado contexto, em um sentido figurado e simbólico”.(RODRIGUES,
2007, p. 69). A partir dessa constatação, Rodrigues (2007) estabelece que devem
ser considerados os seguintes elementos para uma análise apropriada: um determinado contexto, em um sentido figurado e simbólico”.(RODRIGUES,
2007, p. 69). A partir dessa constatação, Rodrigues (2007) estabelece que devem
ser considerados os seguintes elementos para uma análise apropriada: a) descrição física, ou o “[...] formato e tamanho da imagem fotográfica,
tipo de suporte, autor, transformações ocorridas a partir do original etc”. (RODRIGUES, 2007, p.75);igo de periódico NBR 6022/03; b) composição, ao considerar “[...] tipo de luz, nível de nitidez dos
assuntos, ponto de vista do fotógrafo, profundidade de campo e
hierarquia das figuras, enquadramento etc”.(RODRIGUES, 2007, p. 75); c) contexto arquivístico, ou os locais e fatos, históricos ou não,
correspondentes àquela fotografia; d) sentidos denotativos e conotativos, correspondendo ao que imagem
mostra e ao que ela representa, respectivamente; e) tematização, ao atribuir a imagem a um contexto diferente do que é
mostrado, mas que possui relação direta aos elementos denotativos
apresentados. Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 Q
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doi: http://dx.doi.org/10.19132/1808-5245212.7-30 1 Introdução Após traçar um quadro comparativo observou-se que, para uma análise
de imagens coletadas em ambiente web, a metodologia proposta por Rodrigues
(2007) pode ser considerada a mais apropriada, já que enfatiza os elementos
conotativos identificados numa imagem, considerando-se que uma imagem
coletada na internet tem grande possibilidade de não apresentar informações
técnicas referentes a esta. A partir das constatações obtidas neste estudo, buscaremos obter um
breve panorama sobre a indexação realizada livremente pelos usuários de redes
sociais por meio da aplicação prática da metodologia de Rodrigues (2007) e da
interpretação dos dados obtidos. O banco de imagens escolhido para a pesquisa
foi a rede Flickr, por se tratar de uma rede social amplamente utilizada e ter seu
foco no compartilhamento de recursos visuais. Desde já delimitaremos o objeto
de estudo apenas às imagens armazenadas pela rede Flickr, embora a rede
atualmente permita também o compartilhamento de vídeos. | 9 Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital
E-ISSN 1808-5245 Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital
E-ISSN 1808-5245
2 Flickr: uma breve análise Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital
E-ISSN 1808-5245 Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital [...] que podem ser definidas como sistemas orgânicos baseados na
atribuição livre e pessoal de [termos] à informações ou objetos
visando à organização e recuperação. O neologismo folksonomia –
formado pelas palavras, em inglês, folks (pessoas) e taxonomy
(taxonomia) – foi cunhado [...] como forma de expressar
contraposição às classificações do conhecimento tradicionais,
elaboradas por especialistas e construídas baseando-se em arranjos
hierárquicos. (GUEDES; DIAS, 2010, p.48). 2 Flickr: uma breve análise 2 Flickr: uma breve análise O Flickr é uma rede de compartilhamento de imagens hospedada na internet. O
Flickr, segundo os próprios idealizadores,propõe novas formas para organizar
fotografias, que poderão ser acessadas livremente por qualquer pessoa. Parte da solução é tornar colaborativo o processo de organizar fotos
[...] No Flickr, é possível permitir que seus amigos, família e outros
contatos organizem suas coisas - não apenas adicionem
comentários, mas também notas e tags. [...] E, à medida que essas
informações crescem como metadados, você poderá encontrar as
coisas facilmente mais tarde, uma vez que toda essa informação
pode ser buscada. (FLICKR, 2014c, documento eletrônico sem
paginação). Embora seja considerado uma rede social, o Flickr permite que mesmo
aqueles que não possuam cadastro na base tenham acesso às imagens
disponibilizadas. No entanto, o usuário tem a liberdade de restringir o acesso a
algumas de suas fotografias apenas àqueles usuários que lhes forem mais
convenientes. O Flickr é um claro exemplo de ambienteque permite a utilização
e o emprego daquilo que a literatura científica nomeou de folksonomias, [...] que podem ser definidas como sistemas orgânicos baseados na
atribuição livre e pessoal de [termos] à informações ou objetos
visando à organização e recuperação. O neologismo folksonomia –
formado pelas palavras, em inglês, folks (pessoas) e taxonomy
(taxonomia) – foi cunhado [...] como forma de expressar
contraposição às classificações do conhecimento tradicionais,
elaboradas por especialistas e construídas baseando-se em arranjos
hierárquicos. (GUEDES; DIAS, 2010, p.48). No Flickr podemos identificar dois tipos de usuários: aqueles que
possuem cadastro na base e, portanto, podem publicar fotos e se utilizar de todas
as ferramentas que o Flickr oferece; e aqueles que não possuem qualquer
vínculo com o sistema, apenas o utilizam para fazer pesquisas no conjunto de
fotografias disponibilizadas para consulta pública. Ao primeiro tipo,
chamaremos de usuário regular; ao segundo, usaremos a definição usuário-
consultor. | 10 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015
doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital
E-ISSN 1808-5245 Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 E-ISSN 1808-5245 Apesar de ser descrito como um ambiente que utiliza
folksonomias (HIDDERLEY; RAFFERTY, 2007), na prática o Flickr não
permite que a indexação seja feita livremente pelo usuário-consultor, como
acontece na maioria desses ambientes. Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 Q
,
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doi: http://dx.doi.org/10.19132/1808-5245212.7-30 m Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015
i h
//d d i
/10 19132/1808 5245212 7 30 As tags são como palavras-chave ou títulos que você adiciona a uma
foto para facilitar encontrá-la posteriormente. Você pode criar uma
tag para uma foto com frases como "Catarina caminhada trilha
montanha Yosemite." Posteriormente, se você procurar por imagens
de Catarina, bastará clicar nessa tag e obter todas as fotos marcadas
dessa maneira. (FLICKR, 2014a, documento eletrônico sem
paginação). 2 Flickr: uma breve análise Tags, ao contrário de termos de indexação (descritores);
geralmente utilizados em unidades de informação, não possuem um mediador | 11 Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital
E-ISSN 1808-5245 Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital
E-ISSN 1808-5245 Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 que as controle. O Flickr diferencia-se de outros ambientes que utilizam
folksonomias por estabelecer que apenas o usuário regular possua tal liberdade
(embora alguns ambientes que empregam folksonomiastambém utilizem esse
recurso), provavelmente como uma forma de minimizar tags ‘inúteis’. O Flickr
também propõe uma lista de “hot tags”, ou “etiquetas quentes”, que consistem
numa lista das tags mais utilizadas pelos usuários regulares. Essas são,
possivelmente, estratégias desenvolvidas pelos administradores da base para
aprimorar as formas de indexação, ou tagging, das imagens. que as controle. O Flickr diferencia-se de outros ambientes que utilizam
folksonomias por estabelecer que apenas o usuário regular possua tal liberdade
(embora alguns ambientes que empregam folksonomiastambém utilizem esse
recurso), provavelmente como uma forma de minimizar tags ‘inúteis’. O Flickr
também propõe uma lista de “hot tags”, ou “etiquetas quentes”, que consistem
numa lista das tags mais utilizadas pelos usuários regulares. Essas são,
possivelmente, estratégias desenvolvidas pelos administradores da base para
aprimorar as formas de indexação, ou tagging, das imagens. Problemas no processo de indexação e as consequências na
recuperação das imagens foram identificados por alguns autores. Rafferty e
Hidderley (2007), numa pesquisa controlada, mostram como os índices de
revocação podem variar imensamente de acordo com o termo atribuído, quando
explicam que No exemplo mostrado, o termo WEDDING é considerado muito geral
pelos autores, recuperando um número de imagens considerado absurdo. Já o
termo IIJSSELMEER, segundo os autores, é específico demais para uma
imagem disponibilizada numa base para uso público, recuperando apenas uma
imagem. Analisar os resultados da recuperação, no entanto, não é suficiente para
deduzir que a causa do problema seja a forma como os usuários indexam as
imagens no Flickr. Percebemos, inclusive, que há um interesse por parte dos
próprios administradores em educar os usuários a controlar os termos utilizados. 2 Flickr: uma breve análise Apenas o usuário regular pode fazê-lo e,
no máximo, outros usuários regulares que receberam do proprietário das
imagens uma permissão para tanto. Esses poderão indexar as imagens
utilizando-se do recurso detagging (do inglês, etiquetagem), que consiste na
atribuição
de
termos
que
representem
os
conteúdos
informacionais
disponibilizados na web. As tags são como palavras-chave ou títulos que você adiciona a uma
foto para facilitar encontrá-la posteriormente. Você pode criar uma
tag para uma foto com frases como "Catarina caminhada trilha
montanha Yosemite." Posteriormente, se você procurar por imagens
de Catarina, bastará clicar nessa tag e obter todas as fotos marcadas
dessa maneira. (FLICKR, 2014a, documento eletrônico sem
paginação). As tags são como palavras-chave ou títulos que você adiciona a uma
foto para facilitar encontrá-la posteriormente. Você pode criar uma
tag para uma foto com frases como "Catarina caminhada trilha
montanha Yosemite." Posteriormente, se você procurar por imagens
de Catarina, bastará clicar nessa tag e obter todas as fotos marcadas
dessa maneira. (FLICKR, 2014a, documento eletrônico sem
paginação). As tags atribuídas a determinado conteúdo servirão como uma referência
conceitual aos usuários que terão acesso posterior àquela informação. No caso
do Flickr, o usuário regular disponibiliza a imagem na base e atribui, a partir da
sua interpretação, termos que representem a imagem. Em seguida, o usuário-
consultor, ou outros usuários regulares, poderão pesquisar as imagens
disponibilizadas através de palavras-chave e a base naturalmente utilizará as
tags atribuídas a cada imagem como ferramenta de busca. Alguns autores
preferem o termo indexação social para identificar esse processo. Cada imagem pode ter um máximo de 75 tagsatribuídas a ela. No
entanto, o usuário regular tem, além das tags, outra forma de descrever uma
imagem: o texto. Este pode conter, como sugere o próprio Flickr (2014a), a
história da fotografia e/ou notas explicativas sobre esta. É importante ressaltar
que o Flickr não se utiliza exclusivamente das tags atribuídas às imagens para
fazer a recuperação, mas também de termos livres contidos nas descrições. Na
hora da busca, o usuário pode escolher entre pesquisar apenas nas tags, ou por
meio de termos livres, incluindo assim os textos. A […] tag […] “casamento” recuperou 795.280 imagens, quando
utilizada no Flickr em 14 de fevereiro de 2006, sugerindo que este
seria um exemplo de uma tag talvez ampla demais. A […] tag
“Iijsselmeer” recuperou apenas uma imagem, e é um exemplo de
uma tag que talvez seja específica demais para o propósito de
pesquisa pública. (RAFFERTY; HIDDERLEY, 2007, p. 403,
tradução nossa)1. 2 Flickr: uma breve análise É possível que, apesar de os índices de recuperação no Flickr não terem sido
considerados satisfatórios pelos autores, os termos utilizados pelos usuários na
atribuição de tags representem de forma efetiva os elementos apresentados na
imagem. | 12 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015
doi: http://dx doi org/10 19132/1808 5245212 7 30 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015
doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital
E-ISSN 1808-5245 Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 E-ISSN 1808-5245 Partindo desse impasse, coletamos algumas imagens no Flickr
para analisá- las com base na metodologia proposta por Rodrigues (2007),
anteriormente considerada a mais adequada para o ambiente web dentre as
metodologias para representação imagética encontradas, e comparar os
resultados obtidos com as tags atribuídas pelos usuários. Assim, é possível
perceber se a indexação de imagens que está sendo realizada por meio das
ferramentas da Web 2.0 condiz qualitativamente com aquela indexação proposta
pelos estudiosos da área. [...]constitui o menos rigoroso de todos os tipos de amostragem. Por
isso mesmo é destituída de qualquer rigor estatístico. O pesquisador
seleciona os elementos a que tem acesso, admitindo que estes
possam, de alguma forma, representar o universo. Aplica-se este
tipo de amostragem em estudos exploratórios ou qualitativos, onde
não é requerido elevado nível de precisão. (GIL, 2008, p. 94, grifo
nosso). Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015
doi: http://dx.doi.org/10.19132/1808-5245212.7-30 3 Procedimentos metodológicos Entendemos este estudo como sendo de caráter qualitativo, já que não buscamos
uma coleta precisa de dados estatísticos, mas uma análise interpretativa, baseada
essencialmente no que a literatura científica diz a respeito do tema. Através
deste estudo, buscamos compreender um pouco mais sobre as modalidades de
indexação imagética. O Flickr possui uma quantidade de fotos armazenadas que cresce de
forma rápida e significativa. Dessa forma, percebemos de imediato que o Flickr
constitui um universo de amplitude infinita. Sendo esta pesquisa de caráter qualitativo, recorremos a Gil (2008) para
obter alguns esclarecimentos a esse respeito. O autor explica, dentre outras
formas de coletar amostras, que a amostragem por acessibilidade ou por
conveniência [...]constitui o menos rigoroso de todos os tipos de amostragem. Por
isso mesmo é destituída de qualquer rigor estatístico. O pesquisador
seleciona os elementos a que tem acesso, admitindo que estes
possam, de alguma forma, representar o universo. Aplica-se este
tipo de amostragem em estudos exploratórios ou qualitativos, onde
não é requerido elevado nível de precisão. (GIL, 2008, p. 94, grifo
nosso). [...]constitui o menos rigoroso de todos os tipos de amostragem. Por
isso mesmo é destituída de qualquer rigor estatístico. O pesquisador
seleciona os elementos a que tem acesso, admitindo que estes
possam, de alguma forma, representar o universo. Aplica-se este
tipo de amostragem em estudos exploratórios ou qualitativos, onde
não é requerido elevado nível de precisão. (GIL, 2008, p. 94, grifo
nosso). Dessa forma, buscamos tornar possível a análise da metodologia em
ambiente web,mesmo admitindo a infinidade de imagens que podem ser
encontradas nesse contexto.Devemos destacar, antes de explanar os | 13 Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital
E-ISSN 1808-5245 Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital
E-ISSN 1808-5245 Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 procedimentos, que, visando delimitar o campo de estudo, fica estabelecido que
apenas as tags atribuídas na língua portuguesa serão consideradas na análise. Tags em quaisquer outras línguas serão desconsideradas, assim como
aquelasque apresentem composição confusa devido a possíveis erros de grafia. Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015
doi: http://dx.doi.org/10.19132/1808-5245212.7-30 3 Procedimentos metodológicos Em vista disso, foram realizados os seguintes procedimentos para
a coleta das imagens utilizadas neste estudo: a) optamos por fazer a pesquisa como usuário consultor, uma vez que,
desta forma, o cadastro na base não interfere significativamente na
busca; b) na página inicial do Flickr, utilizamos os termos TAGS (para realizar a
pesquisa inicial de imagens, por se tratar de um termo bastante amplo, de
certa forma desprovido de qualquer conceituação, e, como já visto, muito
utilizado na base); e BRASIL (para delimitar ao máximo as imagens
recuperadas à imagens com tags em língua portuguesa). Dessa forma, as
imagens foram recuperadas de forma aleatória no que diz respeito à sua
temática; c) após a pesquisa inicial, a base dá a possibilidade de escolha entre a
pesquisa em todos os textos atribuídos a uma imagem ou apenas em suas
tags. Visando uma maior generalidade de imagens recuperadas, optamos
por permanecer na busca realizada em todos os textos; d) tendo em vista uma maior aleatoriedade na coleta, foi selecionada uma
imagem de cada página dos resultados de busca, sendo analisadas quatro
imagens ao final; e) buscando uma conceituação diferenciada e equilibrada nas imagens
coletadas, duas dessas foram selecionadas por conterem seres humanos,
e as duas restantes por não conterem humanos representados. Coletados os dados, aplicamos a cada imagem a metodologia proposta
por Rodrigues (2007) e, em seguida, comparamos os resultados com as tags
atribuídas, visando analisar qualitativamente a indexação realizada pelos
usuários. | 14 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015
doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital
E-ISSN 1808-5245 Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital 4 Resultados obtidos Utilizando os procedimentos metodológicos estabelecidos para a coleta e análise
das imagens no Flickr, obtivemos as seguintes imagens: Figura 1 – Praia de Jurerê – Florianópolis, SC
Fonte: Flickr(2014b). Figura 1 – Praia de Jurerê – Florianópolis, SC Figura 1 – Praia de Jurerê – Florianópolis, SC Fonte: Flickr(2014b). Na área reservada para o texto descritivo da imagem, o autor indica que
esta imagem refere-se à Praia de Jurerê, localizada em Florianópolis, estado de
Santa Catarina. As tagsatribuídas a esta imagem foram, em ordem de aparição: Quadro 1 – Tags atribuídas à Figura 1 por usuário da redeFlickr
Rainbow
top20travel
Arco-íris
A Plus Photo
Jurerê
The World Throgh a Lens
Florianópolis
Platinum Photo
Santa Catarina
Geo-tagged
Brasil
100v+10f
Brazil
Novas Jurerê
Fonte: elaborado pelos autores Aplicando a metodologia de Rodrigues (2007), chegamos à seguinte
análise: | 15 | 15 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015
doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 Quadro 2 – Aplicação da metodologia de Rodrigues (2007) para a Figura 1
Fonte: elaborado pelos autores. DESCRIÇÃO FÍSICA
COMPOSIÇÃO
CONTEXTO ARQUIVÍSTICO
---
---
Jurerê
Florianópolis
Santa Catarina
SENTIDO DENOTATIVO
SENTIDOS CONOTATIVOS
TEMATIZAÇÃO
Praia
Arco-íris
Chuva
Tranquilidade
Meteorologia Através da metodologia de Rodrigues (2007), atribuímos oito termos que
poderiam recuperar essa imagem. No Contexto Arquivístico, os termos
JURERÊ, FLORIANÓPOLIS e SANTA CATARINA referem-se à procedência
da imagem, ou seja, ao local apresentado na fotografia, e, consequentemente,
onde esta foi produzida. No Sentido Denotativo, os termos PRAIA, ARCO-ÍRIS
e CHUVA representam os objetos identificados pelos autores, enquanto nos
Sentidos Conotativos o termo TRANQUILIDADE expressa a sensação
transmitidaaos autores no momento da observação. Entretanto, por se tratar de
um elemento essencialmente subjetivo, outros termos podem ser atribuídos por
observadores diferentes. Por fim, no campo Tematização, o termo
METEOROLOGIA reproduz um enquadramento temático feito pelos autores
diante dos elementos objetivos apresentados. Apesar desse enquadramento
temático ser subjetivo, é mais fácil apontar os elementos que levaram a tal
determinação, que, no caso da fotografia em questão, os elementos que levaram
à atribuição das tags PRAIA e CHUVA. Entretanto, da mesma forma que os
Sentidos Conotativos, a Tematização pode variar de um observador para outro. Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015
doi: http://dx.doi.org/10.19132/1808-5245212.7-30 4 Resultados obtidos Como ficou claro nas tags associadas a essa imagem, alguns
termos não correspondem à língua portuguesa, enquanto outros apresentam
composição confusa e, portanto, deverão ser desconsiderados. Traçando um
comparativo entre as tagsválidas e os termos adquiridos por meio da
metodologia, temos: | 16 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015
doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 Quadro 3 – Comparação entre as tagsconsideradas válidas e os resultados obtidos na análise
TAGS
TERMOS
Arco-íris
Jurerê
Jurerê
Florianópolis
Florianópolis
Santa Catarina
Santa Catarina
Praia
Brasil
Arco-íris
Novas Jurerê
Chuva
---
Tranquilidade
---
Meteorologia
Fonte: elaborado pelos autores Dentre as tags consideradas válidas para esta pesquisa, apenas quatro
coincidem com os termos adquiridos através da metodologia. Termos
considerados indispensáveis na indexação desta imagem, como PRAIA, não
aparecem nas tags. A tag BRASIL, por sua vez, apesar de corresponder a um
dos critérios de busca para a presente pesquisa e ser relevante para situar a
localização do ambiente apresentado na ilustração, é excessivamente genérica
para descrever, por si só, essa imagem, enquanto NOVAS JURERÊ parece ser
uma indicação individual de localização para o próprio autor da fotografia, não
podendo ser considerado relevante para descrevê-la. Avançando para a próxima página de busca, coletamos a seguinte
imagem: | 17 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015
doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital
E-ISSN 1808-5245
Figura 2 – Maragogi, AL
Fonte: Flickr (2014b). Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital
E-ISSN 1808-5245 Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital Figura 2 – Maragogi, AL
Fonte: Flickr (2014b). Figura 2 – Maragogi, AL Fonte: Flickr (2014b). Nesta imagem é possível identificar um grupo de jovens observando o
mar. Segundo as informações do autor da fotografia, ela foi tirada na Praia de
Peroba, em Maragogi, Estado de Alagoas. Diversas tags referenciam ess
imagem, como as listadas a seguir: | 18 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015
doi: http://dx.doi.org/10.19132/1808-5245212.7-30 4 Resultados obtidos 2015
doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 Quadro 4 – Tags atribuídas à Figura 2por usuário da rede Flickr Quadro 4 – Tags atribuídas à Figura 2por usuário da rede Flickr
Colors
Miramar
Caribe
Color
Boa viagem
Brasileiro
Maragogi
Contraste
O Caribe brasileiro
Alagoas
Cores
Viagem
Peroba
Fortes
Dicas
Praia
Vibrantes
Ficar
Beach
Viva
Salinas
Red
Paraíso
Férias
Yellow
Paradise
Piscinas
Mar
Geotagged
Naturais
Céu
Beaches
Maceió
Azul
Ondas
Turismo
Nuvens
Rio
Brasilviagem
Pentax
Lago
Pousada
Ist
Vivid
Hotéis
D
Praias
Brasil azul
17mm
Maragogi fotos
Pousadas em Maragogi
Fisheye
Fotos de Maragogi
Miramar Maragogi
Brasil
Praia de Peroba
Praia de Maragogi
Brazil
Maragogi online
Porto de Galinhas
Postal
Aventura
Japaratinga
Tamandaré
Galés
Catamarã
Piscinas naturais de Maragogi,
Alagoas
Piscinas naturais de Maragogi
Passeio
Hotel Salinas
Fotos Maragogi
Referência no Turismo
Fonte: elaborado pelos autores. Um olhar não muito atento é o suficiente para perceber que muitas
palavras se repetem em tags diferentes; ainda, percebemos que palavras que
deveriam formar uma única tag acabam tornando-se tags distintas, como, por
exemplo, CARIBE e BRASILEIRO, seguidas da
tag
O CARIBE
BRASILEIRO. Aplicando a metodologia de Rodrigues (2007) obtemos: | 19 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015
doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 Quadro 5–Aplicaçãoda metodologia de Rodrigues (2007) para a Figura 2
Fonte: elaborado pelos autores. Fonte: elaborado pelos os autores. 4 Resultados obtidos DESCRIÇÃO FÍSICA
COMPOSIÇÃO
CONTEXTO ARQUIVÍSTICO
---
Azul
Azul turquesa
Peroba
Maragogi
Alagoas
SENTIDO DENOTATIVO
SENTIDOS CONOTATIVOS
TEMATIZAÇÃO
Praia
Mar
Céu azul
Jovens
Curiosidade
Aventura
Férias
Turismo
Pontos turísticos Na tabela comparativa entre as tags consideradas válidas e os
termos obtidos teremos: Quadro 6–Comparaçãoentre as tags consideradas válidas e os resultados obtidos na análise
TAGS
TERMOS
Maragogi
Azul
Alagoas
Azul turquesa
Peroba
Peroba
Praia
Maragogi
Mar
Alagoas
Céu
Praia
Azul
Mar
Nuvens
Céu azul
Contraste
Jovens
Cores
Curiosidade
Fortes
Aventura
Vibrantes
Férias
Viva
Turismo
Paraíso
Pontos turísticos
TAGS
TERMOS
Brasil
---
Postal
---
Ondas
---
Rio
---
Lago
---
Praias
---
Fotos
--- | 20 Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 E-ISSN 1808-5245
Maragogi
---
Fotos de Maragogi
---
Praia
---
Peroba
---
Caribe
---
Brasileiro
---
O Caribe brasileiro
---
Pousada
---
Hotéis
---
Viagem
---
Dicas
---
Ficar
---
Salinas
---
Boa viagem
---
Brasilviagem
---
Aventura
---
Férias
---
Piscinas
---
Naturais
---
Maceió
---
Turismo
---
Brasil azul
---
Pousadas em Maragogi
---
Praia de Maragogi
---
Porto de Galinhas
---
Japaratinga
---
TAGS
TERMOS
Tamandaré
---
Piscinas naturais de Maragogi, Alagoas
---
Piscinas naturais de Maragogi
---
Referência no Turismo
---
Hotel Salinas
---
Catamarã
---
Galés
---
Passeio
---
Fotos Maragogi
---
Fonte: elaborado pelos os autores. | 21 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015
doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital
E-ISSN 1808-5245 Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 E-ISSN 1808-5245 Com a metodologia de Rodrigues (2007) atribuímos 14 termos a essa
imagem, os quais abrangem desde a sua composição, com a cor que
notavelmente se destaca, até a sua tematização. Acreditamos ser possível, com
esses termos, descrever eficientemente a fotografia e realizar uma recuperação
eficiente. As tags consideradas válidas somam 56, dentre as quais oito
coincidem com os termos atribuídos. 56poderia ser considerada uma quantidade
de termos elevada, especialmente quando comparada com o resultado obtido
através da metodologia de Rodrigues (2007). 4 Resultados obtidos Entretanto, é preciso levar em
conta os aspectos conotativos e toda a gama de possibilidades de interpretação
que a imagem pode despertar ao observador, de modo que, dependendo do
contexto em que o documento seja analisado e representado, um número grande
de termos pode ser considerado ideal.No entanto, é necessário estar atento a
equívocos na interpretação que poderiam levar a conceituações distantes e
passíveis de confusão com outras imagens.Algumas tagsatribuídas à imagem em
questão podem exemplificar isso, como é o caso de PORTO DE GALINHAS ou
TAMANDARÉ, que são praias localizadas no estado de Pernambuco. Ainda, a
tag MACEIÓ refere-se à capital do estado de Alagoas, que não possui relação
direta com a fotografia apresentada, o que poderia provocar confusão no
momento da busca, visto que esse termo não corresponde corretamente à
procedência da fotografia. Na terceira página de busca encontramos a seguinte imagem: Figura 3 – Ponte de madeira – Itapuã, RS
Fonte: Flickr (2014b). Figura 3 – Ponte de madeira – Itapuã, RS Fonte: Flickr (2014b). | 22 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015
doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital
E-ISSN 1808-5245 Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 Nesta imagem temos apenas um panorama, sem a presença de seres
humanos. Segundo a descrição do autor, esta fotografia representa uma região
da cidade de Itapuã, no Estado do Rio Grande do Sul. As tags atribuídas pelo
autor foram: Quadro 7 – Tags atribuídas à Figura 3 por usuário da rede Flickr
Itapuã
PB
Bem Flickr... Bem Brasil! RS
Noiretblanc
Gutemberg Ostemberg
Interior
Bianco
Gutemberg
Gaúcho
Nero
Porto Alegre
Guaíba
Blanco
Mywinners
Rio Grande do Sul
Negro
Bem Flickr... Bem Brasil! BW
Brazil
---
Fonte: elaborado pelos autores. Mais uma vez percebemos, em INTERIOR e GAÚCHO, palavras que
provavelmente deveriam formar uma única tag, mas acabam tornando-se
tagsdistintas. Aplicando a metodologia de Rodrigues (2007) obtemos: Quadro 8–Aplicaçãoda metodologia de Rodrigues (2007) para a Figura 3
Fonte: elaborado pelos autores. DESCRIÇÃO FÍSICA
COMPOSIÇÃO
CONTEXTO ARQUIVÍSTICO
---
Preto e branco
Itapuã
Guaíba
Rio Grande do Sul
SENTIDO DENOTATIVO
SENTIDOS CONOTATIVOS
TEMATIZAÇÃO
Ponte de madeira
Lago
Dique
Imensidão
Infinito
Solidão
Preservação ambiental
Ecossistema Fonte: elaborado pelos autores. Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015
doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 Em Questão, Porto Alegre, v. 21, n. 2, p. 7 30, mai/ago. 2015
doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015
doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015
doi: http://dx.doi.org/10.19132/1808-5245212.7-30 4 Resultados obtidos Adotamos 12 termos para representar esta imagem, que buscam englobar
tanto o que a imagem mostra, o que ela transmiteaos autores, e os contextos em
que pode estar inserida de acordo com a história do local. Itapuã é o nome de
uma reserva florestal localizada no município de Guaíba, Rio Grande do Sul. Percebe-se, assim, que alguns destes termos só puderam ser atribuídos porque o
autor informa através dos textos onde a fotografia foi tirada. A partir daí o
indexador pode investigar o que mais pode ser relevante na sua conceituação,
indo além do que a imagem revela. É importante destacar que Lancaster (2004) | 23 Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital
E-ISSN 1808-5245 Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 prevê isso em seus estudos, quando aborda as questões relacionadas à indexação
de imagens. No quadro comparativo teremos: Quadro 9–Comparaçãoentre as tags consideradas válidas e os resultados obtidos na análise
Fonte: elaborado pelos autores. Fonte: elaborado pelos autores. TAGS
TERMOS
Itapuã
Preto e branco
Interior
Itapuã
Gaúcho
Rio Grande do Sul
Guaíba
Ponte de madeira
Rio Grande do Sul
Lago
Negro
Dique
Porto Alegre
Imensidão
---
Infinito
---
Solidão
---
Guaíba
---
Preservação ambiental
---
Ecossistema Dentre as sete tags relacionadas, apenas três coincidem com os termos
atribuídos
pela
metodologia
de
Rodrigues. Identificamos
umasituaçãosemelhante à encontrada na imagem analisada anteriormente,
quando se adotou a tag PORTO ALEGRE, referente à capital do Estado do Rio
Grande doSul, auma imagem que representa uma localidade situada no
município de Guaíba, também no Rio Grande do Sul. No entanto, nesse caso é
possível afirmar que, apesar de não haver uma relação direta entre a tag e a
localidade apresentada, visto que são municípios diferentes, ainda assim seria
admissível reconhecer uma relação relevante, já que o município de Guaíba fica
localizado na Região Metropolitana de Porto Alegre. Até agora analisamos três imagens, sendo que em duas delas não
aparecem seres humanos. Cumprindo os procedimentos metodológicos
estabelecidos, a última imagem a ser analisada deve necessariamente conter um
ser humano. Na quarta página dos resultados de busca encontramos a seguinte
imagem: | 24 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 4 Resultados obtidos 2015
doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital
E-ISSN 1808-5245 Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital Figura 4 – Chapada dos Veadeiros, GO
Fonte: Flickr(2014b). Figura 4 – Chapada dos Veadeiros, GO Fonte: Flickr(2014b). Na imagem há apenas um rapaz com a sua câmera fotográfica em meio a
uma imensa paisagem verde. Segundo os textos relacionados à fotografia, ela foi
tirada na Chapada dos Veadeiros, localizada no estado de Goiás. As tags
atribuídas a essa imagem, diferentemente das outras analisadas, não foram
muitas, como relacionado abaixo: Quadro 10 – Tags atribuídas à Figura 4 por usuário da rede Flickr
Chapada dos Veadeiros
Eu
Vale da Lua
Borguetti
Alto Paraíso
Top-v1111
Me
---
Fonte: elaborada pelos autores. Com a metodologia de Rodrigues (2007), obtemos os seguintes
resultados: | 25 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015
doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 Quadro 11–Aplicaçãoda metodologia de Rodrigues (2007) para a Figura 4 Quadro 11–Aplicaçãoda metodologia de Rodrigues (2007) para a Figura 4
Fonte: os autores
DESCRIÇÃO FÍSICA
COMPOSIÇÃO
CONTEXTO ARQUIVÍSTICO
---
Verde
Chapada dos Veadeiros
Goiás
SENTIDO DENOTATIVO
SENTIDOS CONOTATIVOS
TEMATIZAÇÃO
Floresta
Homem
Aventura
Pontos turísticos
Ecossistema Na tabela comparativa, considerando os critérios determinados
anteriormente para a validade das tags, teremos: Quadro 12–Comparaçãoentre as tagsconsideradas válidas e os resultados obtidos na análise
TAGS
TERMOS
Vale da Lua
Verde
Alto Paraíso
Chapada dos Veadeiros
Eu
Goiás
Chapada dos Veadeiros
Floresta
---
Homem
---
Aventura
---
Liberdade
---
Pontos turísticos
---
Ecossistema
Fonte: elaborado pelos autores. Quadro 12–Comparaçãoentre as tagsconsideradas válidas e os resultados obtidos na análise Quadro 12–Comparaçãoentre as tagsconsideradas válidas e os resultados obtidos na análise A Chapada dos Veadeiros é uma vasta região que abrange diversos
municípios, dentre eles Cavalcante, Alto Paraíso de Goiás e Teresina de Goiás. Possivelmente por isso o autor atribuiu a tag ALTO PARAÍSO, referindo-se ao
município em que, talvez, localizava-se no momento da fotografia. No entanto,
sendo este termo específico demais, não foi considerado na sua análise. Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 doi: http://dx.doi.org/10.19132/1808-5245212.7-30 4 Resultados obtidos Da
mesma forma, VALE DA LUA refere-se a uma região específica da Chapada
dos Veadeiros, mas que não é possível identificar nem na descrição textual, nem
na imagem em si. Assim, consideramos apenas o termo genérico CHAPADA
DOS VEADEIROS. Nesse sentido, apenas uma tag coincidiu com os termos
atribuídos na análise, que foram nove no total. Sobre a análise qualitativa de dados, podemos fazer uma breve
comparação entre os termos e as tags, e verificar como os dados que obtivemos | 26 Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital
E-ISSN 1808-5245 Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital
E-ISSN 1808-5245 Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 nas análises se comportam. Todas as tagsatribuídas às quatro imagens analisadas
e consideradas válidas somam o total de 83, enquanto os termos adquiridos por
meio da metodologia de Rodrigues (2007) somam 43. Do total de tags, apenas
16 coincidem com o total de termos, o que resulta em aproximadamente 39,02%
de compatibilidade entre as tags e os termos. Além disso, analisando as tabelas comparativas podemos
perceber que, em geral, os termos que coincidiram foram apenas os que dizem
respeito à localização geográfica do ponto em que a fotografia foi tirada, o que
não garante uma riqueza na conceituação por meio das tags. Observamos neste estudo, também, que é essencial para o
indexador identificar e considerar os diversos aspectos que uma imagem pode
conter. Durante as análises, percebemos que as imagens coletadas possuem a
maior parte das características propostas por Rodrigues (2007) como sendo as
mais relevantes. No entanto, à exceção dos termos PASSEIO, FÉRIAS,
PARAÍSO, TURISMO e REFERÊNCIAS NO TURISMO (Figura 2), que
remetem à tematização da imagem, não encontramos nenhuma tag que
correspondesse a elementos conotativos ou temáticos, e poucas as tags
correspondentes a elementos denotativos. Entende-se, assim, que não há um
equilíbrio entre os tipos de elementos descritos, sendo esses predominantemente
relativos à sua composição e ao seu contexto arquivístico (RODRIGUES, 2007). Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 Q
,
g
,
,
, p
,
/ g
doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Q
,
g
,
,
, p
,
/ g
doi: http://dx.doi.org/10.19132/1808-5245212.7-30 5 Considerações finais Apesar da baixa qualidade identificada na indexação realizada por meio das
tags, o valor da indexação social não se perde. O fato de existirem ferramentas
capazes de permitir esse tipo de atividade representa um avanço significativo na
forma como os novos profissionais da informação devem encarar a
representação da informação, seja ela imagética ou não. Alguns estudos
inclusive já apontam para propostas de metodologias para a atribuição das tags
(HIDDERLEY; RAFFERTY, 2007). Isso remonta, de alguma forma, aos
estudos iniciais de Panofsky (1979) sobre a conceituação de imagens. Os
conceitos iniciais evoluíram de tal forma que hoje somos capazes de analisar | 27 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 Q
g
p
g
doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital
E-ISSN 1808-5245 Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 minuciosamente imagens disponibilizadas em ambiente web, algo impensável
algumas décadas atrás. É possível que, em alguma medida, estejamos no mesmo
marco inicial em que se encontrava Panofsky (1979) quando iniciou o estudo
sobre essas questões. Ao lado de grandes ferramentas da Web 2.0, como a Wikipedia, por
exemplo, o Flickr faz cada vez mais parte do cotidiano de nossa sociedade. No
entanto, as possibilidades advindas dessa evolução e a velocidade com que essa
ocorreu e ainda ocorre requerem dos profissionais estudos frequentes para
compreender a usabilidade e os potencializar os mecanismos dessas novas
ferramentas. A indexação de imagens é uma atividade complexa e altamente passível
a interpretações e questionamentos diferenciados. Devido a isso, foi necessário
fazer um estudo aprofundado sobre o tema através da literatura existente, para
entender realmente a problemática e buscar um embasamento científico para
analisar qualitativamente as imagens. Buscamos, a partir desse estudo, criar uma reflexão em torno das
problemáticas que estão surgindo no ambiente de tratamento, organização e
disseminação da informação. Por mais que diversos estudos proponham formas
de tratamento de imagens, é preciso estar atento à eficiência desses métodos em
ambientes que não sejam restritos a bibliotecas e centros de documentação. FLICKR. Ajuda: tags. 2014a. Disponível em: FLICKR. Ajuda: tags. 2014a. Disponível em:
<http://www.flickr.com/help/tags/#37>. Acesso em: 15 out. 2014. <http://www.flickr.com/help/tags/#37>. Acesso em: 15 out. 2014. PANOFSKY, E. Significado nas Artes Visuais. 2. ed. São Paulo: Perspectiva,
1979. RODRIGUES, Ricardo Crisafulli. Análise e tematização da imagem fotográfica. Ciência da Informação, Brasília, v. 36, n. 3, p. 67-76, set./dez. 2007. Q
g
p
g
doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Referências Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015
d i htt
//d d i
/10 19132/1808 5245212 7 30
FLICKR. Ajuda: tags. 2014a. Disponível em:
<http://www.flickr.com/help/tags/#37>. Acesso em: 15 out. 2014. FLICKR. Busca de imagens.2014b. Disponível em:
<http://www.flickr.com/search>. Acesso em: 15 out. 2014. FLICKR. Sobre o Flickr. 2014c. Disponível em:
<http://www.flickr.com/about>. Acesso em: 15 out. 2014. GIL, Antônio Carlos. Métodos e técnicas de pesquisa social. 6. ed.São Paulo:
Atlas, 2008. FLICKR. Ajuda: tags. 2014a. Disponível em: <http://www.flickr.com/help/tags/#37>. Acesso em: 15 out. 2014. FLICKR. Busca de imagens.2014b. Disponível em:
<http://www.flickr.com/search>. Acesso em: 15 out. 2014. FLICKR. Sobre o Flickr. 2014c. Disponível em:
<http://www.flickr.com/about>. Acesso em: 15 out. 2014. GIL, Antônio Carlos. Métodos e técnicas de pesquisa social. 6. ed.São Paulo:
Atlas, 2008. | 28 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015
doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 GUEDES, Roger de Miranda; DIAS, Eduardo José Wense. Indexação social:
abordagem conceitual. Revista ACB: Biblioteconomia em Santa Catarina,
Florianópolis, v. 15, n. 1, p.39-53, jan./jun. 2010. HIDDERLEY, R.; RAFFERTY, P. Flickr and democratic indexing: dialogic
approaches to indexing. Aslib Proceedings, London, v. 59, n. 4/5, p. 397-410,
2007. Disponível em: <www.emeraldinsight.com/0001-253X.htm>. Acesso em:
11 jan. 2012. LANCASTER, F. W. Indexação e resumos: teoria e prática. 2. ed. Brasília:
Briquet de Lemos, 2004. MANINI, Miriam Paula. Análise documentária de fotografias: um referencial
de leitura de imagens fotográficas Para fins documentários. São Paulo, 2002. Tese (Doutorado em Ciências da Comunicação) - Departamento de
Biblioteconomia e Documentação, Escola de Comunicações e Artes,
Universidade de São Paulo, São Paulo, 2002. OLIVEIRA, Rafael Alves de. Metodologias para indexação de imagens
fotográficas em ambiente web. In: ENCONTRO DE ESTUDOS SOBRE
TECNOLOGIA, CIENCIA E GESTÃO DA INFORMAÇÃO. Anais.... Recife:
–Universidade Federal de Pernambuco, 2011. 1 CD-ROM. . OLIVEIRA, Rafael Alves de. Metodologias para indexação de imagens
fotográficas em ambiente web. In: ENCONTRO DE ESTUDOS SOBRE
TECNOLOGIA, CIENCIA E GESTÃO DA INFORMAÇÃO. Anais.... Recife:
–Universidade Federal de Pernambuco, 2011. 1 CD-ROM. . PANOFSKY, E. Significado nas Artes Visuais. 2. ed. São Paulo: Perspectiva,
1979. Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015 Em Questão, Porto Alegre, v. 21, n. 2, p. 7 30, mai/ago. 2015
doi: http://dx.doi.org/10.19132/1808-5245212.7-30 Analysis and index of images on Flickr network Abstract: This article presents a brief overview on the dissemination and
democratization of the collaborative web, and how this process popularized the
use of the image in digital form. A brief analysis of the Flickr social network,
identifying their goals, tools, and some studies on its usability in the context of
information science. Sets out the approach to the images stored on the network,
proposing a qualitative study about the free indexing performed by users. Applies the methodology established in a previous study as the most appropriate | 29 Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital
E-ISSN 1808-5245 Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital Análise e indexação de imagens na rede Flickr
Rafael Alves de Oliveira, Luciane Paula Vital E-ISSN 1808-5245 for indexing images in a web environment, comparing the results obtained with
free indexing performed by users. Keywords: Image. Indexing of images. Flickr. Social networks. Social
indexing. Recebido: 16/10/2014
Aceito: 01/04/2015 Recebido: 16/10/2014
Aceito: 01/04/2015 1No original: “The [...] tag [...] “wedding” retrieved 795.280 images, when it was used on Flickr
on 14 February 2006, which suggests that this an example of a tag that is perhaps too broad. The […] tag “Iijsselmeer” only retrieved one image and is an example of a tag that is perhaps
too specific for public searching purposes”. 1No original: “The [...] tag [...] “wedding” retrieved 795.280 images, when it was used on Flickr
on 14 February 2006, which suggests that this an example of a tag that is perhaps too broad. The […] tag “Iijsselmeer” only retrieved one image and is an example of a tag that is perhaps
too specific for public searching purposes”. | 30 Em Questão, Porto Alegre, v. 21, n. 2, p. 7-30, mai/ago. 2015
doi: http://dx.doi.org/10.19132/1808-5245212.7-30
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W3097783670.txt
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https://www.mdpi.com/2309-608X/6/4/255/pdf?version=1606629039
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en
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Comparative Genomic Analysis of Dactylonectria torresensis Strains from Grapevine, Soil and Weed Highlights Potential Mechanisms in Pathogenicity and Endophytic Lifestyle
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Journal of Fungi
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cc-by
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Journal of
Fungi
Article
Comparative Genomic Analysis of
Dactylonectria torresensis Strains from Grapevine,
Soil and Weed Highlights Potential Mechanisms in
Pathogenicity and Endophytic Lifestyle
David Gramaje 1, * , Carmen Berlanas 1 , María del Pilar Martínez-Diz 2,3 , Emilia Diaz-Losada 2 ,
Livio Antonielli 4 , Sabrina Beier 4 , Markus Gorfer 4 , Monika Schmoll 4 and Stéphane Compant 4
1
2
3
4
*
Instituto de Ciencias de la Vid y del Vino (ICVV), Consejo Superior de Investigaciones Científicas,
Universidad de la Rioja, Gobierno de La Rioja, Ctra. LO-20 Salida 13, 26007 Logroño, Spain;
carmen.berlanas@icvv.es
Estación de Viticultura e Enoloxía de Galicia (AGACAL-EVEGA), Ponte San Clodio s/n,
32427 Ourense, Spain; pilar.martinez.diz@xunta.gal (M.d.P.M.-D.); emilia.diaz.losada@xunta.gal (E.D.-L.)
Facultade de Ciencias, Universidade da Coruña, Zapateira, 15071 A Coruña, Spain
Bioresources Unit, Center for Health & Bioresources, AIT Austrian Institute of Technology GmbH, Konrad
Lorenz Straße 24, 3430 Tulln, Austria; Livio.Antonielli@ait.ac.at (L.A.); sabrina.beier@ait.ac.at (S.B.);
Markus.Gorfer@ait.ac.at (M.G.); Monika.Schmoll@ait.ac.at (M.S.); Stephane.Compant@ait.ac.at (S.C.)
Correspondence: david.gramaje@icvv.es
Received: 29 September 2020; Accepted: 27 October 2020; Published: 29 October 2020
Abstract: The soil-borne fungus Dactylonectria torresensis is the most common causal agent of black-foot
disease in Europe. However, there is a lack of understanding on how this fungus can provoke plant
symptoms. In this study, we sequenced, annotated and analyzed the genomes of three isolates of
D. torresensis collected from asymptomatic vine, weed and soil. Sequenced genomes were further
compared to those of 27 fungal species including root and aerial pathogens, white rot degraders,
indoor biodeterioration agents, saprotrophs, dark septate endophytes and mycorrhiza. Strains of
D. torresensis present genomes with between 64 and 65 Mbp and with up to 18,548 predicted genes for
each strain. Average Nucleotide Identity (ANI) shows that strains are different according to genome
contents. Clusters of orthologous groups were compared, and clusters of genes related to necroses
were particularly detected in all strains of D. torresensis (necrosis inducing peptides and proteins,
and ethylene inducing peptides) as well as several genes involved in resistance against fungicides
frequently used in viticulture such as copper. Interestingly, an expanded high number of genes
related to carbohydrate-active enzymes were detected in each Dactylonectria strain, especially those
related to glycoside hydrolases that could be involved in penetration of plant tissues or pathogenicity.
An increased number of candidate genes for CAZyme classes AA9 and AA3-1 supports the ability
of strains to efficiently degrade plant material. High numbers of genes of D. torresensis related to
secretome and small secreted proteins were further characterized. Moreover, the presence of several
gene clusters such as fujikurin-like genes was detected and were normally found in Fusarium fujikuroi,
that have been linked to fungal pathogenicity. The phenotypes of the three strains investigated
showed further difference in light response. We found that Dactylonectria strains have an increased
number of photoreceptor encoding genes and we showed sequence alterations. Altogether, the results
highlight several gene clusters present in D. torresensis strains that could be linked to endophytic
lifestyle, pathogenicity, plant maceration and degradation of plant tissues as well as adaptation to soil
contaminated with metals and metalloids and light response.
Keywords: black-foot disease; high-throughput next generation sequencing; Vitis vinifera L.
J. Fungi 2020, 6, 255; doi:10.3390/jof6040255
www.mdpi.com/journal/jof
J. Fungi 2020, 6, 255
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1. Introduction
The soil-borne fungus Dactylonectria torresensis is the most common causal agent of black-foot
disease in Europe [1–3], one of the most important destructive diseases in grapevine (Vitis vinifera L.),
which has a devastating effect on grapevine production worldwide [4]. It is well known that D. torresensis
is common in the soil and causes infection of grafted vines after some months of growth in nursery
soils and in young vineyards, especially during the first five years after planting [1,2]. Young vines
affected by D. torresensis generally appear normal at planting but differences in vigour become marked
with reduced trunk growth, shortened internodes, and reduced foliage/canopy. Foliar symptoms
may appear as small leaves with interveinal chlorosis, followed by necrosis and early defoliation [5].
Removal of the rootstock bark of declining plants reveals further black discolouration and necrosis of
wood tissues that develop from the base of the rootstock. Below ground, symptoms include reduced
total root biomass, low numbers of feeder roots, and black, sunken and necrotic root lesions [4].
Although the disease cycle of D. torresensis on grapevines has not been specifically studied,
the behavior of Cylindrocarpon-like asexual morphs on other hosts [6,7] has indicated that conidia and
chlamydospores are likely to be produced on the diseased roots and stem bases of infected vines.
The conidia are apparently dispersed in soil water and the chlamydospores can allow the organism to
survive in the soil for a number of years [8]. Previous research reports have shown that contact between
these spores and the grapevine roots or callused stem bases results in high rates of infection [9,10].
Infection can occur through the small wounds made when roots on the callused cuttings break off
during the planting process or through the incomplete callusing of the basal ends of the cuttings [4].
Concerning Dactylonectria, an alternative that has been poorly addressed is that these fungi
associated with black-foot disease have a dual role: a pathogenic lifestyle on certain plants and
a non-pathogenic one on others. For instance, Agustí-Brisach et al. [11] reported isolation of
Dactylonectria macrodidyma complex, which comprehends Dactylonectria alcacerensis, D. estremocensis,
D. macrodidyma, D. novozelandica and D. torresensis, from 26 of 52 asymptomatic weed species growing in
propagation field nurseries and vineyards, with these strains being pathogenic to grapevine seedlings
in potted assays. Langenhoven et al. [12] isolated several black-foot disease fungi from asymptomatic
plants in South Africa, including grapevine, cereals and brasicaceous crops. Recently, Berlanas et al. [13]
reported the occurrence of 13 species associated with black-foot disease from the asymptomatic inner
tissues of surface sterilized secondary roots of grapevine grafted plants ready to be sold to growers
in Spain. The fact that plant pathogens can be non-pathogenic endophytes on other plants has
important implications, such as asymptomatic plants inadvertently serving as reservoirs of inoculum
and potentially initiating epidemics in other crops [14], or even serving as sources of hidden diversity
of plant-pathogenic species.
Regardless of biological, chemical, or cultural measures, no effective management strategies
for D. torresensis are currently available to avoid fungal infection and/or to eliminate this pathogen
once plants are infected [15]. Despite the importance and necessity of controlling black-foot disease,
the molecular mechanisms of pathogenesis in grapevine and other secondary hosts, and the genetic
basis for host specificity are still poorly understood. To date, most investigations into the nature of
host-specific adaptations have focused on differences between species of plant pathogens, while fewer
studies have been conducted to investigate and explain the intraspecific diversity of host-specific
adaptations. In addition, no genomic and transcriptomic studies have been conducted for D. torresensis
on grapevines although the genome sequence of its sister species D. macrodidyma was made public by
Malapi-Wight et al. [16].
Although single genome analysis facilitates better insights into the biology of a pathogen,
comparative analysis of multiple genomes can often reveal a significantly greater amount of information
on the physiology and evolution of a pathogen [17]. In this study, we analyzed the genomes of three
isolates of D. torresensis collected from asymptomatic vine and weed, and soil. The main objectives of
this study were to (i) identify the genomic characteristics of these fungi, (ii) understand the genetic
variation among the sequenced species, (iii) identify genes potentially involved in niche specialization
J. Fungi 2020, 6, 255
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within species, (iv) to identify fungal adaptations to the endophytic or pathogenic lifestyles, and (v) to
identify unique and shared genes and pathways related to virulence in D. torresensis.
2. Materials and Methods
2.1. Fungal Strains and Culture Collection
Fungal strains included in the study were isolated from the weed species Solanum nigrum (BV-745),
grapevine rootstock 110 Richter (BV-666) and soil samples (BV-349) collected in 2017 in a single
grapevine nursery field in Mendavia (Navarra, Spain) (Figure 1A). Isolation from weed and grapevine
were made from the asymptomatic endosphere tissue of roots. Sections of externally symptomless roots
(1–2 cm long and 1–3 mm diameter) were cut, washed under running tap water, surface disinfested
for 1 min in a 1.5% sodium hypochlorite solution, and washed twice with sterile distilled water.
The bark was carefully peeled out and the endosphere tissue was plated onto malt extract agar (MEA;
Biokar-Diagnostics, Zac de Ther, France) supplemented with streptomycin sulfate (MEAS) at 0.4 g.L–1
(Sigma-Aldrich, St. Louis, MO, USA). Isolation from soil samples were performed by plating them
onto the Glucose-Faba Bean Rose Bengal Agar (GFBRBA) semi-selective culture medium as described
by Berlanas et al. [2]. All isolates were single-spored in order to obtain pure cultures and stored in
filter paper at −20 ◦ C.
2.2. DNA Isolation and Fungal Identification
Fungal strains BV-349, BV-666 and BV-745 were grown on potato dextrose agar (PDA;
Conda Laboratories, Madrid, Spain) plates for 7 days at room temperature. Mycelium was scraped
from plates with the scalpel, transferred to the mortar and grinded with pestle in liquid nitrogen to get
fine powder. One hundred mg of powder was taken for the DNA isolation using DNeasy Plant Mini
Kit (Qiagen, Hilden, Germany) following manufacturer procedure as described by Berlanas et al. [13].
Obtained DNA was cleaned and concentrated using Amicon Ultra-0.5 mL Centrifugal Filters for DNA
and Protein Purification and Concentration with cut off 30 kDa (Millipore-Merck, Bedford, MA, USA).
The identification of black-foot pathogens was made by sequencing part of the histone H3
gene. PCR conditions and sequence analysis were performed according to Berlanas et al. [13].
Maximum Likelihood (ML) was performed on the individual gene alignment in MEGA v. 6 [18]
using the best fit model as estimated with the Bayesian information criterion in jModelTest 2.1.10 [19].
Branch support was calculated from 1000 bootstrap replicates for dataset. Campylocarpon fasciculare
(CBS 112613) and Ca. pseudofasciculare (CBS 112679) were used as outgroups in the phylogenetic analysis.
2.3. Genome Sequencing and Assembly
Fungal genomes were sequenced with Illumina NextSeq 500 using the V2 reagent kit (2 × 150 bp)
and generating a total of 50 million PE reads (LGC Genomics GmbH, Berlin). Illumina raw data
were checked with FastQC [20] and possible PhiX contaminant sequences removed with Bowtie2 [21].
The presence of adapters was checked, reads were quality filtered and trimmed using Trimmomatic
v.0.36 [22]. Genome assembly was carried out with SPAdes v.3.11.1 in careful mode, with k-mer size
21, 33, 55, 77, 99, 127 [23]. Contig coverage was assessed by mapping corrected reads against the
assembled contigs with Bowtie 2 and BAM files were read with QualiMap v.2.2.1 [24]. Small contigs
(<500 bp) with low coverage (<2×) were removed. The presence of contaminant contigs was ascertained
using BlobTools v.1.0.1 [25] and the genome assembly quality of filtered contigs was evaluated with
QUAST v.4.6.0 [26]. Genome completeness reconstruction was assessed with BUSCO v.3.0.2 [27].
Taxonomic affiliation was confirmed by targeting and extracting the complete Internal Transcribed
Spacer (ITS) with ITSx v.1.1 [28] and locally aligning the sequence (blastn) against the UNITE web-based
database (https://unite.ut.ee/) [29].
J. Fungi 2020, 6, 255
J. Fungi 2020, 6, x FOR PEER REVIEW
4 of 23
4 of 23
Figure 1. (A) Colony morphology of Dactylonectria torresensis strains isolated from soil, weed or
grapevine. (B) Maximum likelihood phylogeny of Dactylonectria spp. as estimated from the alignment
Figure
1. (A)H3
Colony
morphology
of Dactylonectria
strains isolated
from soil,
weed
or
of
the histone
gene sequences.
Maximum
likelihood torresensis
bootstrap percentages
are indicated
at the
nodes.
grapevine.
(B)
Maximum
likelihood
phylogeny
of
Dactylonectria
spp.
as
estimated
from
the
alignment
Support values less than 70% bootstrap are omitted. The tree was rooted to Campylocarpon fasciculare
of the112613)
histoneand
H3 Ca.
genepseudofasciculare
sequences. Maximum
likelihood
are
indicated
at the
(CBS
(CBS 112679).
Thebootstrap
scale bar percentages
indicates 0.05
expected
changes
nodes.
per
site.Support values less than 70% bootstrap are omitted. The tree was rooted to Campylocarpon
fasciculare (CBS 112613) and Ca. pseudofasciculare (CBS 112679). The scale bar indicates 0.05 expected
changes per site.
J. Fungi 2020, 6, 255
5 of 23
2.4. Average Nucleotide Identity
Previously available genome sequences of other related fungal organisms, later included in the
comparative analysis, were downloaded from the NCBI repository with the ncbi-genome-download
Python script [30]. Whole genome relatedness of de-novo assembled genomes and downloaded genomes
were assessed using Average Nucleotide Identity (ANI) analysis with BLAST method [31]. The selected
fungi had at least one of the following features in common with D. torresensis: root colonization
(including pathogenic, beneficial endophytic and mycorrhizal colonization), plant pathogenesis and
plant biomass degradation. These fungi were: Ilyonectria destructans C1, Ilyonectria mors-panacis g3b [32],
Dactylonectria macrodidyma JAC15-245 [16], Botrytis cinerea B05.10 [33], Fusarium graminearum PH-1 [34],
Leptosphaeria maculans JN3 [35], Magnaporthe oryzae 70-15 [36], Serendipita indica DSM 11,827 [37],
Ustilago bromivora UBRO v3 [38], Botryosphaeria dothidea LW030101 [39], Trichoderma reesei QM6a [40],
Phaeomoniella chlamydospora UCRPC4 [41], Rhizoctonia solani AG-3 [42], Tulasnella calospora AL13/4D [43],
Phaeoacremonium minimum UCRPA7 [44], Phaeoacremonium sp. FL0889 (Joint Genomics Institute [JGI],
http://fungalgenomes.org), Serpula lacrymans S7.3 [45], Phanerochaete chrysosporium ATCC 20,696 [46],
Rhizophagus irregularis DAOM 181,602 [47], Laccaria bicolor S238N-H82 [48], Tuber melanosporum
Mel28 [49], Phaeomoniella chlamydospora RR-HG1 [50], Cadophora malorum Mo12 [51], Oidiodendron maius
Zn [43], Periconia macrospinosa DSE2036 [52], Verticilium dahliae Vdsl17 [53] and Nectria haemotococca
mpVI [54].
2.5. Gene Prediction and Genome Annotation
The gene prediction of de-novo assembled genomes was conducted in Maker v.3.00.0 [55] generating
a consensus prediction based on the following strategy: (1) low-complexity (simple) and interspersed
(complex) repetitive elements were masked with RepeatMasker v.4.0.5; (2) ab-initio unsupervised
gene finding was made with GeneMark-ES v.4.32 [56]; (3) a gene prediction based on precomputed
Fusarium graminearum model was performed with Augustus v.3.2.3 [57]. The sequences of the previously
available fungal genomes were either de-novo annotated or re-annotated, using data accessible from
the JGI (https://jgi.doe.gov/). The gene prediction was executed in Maker, combining the following
information: GeneMark-ES ab-initio gene prediction, available proteins from same or related organisms,
existing Augustus gene prediction models from related fungal taxa.
Amino acid FASTA files were then annotated with the accelerated blastp implemented in
DIAMOND v.0.9.24.125 using the NCBI fungal protein RefSeq database.
2.6. Functional Annotation of Predicted Genes
Functional annotation was carried out using a local installation of the whole eukaryotic
orthologous eggNOG v.4.5.1 database [58]. Carbohydrate-active enzymes (CAZymes) were classified in
Dactylonectria strains BV-349, BV-666 and BV-745 using the dbCAN Hmmer-based classification system
with E-Value < 1 × 10−15 , coverage > 0.35 [59] at http://bcb.unl.edu/dbCAN2/. Similar annotation was
done for 27 fungal genomes including root pathogens, aerial pathogens, trunk or cane pathogens,
white rot degraders, indoor biodeterioration agents, saprotrophs, dark septate endophytes and
mycorrhiza. All positive hits were manually examined for final validation. All CAZyme classes,
including Glycoside Hydrolases (GH), Carbohydrate Esterases (CE), Glycoside Transferases (GT),
Polysaccharide Lyases (PL) and Carbohydrate-Binding Modules (CBM) were considered. Circos online
software at http://mkweb.bcgsc.ca/tableviewer/was further used for data visualization and comparison
of CAZymes with other fungi.
J. Fungi 2020, 6, 255
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The secretome tool software http://genomics.cicbiogune.es/SECRETOOL/ was also utilized for
general secretome analysis and small secreted proteins (ssp) using the default settings for eucaryotic
genomes. Comparisons were further carried out with the data from the 27 other fungal genomes.
Antismash analysis was finally performed for prediction of genes related to secondary metabolites
(https://fungismash.secondarymetabolites.org/).
2.7. Analysis of Growth and Light Response
Strains were cultivated on malt extract (3% w/v), Mandels Andreotti minimal medium [60] or
synthetic nutrient poor agar (SNA) medium [61] in daylight (12 h:12 h cycles of light and darkness) or
constant darkness for 72 h for assessment of hyphal extension and light response. Fungal growth on
Petri dishes were documented photographically.
3. Results and Discussion
3.1. Fungal Strains
A phylogenetic analysis was performed with Dactylonectria strains isolated from the weed species
Solanum nigrum (BV-745), grapevine rootstock 110 Richter (BV-666) and soil samples (BV-349) to identify
them at species-level. The Bayesian Information Criterion (BIC) best-fit nucleotide substitution model
identified by jModelTest was Hasegawa-Kishino-Yano model (HKY) with gamma distributed with
invariant sites rates (G + I) for the Dactylonectria analysis. Alignment of 31 Dactylonectria sequences
resulted in a 540-character dataset. The three isolates clustered strongly (>98%) with the type specimens
of D. torresensis (CBS 129,086 and CBS 119.41) (Figure 1B). Strains BV-666 and BV-745 were isolated from
asymptomatic vine and weed, respectively. Recent studies have suggested that black-foot fungi have a
non-pathogenic endophytic phase [12,13] and may become pathogenic to grapevine after different
abiotic and/or biotic stresses conditions and thus, they are considered as latent pathogens in grapevine.
Several factors have been reported to be determinants in triggering pathogenicity in an endophyte that
was previously asymptomatic, such as the nutrient status, changes in plant gene expression, habitat,
host genotype or the locally occurring abiotic stresses that might reduce host fitness, resulting in bias
of this delicate equilibrium and thus influencing the symptom expression in plants [62]. Abiotic stress
factors in new plantations and grapevine nursery fields include water stress, J-rooting, winter-kill,
waterlogging, soil compaction, nutrition deficiency and/or overcropping [15].
3.2. Genomes
The genomes of three D. torresensis strains isolated from grapevine, weed or soil were sequenced
and annotated. Strain BV-349 has a genome size of 64.42 Mb (GC content: 50.67%) while strain
BV-666 and strain BV-745 have 65.33 Mb (GC content: 50.17%) and 64.21 Mb (GC content: 50.23%),
respectively and with 19,102 predicted proteins for strain BV-349, 19,090 for strain BV-666, and 18,724
for strain BV-745 (Table 1). Single-copy ortholog analysis reported a genome assembly completeness of
98.6% for strain BV-349, 98.3% for BV-666 and 99.0% for BV-745. ANI analysis showed that strains
are different at genome level (Supplementary File 1: Figure S1). The number of transposon-related
proteins in the three strains is 117, 139 and 143, respectively.
J. Fungi 2020, 6, 255
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Table 1. Genome statistics of Dactylonectria strains.
Strain
Genome Statistics
BV-349
BV-666
BV-745
Genome assembly size (Mbp)
Number of reads
Mapped reads
Unmapped reads
Coverage
Completeness
Duplication ratio
Largest alignment
Total aligned length
Contigs
Largest contig
GC (%)
Proteins encoding genes
64.42
19,695,918
98.23%
1.77%
42.23×
98.6%
1.008
242,734
48,129,906
2809
377,758
50.67
19,102
65.33
32,400,268
97.44%
2.56%
67.88×
98.3%
1.008
329,511
48,518,112
2598
402,120
50.17
19,090
64.21
25,108,162
98.65%
1.35%
53.58×
99%
1.007
254,604
48,295,324
2396
458,724
50.23
18,724
3.3. COG Categories and Eggnog Analysis
Using COG categories and eggnog database, we analyzed each group of gene categories (Table 2)
and searched which potential genes could be involved in pathogenicity or endophytism. Each strain
contains genes related to ethylene induced peptide-related gene and necrosis inducing protein (NPP1)
(Table 3). A high number of genes related to carbohydrate-actives enzymes were further detected in each
strain. Other interesting genes to highlight were genes related to heavy metal or metalloid resistance
such as copper, cadmium and others that could be detected in a vineyard (Table 3). Other genes of
interest were those related to salicylate hydrolase, which degrades salicylic acid normally involved in
signalling of plant defense reaction, siderophore as an iron chelator and auxin as a phytohormone
(Table 3). Only a few fungal salicylate hydroxylase enzymes have been reported such as in the
endophyte Epichloë festucae or in the pathogen Fusarium graminearum causing Fusarium head blight
(FHB) [63]. Salicylate hydroxylase enzymatic activities have been also found in Trichosporon cutaneum
and other Fusarium spp. [64,65]. Interestingly, no symptom was recorded in the weed and vine and the
strains were established as non-pathogenic endophytes, suggesting the possibility that degradation of
salicylic acid is a factor on how Dactylonectria strains avoid plant defense reaction.
3.4. CAZymes
A high number of genes related to carbohydrate metabolism was detected in the genomes of all
three Dactylonectria strains investigated. Previously, CAZyme encoding genes were shown to have
roles during infection of plants by fungal pathogens [39]. Therefore, we further analyzed the genomes
of Dactylonectria strains BV-349, BV-666, BV-745 for CAZymes related genes. The strains contain 1140,
1116, and 1133 genes encoding putative CAZymes, respectively (Table 3).
As for many fungi, among CAZymes related genes, the Glycoside Hydrolase (GH)
superfamily is the most represented class in the three Dactylonectria strains (Figure 2). We found
that some GH genes were represented by a number of genes >20 as for GH78 (Figure 2)
corresponding to α-L-rhamnosidase/rhamnogalacturonan α-L-rhamnohydrolase/L-Rhap-α-1,3-D
-Apif-specific α-1,3-L-rhamnosidase). Also, GH43 was over-represented. GH43 has functions like
β-xylosidase/α-L-arabinofuranosidase/xylanase/α-L-arabinofuranosidase/exo-β-1,3-galactanase/β-D
-galactofuranosi-dase. GH3 was also highly represented in each genome and corresponds
to β-glucosidase/xylan 1,4-β-xylosidase/β-glucosylceramidase/β-N-acetylhexosaminidase/α-Larabinofuranosidase/isoprime-verose-producing oligoxyloglucan hydrolase/coniferin β-glucosidase/
exo-1,3-1,4-glucanase or β-N-acetylglucosaminide phosphorylases (Figure 2). Additionally,
GH28 was highly present (Figure 2). GH28 has potential activities like polygalacturonase/α
-L-rhamnosidase/rhamno-galacturonase/rhamnogalacturonan α-1,2-galacturono-hydrolase/endo-
J. Fungi 2020, 6, 255
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xylogalacturonan hydrolase. Lastly, GH18 (chitinase/lysozyme/endo-β-N-acetylglucosaminidase/
peptidoglycan hydrolase/Nod factor hydrolase/xylanase inhibitor/concanavalin B or narbonin),
GH16 (xyloglucan/xyloglucosyltransferase/β-agarase/κ-carrageenase/xyloglucanase/β-porphyranase/
hya-luronidase/endo-β-1,4-galactosidase/chitin β-1,6-glucanosyltransferase), and GH109 (α-Nacetylgalactosaminidase) were also represented by a number of genes >20 (Figure 2).
Table 2. Clusters of ortholog groups of Dactylonectria strains.
COG Categories
BV-349
BV-666
BV-745
RNA processing and modification [A]
Chromatin structure and dynamics [B]
Energy production and conversion [C]
Cell cycle control, cell division, chromosome partitioning [D]
Amino acid transport and metabolism [E]
Nucleotide transport and metabolism [F]
Carbohydrate transport and metabolism [G]
Coenzyme transport and metabolism [H]
Lipid transport and metabolism [I]
Translation, ribosomal structure and biogenesis [J]
Transcription [K]
Replication, recombination and repair [L]
Cell wall/membrane/envelope biogenesis [M]
Cell Motility [N]
Posttranslational modification, protein turnover, chaperones [O]
Inorganic ion transport and metabolism [P]
Secondary metabolites biosynthesis, transport and catabolism [Q]
General function prediction only [R]
Function unknown [S]
Signal transduction mechanisms [T]
Intracellular trafficking, secretion, and vesicular transport [U]
Defense mechanisms [V]
Extracellular structures [W]
Nuclear structure [X]
Cytoskeleton [Y]
TOTAL
342
125
657
370
774
207
1287
213
548
412
536
424
399
3
729
359
1018
0
7790
669
427
113
8
28
194
17,632
321
121
671
347
790
191
1321
222
542
419
558
432
356
3
736
369
1062
0
7853
630
415
123
9
24
177
17,692
336
131
665
329
780
186
1300
216
536
415
541
439
345
3
702
357
1020
0
7720
595
423
117
8
24
189
17,377
Table 3. Genes related to pathogenicity, metal resistance or other properties in Dactylonectria genomes.
Clusters of Genes Related to Pathogenicity
Strain
Metal Resistance or Other Properties
BV-349
BV-666
BV-745
Genes related to necrosis
Necrosis and ethylene inducing peptide
Necrosis inducing protein (NPP1)
Genes related to carbohydrate-actives enzymes
Carbohydrate-binding modules
Carbohydrate esterases
Glycoside hydrolases
Glycosyl transferases
Polysaccharide lyases
Auxiliary activities
Genes related to heavy metal/metalloid resistance
Copper
Cadmium
Other heavy metal/metalloids
Genes related to other properties
Salicylate hydroxylase
Siderophore
Auxin
11
2
9
1140
134
210
474
129
42
151
48
22
2
8
23
18
4
1
10
2
8
1116
124
207
459
128
43
155
37
23
5
5
18
13
4
1
10
2
8
1133
117
221
466
130
44
155
36
23
2
3
22
17
4
1
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9 of 23
10 of 23
Figure
2. Numbers
Numbers of
of genes
genesrelated
relatedtotoglycoside
glycosidehydrolases
hydrolases
Dactylonectria
torresensis
strains
BVFigure 2.
in in
Dactylonectria
torresensis
strains
BV-349,
349,
BV-666,
BV-745.
BV-666, BV-745.
The
fact thattothese
three genomes lyase
possess
a high
number
of CAZymes
and
PCWDE
domains
In addition
GH, polysaccharide
related
genes
(PCWDE)
were also
detected
in the
three
suggests
strains have
broad
spectrum
enzymes
as weapons
plant
cell-wall
genomes that
of D.the
torresensis
with aPL1
being
the mostofpresent
family
followedfor
bydegrading
PL3, PL4, PL9
and
others
and to7, establish
as3).
endophytes
inside
plant tissues
as well
as to
macerate
root tissues.
(PL6,
11, 14, 20,themselves
22, 26; Figure
Genes related
to glycosyl
transferase
(GT1,
GT2,
GT4), carbohydrate
Several
studies have indeed
that a high
amount
of auxiliary
CAZymeactivities
and PCWDE-related
esterasegenomic
(CE10), carbohydrate
bindingindicated
module (CBM67,
CBM50)
and
(AA7, AA3)
genesalso
is linked
non-pathogenic
or pathogenic
endophytes
[43,52,67].
were
furthertohighly
present in the
three genomes
(Figures 3–5).
Of those, particularly the expansion
in GT1, PL1 and PL3 gene families was found to be characteristic for plant pathogenic fungi [66].
J. Fungi 2020, 6, 255
J. Fungi 2020, 6, x FOR PEER REVIEW
Figure 3.
10 of 23
11 of 23
Numbers of genes related to auxiliary activities and polysaccharide lyases in
Figure 3. Numbers of genes related to auxiliary activities and polysaccharide lyases in Dactylonectria
Dactylonectria torresensis strains BV-349, BV-666, BV-745.
torresensis strains BV-349, BV-666, BV-745.
3.5. Genes Involved in Oxidative Degradation of Plant Biomass
CAZyme gene analysis revealed the characteristic expansions in gene families implicated in
infection and pathogenicity [39,66]. Here, in particular the increased number of GH43 encoding
genes, but also of auxiliary functions suggest a role of the competence to degrade lignocellulose in
pathogenicity of Dactylonectria spp. Therefore, we investigated the genomic content for the presence
of AA3-1, AA9 and AA14 members. The AA9 family represents an efficient group of lytic
polysaccharide monooxygenases (LPMOs) of high biotechnological relevance [68] due to their
contribution to cellulose degradation, but also xyloglucan degradation was shown for this family
[69]. A contribution of LPMOs to lignin degradation was reported as well [70] For AA14 LPMOs,
only recently a high efficiency in boosting wood saccharification was shown [71]. LPMOs degrade
J. Fungi 2020, 6, 255
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11 of 23
12 of 23
Figure 4. Numbers of genes related to carbohydrate-binding modules and carbohydrate esterases in
Figure 4.
Numbers of genes related to carbohydrate-binding modules and carbohydrate esterases in
Dactylonectria torresensis strains BV-349, BV-666, BV-745.
Dactylonectria torresensis strains BV-349, BV-666, BV-745.
For AA14, a Blastp search of the 321 Sordariomycetes genomes in Mycocosm
(https://mycocosm.jgi.doe.gov) with the characterized Pyconoporus coccineus AA14 proteins [71]
revealed putative homologues (e-values around 1E-38) in only about half of the Sordariomycetes
species so far sequenced. In only a few cases, two putative homologues were detected. In the three
Dactylonectria strains investigated here, we found one putative homologue each, but with amino acid
identities around 25% and similarities around 31% to P. coccineus AA14a and AA14b. Since for those
proteins no functional domains were characterized yet, a comparable functionality of the
Dactylonectria homologues remains to be confirmed.
Members of the class AA9 (formerly GH61) are particularly important for oxidative degradation
of plant biomass. Dactylonectria strains BV-666 and BV-745 contain twelve homologues with the
J. Fungi 2020, 6, 255
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12 of 23
13 of 23
Figure 5. Numbers
Numbers of
of genes
genes related
related to
to glycosyl
glycosyl transferases
transferases in
inDactylonectria
Dactylonectria torresensis
torresensis strains
strains BV-349,
BV-349,
BV-666, BV-745.
BV-745.
BV-666,
As
only small
differences
were recorded
between the
strainsfuel
for LPMO
either genes
relatedwas
to glycoside
Analysis
of putative
cellobiose
dehydrogenases,
which
efficiency,
done in
hydrolases
2), auxiliary
activities and
polysaccharide
lyases
(Figure 3),
carbohydrate-binding
comparison(Figure
of characterized
homologues
from
www.cazy.org.
A strikingly
high
number of putative
modules,
carbohydrate
esterases
(Figure 4) orstrains,
glycosyl
transferases
(Figure
moreand
information
homologues
was detected
in the Dactylonectria
with
four in BV-349
and 5),
BV-666
even five
were
searched
in relation/differences
of contains
Dactylonectria
tonone.
other However,
fungi. Circos
simulation
shows
in BV-745,
while
for example N. crassa
2 andstrains
T. reesei
their
functionality
as
relationship
between
the
percentages
of
CAZymes
genes
shared
by
Dactylonectria
strains
and
also
with
cellobiose dehydrogenases remains to be confirmed.
otherInblack-foot
fungi
(Supplementary
File
S2). CAZyme
numbers
appears
as significantly
summary,
while
the functionality
of 2:
theFigure
additional
homologues
remains
to be experimentally
higher
in Dactylonectria
spp. expression
than the average
forthe
thehigh
other
27 fungal
genomesCDH
(Figure
which
agrees
confirmed
along with their
in vivo,
number
of putative
and6),AA9
encoding
with
characteristics
of
plant
pathogens
[66].
genes supports the role of Dactylonectria as a pathogen with an efficient machinery for wood
degradation. Despite important possible contributions of other factors to virulence, a clear difference
for the three strains according to their isolation site (soil, grapevine, weed) could not be deduced from
abundance of genes predominantly associated with wood degradation.
J. Fungi 2020, 6, 255
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13 of 23
14 of 23
Figure 6. Comparative analyses of genome size, numbers of genes related to carbohydrate-active
Figure 6. Comparative analyses of genome size, numbers of genes related to carbohydrate-active
enzymes
torresensisstrains
strainsBV-349,
BV-349,BV-666,
BV-666,
BV-745
enzymes(CAZyme)
(CAZyme)and
andsecretome
secretome in
in Dactylonectria
Dactylonectria torresensis
BV-745
to to
known
fungal
pathogens,
saprotrophs
or
symbionts.
CBM:
Carbohydrate-Binding
Module,
known fungal pathogens, saprotrophs or symbionts. CBM: Carbohydrate-Binding Module, CE:CE:
Carbohydrate
Esterase,
GH:
Glycoside
Hydrolase,
GT:GT:
Glycosyl
Transferase,
PL: Polysaccharide
lyases,
Carbohydrate
Esterase,
GH:
Glycoside
Hydrolase,
Glycosyl
Transferase,
PL: Polysaccharide
AA:
Auxiliary
Activity,
Total:
total
number
of
CAZymes,
Sec:
number
of
secreted
proteins.
lyases, AA: Auxiliary Activity, Total: total number of CAZymes, Sec: number of secreted proteins.
comparison
to Dactylonectria
strains
described
3.6.InAnalyses
of Secretome
and Small Secreted
Proteins
(SSP) in this study, the dark septate endophyte
Oidiodendron maius Zn has a total of 1111 genes related to CAZymes and the trunk disease pathogen
High numbers of genes of D. torresensis corresponding to secretome and a larger size of small
Phaeoacremonium sp. FL0889 1172 (Figure 6). The highest score was also related to black-foot pathogens
secreted proteins were further characterized in the three genomes of Dactylonectria (with number
such as Dactylonectria macrodidyma JAC15-245 (1080 genes), and Ilyonectria destructans C1 (1088).
between 683 and 687 secreted proteins and 251–260 for SSP) (Figures 6 and 7). Comparison showed
Other genomes had genes numbers between 237 and 992.
J. Fungi 2020, 6, 255
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The fact that these three genomes possess a high number of CAZymes and PCWDE domains
suggests that the strains have a broad spectrum of enzymes as weapons for degrading plant cell-wall
and to establish themselves as endophytes inside plant tissues as well as to macerate root tissues.
Several genomic studies have indeed indicated that a high amount of CAZyme and PCWDE-related
genes is linked to non-pathogenic or pathogenic endophytes [43,52,67].
3.5. Genes Involved in Oxidative Degradation of Plant Biomass
CAZyme gene analysis revealed the characteristic expansions in gene families implicated in
infection and pathogenicity [39,66]. Here, in particular the increased number of GH43 encoding
genes, but also of auxiliary functions suggest a role of the competence to degrade lignocellulose in
pathogenicity of Dactylonectria spp. Therefore, we investigated the genomic content for the presence of
AA3-1, AA9 and AA14 members. The AA9 family represents an efficient group of lytic polysaccharide
monooxygenases (LPMOs) of high biotechnological relevance [68] due to their contribution to cellulose
degradation, but also xyloglucan degradation was shown for this family [69]. A contribution of LPMOs
to lignin degradation was reported as well [70] For AA14 LPMOs, only recently a high efficiency
in boosting wood saccharification was shown [71]. LPMOs degrade lignocellulosic biomass via an
oxidative mechanism, that requires an electron donor [72]. Cellulose dehydrogenases (CDHs) of the
family AA3-1 are known to reduce LPMOs in nature and are hence crucial for their function [73].
Hence, the combination of these LPMOs and CDHs along with the fact that LPMOs were also found
to be efficient under anaerobic conditions [74], make them ideal candidates for virulence factors
of Dactylonectria.
For AA14, a Blastp search of the 321 Sordariomycetes genomes in Mycocosm (https://mycocosm.jgi.
doe.gov) with the characterized Pyconoporus coccineus AA14 proteins [71] revealed putative homologues
(e-values around 1E-38) in only about half of the Sordariomycetes species so far sequenced. In only a
few cases, two putative homologues were detected. In the three Dactylonectria strains investigated here,
we found one putative homologue each, but with amino acid identities around 25% and similarities
around 31% to P. coccineus AA14a and AA14b. Since for those proteins no functional domains were
characterized yet, a comparable functionality of the Dactylonectria homologues remains to be confirmed.
Members of the class AA9 (formerly GH61) are particularly important for oxidative degradation of
plant biomass. Dactylonectria strains BV-666 and BV-745 contain twelve homologues with the respective
domain, while BV-349 only has ten putative LPMOs of class AA9 (Supplementary File 3: Figure S3).
Analysis of putative cellobiose dehydrogenases, which fuel LPMO efficiency, was done in
comparison of characterized homologues from www.cazy.org. A strikingly high number of putative
homologues was detected in the Dactylonectria strains, with four in BV-349 and BV-666 and even five
in BV-745, while for example N. crassa contains 2 and T. reesei none. However, their functionality as
cellobiose dehydrogenases remains to be confirmed.
In summary, while the functionality of the additional homologues remains to be experimentally
confirmed along with their expression in vivo, the high number of putative CDH and AA9 encoding
genes supports the role of Dactylonectria as a pathogen with an efficient machinery for wood degradation.
Despite important possible contributions of other factors to virulence, a clear difference for the three
strains according to their isolation site (soil, grapevine, weed) could not be deduced from abundance
of genes predominantly associated with wood degradation.
3.6. Analyses of Secretome and Small Secreted Proteins (SSP)
High numbers of genes of D. torresensis corresponding to secretome and a larger size of small
secreted proteins were further characterized in the three genomes of Dactylonectria (with number
between 683 and 687 secreted proteins and 251–260 for SSP) (Figures 6 and 7). Comparison showed
that dark septate endophytes Pericornia macrospinosa DSE2036 and Oidiodendron maius Zn as well as
trunk disease pathogen Cadophora malorum, Phaeoacremonium sp. FL0889, and black-foot pathogens
Dactylonectria macrodidyma JAC15-245, as well as Ilyonectria destructans C1 had a similar range of genes
J. Fungi 2020, 6, x FOR PEER REVIEW
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J. Fungi
2020,septate
6, 255 endophytes Pericornia macrospinosa DSE2036 and Oidiodendron maius Zn as well
15 ofas
23
that
dark
trunk disease pathogen Cadophora malorum, Phaeoacremonium sp. FL0889, and black-foot pathogens
Dactylonectria macrodidyma JAC15-245, as well as Ilyonectria destructans C1 had a similar range of genes
related to secretome (650–731) (Figure 6). Small secreted proteins play important roles in pathogenicity
related to secretome (650–731) (Figure 6). Small secreted proteins play important roles in
of fungal-plant interactions and in symbiosis [52,75–77]. The large numbers of secreted proteins in
pathogenicity of fungal-plant interactions and in symbiosis [52,75–77]. The large numbers of secreted
Dactylonectria strains show also potential roles in pathogenicity.
proteins in Dactylonectria strains show also potential roles in pathogenicity.
Figure 7.
7. Numbers
Numbersof
ofgenes
genesin
inDactylonectria
Dactylonectriatorresensis
torresensisstrains
strainsBV-349,
BV-349,BV-666,
BV-666,BV-745
BV-745 corresponding
corresponding
Figure
to
small
secreted
proteins
(ssp).
to small secreted proteins (ssp).
3.7. Antismash Analyses
3.7. Antismash Analyses
One of the crucial weapons for fungal plant pathogen is the production of phytotoxic
One of the crucial weapons for fungal plant pathogen is the production of phytotoxic
compounds [78]. Fungal antismash analysis was used to search in the genomes’ gene clusters
compounds [78]. Fungal antismash analysis was used to search in the genomes’ gene clusters
encoding key enzymes such as NRPS (non-ribosomal peptide synthetase), PKS (polyketide synthase),
encoding key enzymes such as NRPS (non-ribosomal peptide synthetase), PKS (polyketide synthase),
HYBRID PKS-NRPS and others. Dactylonectria strains were found to contain a significant
HYBRID PKS-NRPS and others. Dactylonectria strains were found to contain a significant number of
number of genes encoding key secondary metabolism biosynthesis enzymes (Table 4). Interestingly,
genes encoding key secondary metabolism biosynthesis enzymes (Table 4). Interestingly,
biosynthetic gene clusters for echinocandin B (antifungal lipopeptide inhibiting the synthesis of
biosynthetic gene clusters for echinocandin B (antifungal lipopeptide inhibiting the synthesis of
glucan), brefeldin (antiviral metabolite) and asperfuranone (a polyketide) were detected in the
glucan), brefeldin (antiviral metabolite) and asperfuranone (a polyketide) were detected in the
Dactylonectria genomes, except in BV-349 for echinocandin B (Table 4). Surprisingly, a fujikurin
Dactylonectria genomes, except in BV-349 for echinocandin B (Table 4). Surprisingly, a fujikurin
biosynthetic gene cluster was also detected in all three genomes with percentage of similarity between
biosynthetic gene cluster was also detected in all three genomes with percentage of similarity
83 and 100% to known gene cluster (Table 4). Fujikurin has been isolated from an endophytic
between 83 and 100% to known gene cluster (Table 4). Fujikurin has been isolated from an endophytic
Fusarium species [79] and phytopathogens and highlights were made on a possible role of this
Fusarium species [79] and phytopathogens and highlights were made on a possible role of this
metabolite as a phytopathogenic virulence determinant [80]. Other fujikurin-like clusters were
metabolite as a phytopathogenic virulence determinant [80]. Other fujikurin-like clusters were also
also detected in opportunistic pathogens such as Aureobasidium pullulans and Scedosporium spp.,
detected in opportunistic pathogens such as Aureobasidium pullulans and Scedosporium spp., in the
in the saprophyte Endocalyx cinctus (saprophyte in dead palms), in the ericoid mycorrhizal fungus
saprophyte Endocalyx cinctus (saprophyte in dead palms), in the ericoid mycorrhizal fungus
Cairneyella variabilis, in Paecilomyces hepiali (entomopathogenic fungus associated with plants) and
Cairneyella variabilis, in Paecilomyces hepiali (entomopathogenic fungus associated with plants) and
Ophiostoma sp. responsible of Dutch elm disease [81]. The presence of this gene cluster, but with a
Ophiostoma sp. responsible of Dutch elm disease [81]. The presence of this gene cluster, but with a
configuration different from the one of Fusarium fujikuroi, a species complex belonging to Nectriaceae [82]
configuration different from the one of Fusarium fujikuroi, a species complex belonging to Nectriaceae
(Supplementary File 4: Figure S4) suggests that this secondary metabolite could allow several fungal
[82] (Supplementary File 4: Figure S4) suggests that this secondary metabolite could allow several
species to interact with plants, as phytopathogens or non-pathogenic endophytes.
fungal species to interact with plants, as phytopathogens or non-pathogenic endophytes.
J. Fungi 2020, 6, 255
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Table 4. Antismach analysis of Dactylonectria genomes.
Strain
Antismash Analysis
BV-349
BV-666
BV-745
Terpene (number of gene clusters)
T1pks (number of gene clusters)
Nrps (number of gene clusters)
T1pks-nrps (number of gene clusters)
T3pks (number of gene clusters)
Other (number of gene clusters)
Brefeldin_biosynthetic gene cluster (% similarity)
Echinocandin_B_biosynthetic gene cluster (% similarity)
Asperfuranone_biosynthetic gene cluster (% similarity)
Brefeldin_biosynthetic gene cluster (% similarity)
Fujikurins_biosynthetic gene cluster (% similarity)
8
15
7
2
1
7
50
27
83
9
17
10
2
1
7
20
13
27
40
100
9
16
9
2
1
8
13
40
27
20
83
3.8. Light Response
Light has a profound impact on physiology and metabolism of fungi [83] and in particular
also on regulation of plant cell-wall degradation [84,85], which may be relevant for pathogenicity
of Dactylonectria. Light and photoreceptors impact circadian rhythmicity as well and recently a
connection of fungal circadian rhythmicity of pathogenicity was detected [86,87]. We noticed that the
three strains investigated in this study show phenotypic differences when grown in light (Figure 8A).
Especially, BV-666 showed a clear reaction to changing light conditions when it was grown in daylight
(light:dark 12 h:12 h). Differences in hyphal extension between daylight and darkness were obvious on
xylan (BV-745) or cellulose (BV-349) (Figure 8B). Therefore, we were interested if these differences are
reflected in the genomes of these strains. We analyzed blue light photoreceptor candidates of all three
Dactylonetria strains and found that the photoresponse machinery of these fungi is more complex than
that of N. crassa or T. reesei. Besides homologues of ENV1/VVD and BLR2/WC-2, Dactylonectria spp.
have an additional, close homologue to BLR1/WC-1, representing the crucial PAS/LOV photoreceptors.
Moreover, we found two further proteins related to and sharing similar domains with BLR1/WC1
(Figure 8C). All Dactylonectria proteins containing a putative PAS/LOV (Per-ARNT-Sim/Light, oxygen or
voltage) domain also comprised the conserved sequence NCRFLQ which is considered crucial for light
responses [88], except for one of them (Figure 8D), where the sequence is altered to NCRLLQ. Then,
we tested whether alterations in the sequences of the detected photoreceptors would correlate with the
altered light response we had observed. Indeed, aligning the sequences revealed that in BV-666, three of
the four photoreceptor homologues showed alterations in one or more amino acids (Figure 8E), which is
in accordance with an altered response to daylight (Figure 8A). However, since these alterations did
not occur in functional domains like phosphorylation or myristoylation sites, we could not assign a
possible relevance. Since BV-666 was isolated from grapevine, further work to evaluate whether the
detected alterations may have affected association with the plant and hence virulence, could provide
insight into the relevance of the extended photoresponse system of Dactylonectria on pathogenicity.
Fungi 2020,
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17 of
Figure 8. Light response and photoreceptors in Dactylonectria. (A) Growth characteristics of mycelia
grown
3% (w/v)
malt extract
(MEX) or SNA
in daylight
(DL; 12:12
dark:light of
cycles)
or
Figure on
8. Light
response
and photoreceptors
in medium
Dactylonectria.
(A) Growth
characteristics
mycelia
constant
darkness
(DD)
for
7
days.
For
strain
BV-666
formation
of
rings
is
visible
on
both
media.
grown on 3% (w/v) malt extract (MEX) or SNA medium in daylight (DL; 12:12 dark:light cycles) or
(B)
Analysis
of hyphal
growth
on Mandels
Andreotti
(Mandels
constant
darkness
(DD)extension
for 7 days.upon
For strain
BV-666
formation
of rings minimal
is visiblemedium
on both media.
(B)
1978)
withof1%
(w/v) extension
xylan or cellulose
in daylight
(DL) or
constantminimal
darknessmedium
(DD). Errorbars
Analysis
hyphal
upon growth
on Mandels
Andreotti
(Mandelsreflect
1978)
standard
deviation
biological
replicates.
(C)orPhylogenetic
analysis
of photoreceptor
homologues
with 1% (w/v)
xylanofortwo
cellulose
in daylight
(DL)
constant darkness
(DD).
Errorbars reflect
standard
in
Dactylonectria.
The
evolutionary
history
was
inferred
using
the
minimum
evolution
method
(Eck
deviation of two biological replicates. (C) Phylogenetic analysis of photoreceptor homologues
in
and
Dayhoff
1966).
The
bootstrap
consensus
tree
inferred
from
500
replicates
is
taken
to
represent
the
Dactylonectria. The evolutionary history was inferred using the minimum evolution method (Eck and
evolutionary
history
of the taxaconsensus
analyzed (Felsenstein
1985).
analyses
were
conductedthe
in
Dayhoff 1966).
The bootstrap
tree inferred
fromPhylogenetic
500 replicates
is taken
to represent
MEGA4
(Tamura
et
al.,
2007).
GenBank
accession
numbers
of
reference
proteins
are
given
with
the
evolutionary history of the taxa analyzed (Felsenstein 1985). Phylogenetic analyses were conducted
protein
names
and species.
(D) Encoded
amino
acids numbers
in the genomic
regionproteins
of the NCRFLQ
conserved
in MEGA4
(Tamura
et al. 2007).
GenBank
accession
of reference
are given
with the
motif
in
the
proteins
shown
in
the
phylogenetic
tree.
(E)
Alterations
in
the
amino
acid
sequence
at
protein names and species. (D) Encoded amino acids in the genomic region of the NCRFLQ conserved
different
loci
of
proteins
shown
in
the
phylogenetic
tree.
motif in the proteins shown in the phylogenetic tree. (E) Alterations in the amino acid sequence at
different loci of proteins shown in the phylogenetic tree.
J. Fungi 2020, 6, 255
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The sequence differences in the photoreceptors described above may impact circadian rhythms
already, but in addition we analyzed the sequence of FREQUENCY (FRQ), which is crucial for circadian
rhythmicity in N. crassa [89]. However, we did not detect any differences in FRQ between the strains.
4. Conclusions
In this study, we analyzed genomes of D. torresensis strains from three different habitats such
as soil, weed, and grapevine. Overall, there was a similar genome content in the strains with genes
related to necrosis, heavy metal/metalloid resistance, salicylic acid degradation, and a high number of
CAZymes related to glycoside hydrolases that could be involved in non-pathogenic endophytism or
pathogenicity. Analysis of photoreceptors revealed an increased number as well as specific mutations
in one strain, indicating an increase relevance of light perception and potentially integration with
other signals in Dactylonectria compared to other ascomycetes. High numbers of genes related to
secretome and small secreted proteins were further detected. However, small differences were recorded
between the three genomes. Our analysis also demonstrates the presence of several gene clusters
with some as fujikurin-like genes that have been linked to fungal pathogenicity. We further detected
high numbers of genes related to CAZyme families and high numbers of transposons. This finding is
further represented by CAZymes associated with wood degradation.
The fact that D. torresensis can be a non-pathogenic endophyte on weeds or grapevine has important
implications. The potential role of asymptomatic hosts may include not only preservation of a viable
inoculum source quantitatively during for example, crop rotation in grapevine nursery fields, but also
in shaping the genetic structure of the pathogen population qualitatively, which may have significant
implications for disease management. This finding also highlights the urgent need to implement early,
accurate and specific in planta detection and quantification of D. torresensis to prevent the spread of
black-foot disease in grapevine propagation material. The future direction of research on black-foot
needs to investigate: (i) how these fungi colonize roots of secondary hosts or grapevine and establish
themselves inside, and (ii) what triggers latent black-foot fungi to transition from a non-pathogenic
endophyte to a pathogenic endophyte, and cause disease symptoms in grapevine.
Supplementary Materials: The following are available online at http://www.mdpi.com/2309-608X/6/4/255/s1,
Figure S1: Average Nucleotide Identity (ANI). Figure S2: circular network plot of CAZymes categories and
their percentages shared in Dactylonectria torresensis strains (top) and with other black-foot pathogens (below).
A: CBM, B: CE, C: GH, D: GT, E: PL, F: AA, G: BV-745, H: BV-666, I: BV-349, J: Ilyonectria destructans C1, K:
Ilyonectria mors-panacis G3B, L: Dactylonectria macrodidyma JAC15-245. Figure S3: phylogenetic analysis of AA9
candidates of Dactylonectria. The evolutionary history was inferred using the maximum parsimony method (Eck
and Dayhoff 1966). The bootstrap consensus tree inferred from 500 replicates is taken to represent the evolutionary
history of the taxa analyzed (Felsenstein 1985). Phylogenetic analyses were conducted in MEGA4 (Tamura et al.
2007). GenBank accession numbers of reference proteins are given with the protein names and species. Figure S4:
antismash analyses of fujikurin-like genes in Dactylonectria torresensis strains BV-349, BV-666, BV-745.
Author Contributions: L.A.; M.G., S.C., M.S., S.B. and C.B. performed the bioinformatic analyses; S.B.
performed plate assays for light response and phenotype, L.A., M.G., S.C., M.S., S.B. and D.G. interpreted
the results; S.C. and D.G. drafted the manuscript; all authors read, revised and approved the manuscript; D.G.
acquired the funding; C.B., M.d.P.M.-D. and E.D.-L. maintained the fungal cultures, and generated the DNA for
genome sequencing. All authors have read and agreed to the published version of the manuscript.
Funding: The research was funded by CAR (Government of La Rioja, Spain), under the project “Development of
Microsatellite Markers to Study the Genetic Diversity of Dactylonectria torresensis Populations in Spain” (project
number R-05-18).
Acknowledgments: We are grateful to B. Nikolic from AIT Austrian Institute of Technology for DNA extraction
of the fungi. M.P. Martínez-Diz and C. Berlanas were supported by the FPI-INIA program from the INIA. D.
Gramaje was supported by the Ramón y Cajal Program, Spanish Government (RYC-2017-23098). We acknowledge
support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of
Information Resources for Research (URICI). We would like to thank authors of published or publicly available
genomes that were used in this study, especially the genome data produced by the Joint Genome Institute.
Conflicts of Interest: The authors declare no conflict of interest.
Availability of Data and Materials: This Whole Genome Shotgun (WGS) Project has been deposited at
DDBJ/ENA/GenBank under the accessions VYKH00000000 (D. torresensis isolate BV-349), VYKG00000000 (isolate
J. Fungi 2020, 6, 255
19 of 23
BV-666) and VYKF00000000 (isolate BV-745). The versions described in this paper are version VYKH01000000,
VYKG01000000 and VYKF01000000, respectively. This Dactylonectria torresensis Genome Sequencing and Assembly
Project was submitted under BioProject PRJNA566152.
Abbreviations
Average Nucleotide Identity (ANI); Carbohydrate-Active Enzymes (CAZymes); Carbohydrate Esterases
(CE); Carbohydrate-Binding Modules (CBM); Fusarium Head Blight (FHB); Glucose-Faba Bean Rose Bengal Agar
(GFBRBA); Glycoside Hydrolases (GH); Glycoside Transferases (GT); Hasegawa-Kishino-Yano model (HKY);
Internal Transcribed Spacer (ITS); Malt Extract Agar (MEA); Polysaccharide Lyases (PL); and Potato Dextrose
Agar (PDA).
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Human influence on European winter wind storms such as those of January 2018
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Human influence on European winter wind storms such
as those of January 2018 as those of January 2018
Robert Vautard1, Geert Jan van Oldenborgh2, Friederike E. L. Otto3, Pascal Yiou1, Hylke de Vries2,
Erik van Meijgaard2, Andrew Stepek2, Jean-Michel Soubeyroux4, Sjoukje Philip2, Sarah F. Kew2,
Cecilia Costella5, Roop Singh5, and Claudia Tebaldi6
1Laboratoire des Sciences du Climat et de l’Environnement, UMR 8212 CEA/CNRS/UVSQ, IPSL and
University Paris-Saclay, Gif-sur-Yvette, France
2Royal Netherlands Meteorological Institute (KNMI), De Bilt, the Netherlands
3Environmental Change Institute, University of Oxford, Oxford, UK
4Météo-France, Direction des Services Climatiques, Toulouse, France
5Red Cross Red Crescent Climate Centre, The Hague, the Netherlands
6National Center for Atmospheric Research, Boulder, Colorado, USA
Correspondence: Robert Vautard (robert.vautard@lsce.ipsl.fr)
Received: 3 August 2018 – Discussion started: 20 September 2018
Revised: 3 February 2019 – Accepted: 8 March 2019 – Published: 25 April 2019 Robert Vautard1, Geert Jan van Oldenborgh2, Friederike E. L. Otto3, Pascal Yiou1, Hylke de Vries2,
Erik van Meijgaard2, Andrew Stepek2, Jean-Michel Soubeyroux4, Sjoukje Philip2, Sarah F. Kew2,
Cecilia Costella5, Roop Singh5, and Claudia Tebaldi6 Correspondence: Robert Vautard (robert.vautard@lsce.ipsl.fr) Received: 3 August 2018 – Discussion started: 20 September 2018
Revised: 3 February 2019 – Accepted: 8 March 2019 – Published: 25 April 2019 Abstract. Several major storms pounded western Europe in January 2018, generating large damages and casu-
alties. The two most impactful ones, Eleanor and Friederike, are analysed here in the context of climate change. Near surface wind speed station observations exhibit a decreasing trend in the frequency of strong winds asso-
ciated with such storms. High-resolution regional climate models, on the other hand, show no trend up to now
and a small increase in storminess in future due to climate change. This shows that factors other than climate
change, which are not in the climate models, caused the observed decline in storminess over land. A large part is
probably due to increases in surface roughness, as shown for a small set of stations covering the Netherlands and
in previous studies. This observed trend could therefore be independent from climate evolution. We concluded
that human-induced climate change has had so far no significant influence on storms like the two mentioned. However, all simulations indicate that global warming could lead to a marginal increase (0 %–20 %) in the prob-
ability of extreme hourly winds until the middle of the century, consistent with previous modelling studies. Human influence on European winter wind storms such
as those of January 2018 This
excludes other factors, such as surface roughness, aerosols, and decadal variability, which have up to now caused
a much larger negative trend. Until these factors are correctly simulated by climate models, we cannot give
credible projections of future storminess over land in Europe. Earth Syst. Dynam., 10, 271–286, 2019
https://doi.org/10.5194/esd-10-271-2019
© Author(s) 2019. This work is distributed under
the Creative Commons Attribution 4.0 License. 1
Introduction been found in observations (Smits et al., 2005; Wever, 2012),
consistent with observed large-scale near-surface wind de-
creases found over continental areas (Vautard et al., 2010;
McVicar et al., 2012). By contrast, a more zonal flow is
expected from climate projections (Haarsma et al., 2013),
inducing a mean large-scale circulation more favourable to
winter wind storms. Over the middle of northern Europe,
along the track of highest mean wind speeds, a slight increase
in extreme wind speeds was found in several model stud-
ies (Ulbrich et al., 2009; Mölter et al., 2016; Vautard et al., The influence of climate change on extratropical storms has
been the subject of a number of studies so far (Ulbrich et al.,
2009). It has been demonstrated that with the expansion of
the Hadley cell the jet streams and storm tracks are moving
poleward (Yin, 2005; Bengtsson et al., 2006; Ulbrich et al.,
2008; Li et al., 2018). However, conflicting results regard-
ing wind storm intensities have not allowed a clear under-
standing of expected changes in the evolution of extratropi-
cal wind storms. A decreasing trend in storminess indices has Published by Copernicus Publications on behalf of the European Geosciences Union. 272 R. Vautard et al.: Human influence on European winter wind storms R. Vautard et al.: Human influence on European winter wind storms Storm Friederike led to at least 11 casualties and major
disruptions in the Netherlands and parts of Germany. In ad-
vance of storm Friederike, warnings were issued in both the
Netherlands and Germany for severe wind gusts. On 18 Jan-
uary, the timing of the strongest winds was around 09:00–
11:00 LT (local time, UTC+1) just after the peak of the morn-
ing commute, with many people already on the road and in
some cases caught by the strong winds. In addition to the
wind hazard, snow created icy road conditions, causing car
accidents with casualties. In Germany, according to the In-
surance Journal (2018), storm Friederike is estimated to have
caused around EUR 1.6 billion worth of damage. The authors
estimate that this was the second most expensive storm to
strike Germany in the past 20 years. In the Netherlands, three
people were killed during the storm. For the first time in his-
tory, train traffic was completely shut down across the coun-
try. Amsterdam Airport Schiphol was closed and more than
300 flights were cancelled. 1
Introduction Numerous roads were blocked by
fallen trees and overturned trucks. Due to their height, trucks
were susceptible to being blown off the roads, which caused
disruptions and accidents. 2014), while no consistent changes were found in wind storm
number or intensities over the Mediterranean areas (Nissen et
al., 2014). The frequency of occurrence of “sting jets”, some-
times found in the strongest wind storms in the north-east
Atlantic, has been suggested to be increasing, from climate
model simulations (Martínez-Alvarado et al., 2018), but the
area of concern is mostly over ocean. Attribution of extreme weather events, an emerging sci-
entific area (Stott et al., 2016), attempts to study changes
that occurred for certain classes of events with specific mag-
nitude, spatial scale, and timescale. The link between such
events and climate change is often questioned by the me-
dia and the public when they occur; even though it may not
yet be mature enough for these purposes, event attribution
can also potentially be used for responsibility assessment
when impacts and losses are present. While storm changes
have been studied as a broad category, only few event at-
tribution studies analysed the influence of human activities
on such types of extratropical wind storms. There are few
other studies on observed trends in wind storms over Europe
and those results were mostly inconclusive. Vose et al. (2014)
call trends over land “inconclusive”, but find a trend over sea. Barredo (2010) finds no upward trend in losses indicating in-
significant storminess change. Beniston (2007) finds a sharp
decline in wind storms in Switzerland since about 1980 but a
connection to the North Atlantic Oscillation (NAO) evolution
is proposed. The other major storm, storm Eleanor, led to major dis-
ruptions in France during the ski holiday season and is esti-
mated to have cost as much as EUR 700 million (Insurance
Journal, 2018). Ski resorts were closed for one or two days
in the Alps, with significant economic consequences. Wind
gusts of more than 130 km h−1 and nearing 150 km h−1 were
reported over several flat regions in France and Switzerland. Large waves on the Atlantic coasts of Spain and France killed
two people. Over France, according to the severity index de-
veloped by Météo-France, Eléanor was the sixth most severe
storm since 1995. 1
Introduction In this article we take as an example two of the devastating
wind storms that occurred during January 2018 in western
Europe and analyse, using event attribution techniques, how
the frequency of such storms has been and will be altered by
human activities. For the first time we analyse both obser-
vations and high-resolution regional climate projections for
our analysis, which are shown to fairly well simulate extreme
wind speeds that are present in such storms. The strongest wind gusts estimated from the European
Centre for Medium-Range Weather Forecasts (ECMWF) are
shown in Fig. 1 for both storms, which have a very different
pattern. In Sect. 2, we describe the meteorological context of the
stormy month of 2018 in western Europe, the events stud-
ied, and their impacts. In Sect. 3, a quantitative character-
ization of the events is provided based on the analysis of
observations. In Sect. 4, models and observations used are
described. Sections 5 and 6 develop the analysis of each of
the two storms analysed and Sect. 7 provides a summary and
synthesis of the findings. More storms than these two were reported during Jan-
uary 2018. For instance, storm Carmen, which preceded
storm Eleanor by two days, crossed southern France with
wind gusts exceeding 130 km h−1. On 17 January, another
storm, Fionn, passed over parts of the Mediterranean re-
gion and broke wind speed records, such as at Cap Corse,
the northern edge of Corsica (winds reached 225 km h−1). In terms of number of events, January 2018 is the stormiest
month since 1998 in France. 3
Event definitions Classical event attribution relies on defining an event as an
exceedance of a threshold in the tail of the distribution of
an event indicator. Once the indicator is defined the proba-
bility of exceeding the threshold is calculated for the current
climate and for a hypothetical climate where anthropogenic
influence is not present or largely reduced. Once this is done,
the ratio of the probabilities (probability ratio denoted here-
after as “PR”) is estimated. It indicates how much more likely
(PR >1) or unlikely (PR <1) the event is between the two
climates. We define in this section the indicators associated
with the two studied storms. This area contains 68 stations observing wind speed. The
area average cannot be exactly calculated using the sta-
tions because the distribution of the stations is not even or
dense enough, but we take the station average as a reason-
able approximation. Using this indicator, storm Friederike
is the eleventh strongest storm in the area since 1 Jan-
uary 1976, with an indicator value of 16.0 m s−1 max daily
wind. The 2017–2018 winter season (DJF) becomes the
seventh strongest in terms of strongest winter winds over
this station network. However, this storm was not the sev-
enth strongest storm as some seasons had multiple stronger
storms. We also considered the daily mean wind for models
that did not store higher-frequency data. In terms of that indi-
cator, Friederike was not remarkable with 8.7 m s−1, as it was
a very short duration storm with a calm period immediately
following it, bringing the daily mean to a moderate value. Storm Friederike was the result of rapidly developing cy-
clogenesis and the area with highest wind speeds, located
south of the trough centre, moved rapidly from west to east. It crossed the Netherlands and central Germany in about half
a day. In this analysis, the salient event characteristics will
be represented by an indicator defined on the basis of daily
maximum wind speed, derived from observations available
from the Integrated Surface Database (“Lite” version, ISD-
Lite; Smith et al., 2011). The database contains global hourly
weather data for eight variables. Many of these observations
are made at airports from cup anemometers. However, many
stations only contain three-hourly data for the earlier part
of the record. 2
The stormy month of January 2018 and the
studied storm cases This exceptional storm activity was due to a strong west-
erly flow that persisted throughout the month (as shown in
Fig. 2, first row) was enhanced by the jet stream extension
eastward of its normal position. The persistence of the flow is
also characterized by the frequency of occurrence of the so-
called “zonal weather regime” (ZO), as defined by Michelan-
geli et al. (1995) using cluster analysis on sea level pres-
sure (SLP) data from the NCAR/NCEP reanalysis. Approxi-
mately 45 % of the January days were classified in this clus-
ter (Fig. 2, remaining panels), which is characterized by mild The year 2018 started with a series of four strong wind
storms over western Europe. In particular, two major events
pounded the continent: one on 3 January, named storm
Eleanor by the Irish Meteorological Service (Met Éireann),
and another one on 18 January, named storm Friederike by
the Berlin Institut für Meteorologie. Earth Syst. Dynam., 10, 271–286, 2019 www.earth-syst-dynam.net/10/271/2019/ 273 R. Vautard et al.: Human influence on European winter wind storms Figure 1. Strongest wind gusts during the storms Friederike (a) and Eleanor (b) as estimated from the ECMWF deterministic forecasts
starting at 18 January 2018 00:00 UTC and 3 January 2018 00:00 UTC respectively. White contours are used to indicate areas where gusts
exceed 118 km h−1. The boxes indicate the spatial event definitions (Sect. 3). Figure 1. Strongest wind gusts during the storms Friederike (a) and Eleanor (b) as estimated from the ECMWF deterministic forecasts
starting at 18 January 2018 00:00 UTC and 3 January 2018 00:00 UTC respectively. White contours are used to indicate areas where gusts
exceed 118 km h−1. The boxes indicate the spatial event definitions (Sect. 3). speed was calculated only if at least four of the eight sam-
pled observations were available. and wet winter weather. The average frequency of the ZO
weather regime is close to 25 %. Although not exceptional,
this high frequency is significantly higher than normal. In Fig. 3, we plot the values of the daily maximum
wind observed over northwestern Europe on the days of
the storms. The track of storm Friederike (Fig. 3a) can be
seen in the box (50–53◦N, 2–15◦E) where wind speeds
are largest. We therefore selected the seasonal (December–
January–February, DJF) maximum value of this land area
average of daily maximum wind speed as the event indica-
tor (see also Fig. 1a). Earth Syst. Dynam., 10, 271–286, 2019 3
Event definitions Also, when analysing outputs from some of
the models contributing to EURO-CORDEX, the daily max-
imum near-surface wind speed was obtained on the basis of
three-hourly wind speeds. For these reasons, we only sam-
pled observations every 3 h and the daily maximum wind For models, the area average is calculated over land grid
points, which slightly lowers the indicator value (see com-
parisons in Table 1 for model evaluation) as the stations are
concentrated near the coast where the intensity was higher. In
order to calculate seasonal return periods, we take the maxi-
mum value of the indicator over the winter season (DJF). The structure of storm Eleanor was very different. Eleanor
was embedded in a deep large-scale low-pressure system. Its strong winds affected a much broader area than storm www.earth-syst-dynam.net/10/271/2019/ Earth Syst. Dynam., 10, 271–286, 2019 Earth Syst. Dynam., 10, 271–286, 2019 74
R. Vautard et al.: Human influence on European winter wind storm
igure 2. (a) Sea level pressure (left) and anomaly (right) (ECMWF analysis, ERA-Interim climatology); (b) first row: weather regi
uster centroids from NCAR/NCEP reanalysis; second row: occurrence of weather regimes from 1 December 2017 to 28 February 2018;
ertical bars indicate the preferred centroid (NAO-; Atlantic Ridge, AR; Scandinavian Blocking, BLO; Zonal, ZO) and the coloured circ
ndicate the spatial correlation with the preferred centroid; third row: weather regime wintertime frequencies from 1948 to 2018. i d ik
f
I l
d
d th UK
i
t
F
t
f
i d
d b t
d
h
id R. Vautard et al.: Human influence on European winter wind storms R. Vautard et al.: Human influence on European winter wind storms 274 Figure 2. (a) Sea level pressure (left) and anomaly (right) (ECMWF analysis, ERA-Interim climatology); (b) first row: weather re
cluster centroids from NCAR/NCEP reanalysis; second row: occurrence of weather regimes from 1 December 2017 to 28 February 2018 Figure 2. (a) Sea level pressure (left) and anomaly (right) (ECMWF analysis, ERA-Interim climatology); (b) first row: weather regime
cluster centroids from NCAR/NCEP reanalysis; second row: occurrence of weather regimes from 1 December 2017 to 28 February 2018; the
vertical bars indicate the preferred centroid (NAO-; Atlantic Ridge, AR; Scandinavian Blocking, BLO; Zonal, ZO) and the coloured circles
indicate the spatial correlation with the preferred centroid; third row: weather regime wintertime frequencies from 1948 to 2018. Friederike: from Ireland and the UK via western France to
Switzerland and the Riviera coast. Its high wind speeds, un-
usual in western Europe, constituted its most striking aspect. As this storm also passed within a day, we construct the same
indicators as for Friederike, which are daily maximum and mean of wind speed, but averaged over a much wider area,
from 42 to 52◦N and 0 to 10◦E (see Figs. 1b and 3b). The
value of the indicator is 12.3 m s−1 for maximum winds and
8.3 m s−1 for daily mean winds. www.earth-syst-dynam.net/10/271/2019/ Earth Syst. Dynam., 10, 271–286, 2019 www.earth-syst-dynam.net/10/271/2019/ 1 Only daily mean winds available, so statistics only from daily means. 2 For Eleanor, averages made with stations north of 43.5◦N are in
square brackets. R. Vautard et al.: Human influence on European winter wind storms 275 R. Vautard et al.: Human influence on European winter wind storms Figure 3. Daily maximum wind speeds at ISD-Lite stations over northwestern Europe and area defining the event indicator for storms
(a) Friederike and (b) Eleanor. Figure 3. Daily maximum wind speeds at ISD-Lite stations over northwestern Europe and area defining the event indicator for storms
(a) Friederike and (b) Eleanor. Table 1. Mean (WXm), 95th (WX95), and 99th (WX99) percentiles of the distribution of the daily maxima of wind speed averaged over
the 68 stations or the land grid points (for models) for the winter season. Italics indicate that the daily mean rather than the daily maxima of
the wind speed statistic has been used. Observation cells (Obs.) are the first two data rows, the upper one gives the daily maximum of the
wind speed statistic, and the lower one (in italics) gives the daily mean of the wind speed statistic. For the three model ensembles (RACMO,
EURO-CORDEX (pooled), and HadGEM3-A), both station (station) and area (area) averages are shown. For weather@home only the area
average was available. Model/ensemble
WXm
WX95
WX99
WXm
WX95
WX99
(values in metres per second)
Friederike
Eleanor
Obs. (ISD-Lite) daily max2
6.4
11.4
13.5
6.1 [5.8]
9.7 [9.5]
11.1 [11.0]
Daily mean
4.5
8.4
10.1
4.1
6.6
7.7
RACMO (16 members) (station)
6.8
10.9
12.6
5.3
8.2
9.4
RACMO (16 members) (area)
6.6
10.4
12.0
5.5
8.5
9.8
HADGEM3-A1 (15 members) (station)
4.4
8.1
9.6
3.5
5.7
6.7
HADGEM3-A1 (15 members) (area)
3.9
7.6
9.0
3.1
5.4
6.3
bc-EURO-CORDEX (pooled) (station)
5.9
10.2
12.0
4.7
7.5
8.7
bc-EURO-CORDEX (pooled) (area)
5.6
9.7
11.5
4.7
7.8
9.1
bc-ARPEGE (zoomed version)
5.7
9.8
11.9
4.8
7.6
8.9
bc-RACMO+HADGEM
6.2
10.4
12.0
4.9
7.9
9.0
bc-RACMO+EC-EARTH
6.2
10.4
12.2
4.7
7.8
9.2
bc-REMO+MPI
6.0
10.2
12.1
4.7
7.8
9.0
bc-WRF+IPSL
5.9
10.2
12.1
4.7
7.9
9.0
bc-HIRHAM+EC-EARTH
5.8
10.2
11.8
4.7
7.8
9.2
bc-RCA+ARPEGE
5.8
10.0
11.8
4.7
7.9
9.2
bc-RCA+IPSL
5.8
10.0
11.7
4.7
7.8
9.2
bc-RCA+HADGEM
5.7
10.1
11.6
4.7
7.9
9.2
bc-RCA+MPI
5.9
10.0
11.9
4.8
8.1
9.1
bc-RCA+EC-EARTH
5.6
10.0
11.8
4.5
7.8
9.0
weather@home1
6.4
11
11.8
5.22
8.45
9.03
1 Only daily mean winds available, so statistics only from daily means. 2 For Eleanor, averages made with stations north of 43.5◦N are in
square brackets. 1 Only daily mean winds available, so statistics only from daily means. 4
Observations, model ensembles, and evaluation Such a calculation
is made by multiplying wind speeds by “exposure correction
factors” which to first order account for changes in the el-
evation of the wind anemometer and changes in roughness
surrounding the station in different directions. These factors
are deduced from the high-frequency variability in the wind
(intra 10 min standard deviation or wind gust). These expo-
sure correction factors are recomputed every 3 years. Three
years of measurements are required to ensure that there are
at least 10 appropriate measurements in each of the wind di-
rection sectors. If a new exposure correction factor is found
to be significantly different (absolute difference >0.05) from
the existing factor the new factor is introduced. The second ensemble is the multimodel EURO-CORDEX
ensemble (Jacob et al., 2014), using a 0.11◦resolution
over Europe. For this ensemble, only 11 simulations were
used and a bias correction was applied using the cumu-
lative distribution function transform (CDFt; Vrac et al.,
2016). These simulations have been evaluated in the context
of the CLIM4ENERGY Copernicus Climate Change Ser-
vice project (http://clim4energy.climate.copernicus.eu, last
access: 25 July 2018). The reference data used for bias cor-
rection is the Watch Forcing Data ERA-Interim (WFDEI;
Weedon et al., 2014). For wind speed, it is essentially an
interpolation of ERA-Interim over a 0.5◦× 0.5◦grid. This
dataset has a relatively low resolution, so extreme winds are
not expected to be accurately represented. This weakness is,
therefore, probably propagated to the EURO-CORDEX en-
semble. The ensemble is pooled, which is formally possible
because the bias correction method corrects data making it
homogeneous across the multimodel distribution. We used four complementary ensembles of climate model
simulations. Two of them are made of regional climate simu-
lations by downscaling low-resolution global climate models
(GCMs) with a high resolution (12.5 km). One of these is
using the same model chain with different members for the
GCM, while the second one is a multimodel ensemble mem-
ber. We therefore cover several aspects of the uncertainty. The other two ensembles were available at the time of the
study and also used, one of which consists of a very large en-
semble. However, for these latter ensembles only daily mean
wind speed was available while for the former daily maxi-
mum wind speed was available. 4
Observations, model ensembles, and evaluation analysed. In RACMO, the near-surface wind speed is diag-
nosed from the model wind and stability vertical profile as
the wind speed at 10 m, applying a roughness length of at
most 3 cm for land grid points, and a Charnock-type rela-
tion for sea grid points (van Meijgaard et al., 2008). This
ensemble was previously used to estimate the change in the
odds of wind stagnations in northwestern Europe (Vautard
et al., 2017) and was found to simulate monthly wintertime
wind speeds over western Europe in a satisfactory manner. RACMO simulations are available for the 1950–2100 period. As in previous analyses (e.g. Philip et al., 2018), we use a
20th century early 30-year period (1951–1980) to estimate
odds in the past climate, and the 2001–2030 period to esti-
mate odds in the current climate. We also use two future pe-
riods, a period called “near future” (2021–2050) and a period
called “mid-century” (1941–1970). We only used the simula-
tions using the RCP8.5 radiative forcing scenario. As a cross-
check we fitted a time-dependent generalized extreme value
(GEV) function to the whole period 1971–2070, as described
in van der Wiel et al. (2017). For the observational part of the attribution analysis, we
used two sources of station data. Unfortunately, the available
quantities were slightly different in the different datasets. The
analysis is mainly based on the ISD-Lite database described
above, in which we used the daily maximum of three-hourly
instantaneous wind speed. Additional results are based on
the KNMI climatological service database, which provides
the daily maximum of the hourly averaged wind speed at
34 weather stations in the Netherlands. The highest hourly
wind of the year series were visually quality controlled. For
three series, early data were discarded for obvious inho-
mogeneities supported by the metadata (Leeuwarden before
1990; De Bilt before 2002; Lichteiland Goeree before 1995). Most series start in 1981, but they are notably more variable
and possibly unreliable before circa 1990. The KNMI data at these stations plus 22 sea stations were
also converted to potential winds, i.e. the wind speed at 10 m
that would have occurred assuming a roughness length of
3 cm over land and 2 mm over water, and assuming neu-
tral stability (Wever and Groen, 2009). R. Vautard et al.: Human influence on European winter wind storms 2 For Eleanor, averages made with stations north of 43.5◦N are in
square brackets. Earth Syst. Dynam., 10, 271–286, 2019 www.earth-syst-dynam.net/10/271/2019/ R. Vautard et al.: Human influence on European winter wind storms 276 European winter wind storms 4
Observations, model ensembles, and evaluation 4
Observations, model ensembles, and evaluation tics. For EURO-CORDEX, we calculated both individual
model and pooled statistics. Results are presented in Table 1
for the average over all grid points closest to the 68 ISD-Lite
stations, together with equivalent statistics when the average
is made over all land grid points, instead of the positions
of the stations. Results show that the models reproduce the
indicator with success along the distribution. Comparisons
to station data indicate a general underestimation of mod-
els within a 10 % range. EURO-CORDEX simulations are
bias corrected, so the bias is essentially reflecting the WFDEI
(ERA-Interim based) bias. The fact that statistics do not dif-
fer from one model to the other supports pooling the mod-
els’ simulations together in a common distribution. This bias
is consistent with models not simulating observational noise
due to remaining turbulence. For weather@home, we only
have daily values for mean wind speed, so we calculate the
maximum mean area-averaged daily wind speed in a winter
season. The simulated values are higher than the observed
values for this quantity, especially for the mean, while the
95th and 99th percentiles are comparable to observations in
particular for storm Friederike. the Met Office Hadley Centre for Climate Science and Ser-
vices regional climate model HadRM3P at 25 km resolution
over Europe embedded in the atmosphere-only global circu-
lation model HadAM3P at N96 resolution. The first set of
ensembles represents possible winter weather under current
climate conditions. This ensemble is called the “all forcings”
scenario and includes human-caused climate change. The
second set of ensembles represents possible winter weather
in a world as it might have been without anthropogenic cli-
mate drivers, using different estimates of pre-industrial SST
deduced from the CMIP5 ensemble and pre-industrial green-
house gas and aerosol concentrations. Land-use in both en-
sembles is identical. This ensemble is called the “natural” or
“counterfactual” scenario (Schaller et al., 2016). The third
set of ensembles represents a future scenario in which the
global mean surface temperature is 1.5 ◦C higher than pre-
industrial global temperatures. The fourth scenario is the
same as the third, but for 2 ◦C of future global mean tem-
perature anomaly. To simulate the third and fourth scenar-
ios, we use atmospheric forcings derived from RCP2.6 and
RCP4.5 and sea surface temperatures that match the atmo-
spheric forcing obtained from CMIP5 simulations (Mitchell
et al., 2017). 4
Observations, model ensembles, and evaluation Our assessment is therefore
rather based on the first two ensembles, which better repre-
sent the January 2018 storms, the other ensembles being used
for consistency checking. The third model ensemble is the HadGEM3-A ensemble
(Ciavarella et al., 2018; Vautard et al., 2018), which includes
a set of 15 realizations of atmospheric simulations using ob-
served sea surface temperatures (SSTs; reflecting the actual
world) and a set using SSTs where the CMIP5 mean patterns
of anthropogenic heat contribution are removed to estimate
the ocean response to a pre-industrial atmospheric compo-
sition (as the natural/counterfactual world). The latter runs
also use pre-industrial greenhouse gas and aerosol concentra-
tions. Land use, and hence roughness, is set to 1850 values in
the counterfactual ensemble. For this model, the wind speed
daily maximum was not available and the daily mean wind
was used instead. No future simulations were available. The first ensemble is the RACMO regional climate model
ensemble downscaling 16 initial-condition realizations of
the EC-EARTH 2.3 coupled climate model in the CMIP5
RCP8.5 scenario (Lenderink et al., 2014; Aalbers et al.,
2017). The RACMO model uses a 0.11◦(12 km) resolu-
tion and the daily maximum of near-surface wind speed is The fourth ensemble is obtained from simulations
using the distributed computing framework known as
weather@home (Massey et al., 2015). We used four different
large ensembles of December–February wind speeds using www.earth-syst-dynam.net/10/271/2019/ Earth Syst. Dynam., 10, 271–286, 2019 R. Vautard et al.: Human influence on European winter wind storms 277 Figure 4. (a) Highest winter value of the Friederike index described in Sect. 2 fitted to a generalized extreme value (GEV) function that
scales with time. The thick line denotes the position parameter µ, the thin lines the 1 in 6 years and 1 in 40 years return values. (b) The GEV
fit as a function of return period for the climate of 1976 (blue, observations have been scaled up with the fitted trend) and 2018 (red). Figure 4. (a) Highest winter value of the Friederike index described in Sect. 2 fitted to a generalized extreme value (GEV) function that
scales with time. The thick line denotes the position parameter µ, the thin lines the 1 in 6 years and 1 in 40 years return values. (b) The GEV
fit as a function of return period for the climate of 1976 (blue, observations have been scaled up with the fitted trend) and 2018 (red). www.earth-syst-dynam.net/10/271/2019/ 4
Observations, model ensembles, and evaluation the winter maximum of the daily maximum wind speed av-
eraged over a specific area depending on the storm studied
(see below for the definition for each case) in the current
climate with the probability of the exceedance in a world
where anthropogenic forcing on climate was not present or
was weaker. For observations, this is done by fitting an ex-
treme value distribution as observations are not numerous. In
this case the parameters of the distribution are taken to be
functions of global temperature, as in previous studies (e.g. Philip et al., 2018). Using model ensembles, this is done in
a nonparametric way by pooling all winter maxima of each
ensemble member into a single pool and computing the prob-
ability by counting the number of exceedances of the thresh-
old. To obtain confidence intervals, this procedure is done
within a bootstrap framework where random drawings are
done including possible repetitions. The 95 % confidence in-
tervals are obtained by taking the 5th and the 95th return peri-
ods of the bootstrap sample, in a procedure similar to Vautard
et al. (2017), while the median value is used as a best esti-
mate. Once a value is selected in the bootstrap, one forces
the whole model series to be selected. The probability ratios
(PRs) are calculated in the same way by calculating ratios
of probabilities for each return value in a bootstrap frame-
work. The reference in this case is taken as the “current cli-
mate” and ratios are calculated relative to this period. When
the confidence interval of this ratio does not include 1, one
concludes that the probability is significantly changing due
to climate change. Results from observations and models are
comparable in terms of climate time period as the parametric
method used for observations accounts for time dependence. The return period of an event like Friederike or worse in
the area in which the indicator value reached 16.0 m s−1 on
18 January in the current climate is 13.5 years as estimated
directly from the data (Table 1). By looking at the GEV fits
(Fig. 4), in the 1970s, this was roughly 5 years, so the event,
defined using our indicator, has become a fairly rare event
due to the decrease in high wind speeds observed during this
period. 4
Observations, model ensembles, and evaluation Grid point averages reach lower values than station aver-
ages, which is a probable consequence of the higher density
of stations near the North Sea coast where winds are stronger,
which is reflected in the observed area average. The factor
between observation statistics and model statistics for sta-
tion averages is rather uniform across the distributions, even
though the distribution is more heavy-tailed for observations
than for simulations. In order to homogenize attribution re-
sults among models and observations and compare return pe-
riods with observations, we scaled all simulations by the ra-
tio between 99th centiles of observed station averages and
simulated grid-point averages. These bias corrections are a
factor of 1.13 for RACMO (for both storms), 1.17 (1.28) for The evaluation of the models’ ability to simulate the in-
dicator is made using the ISD-Lite observations, which are
available in near-real time. In order to evaluate the capacity
of the models to simulate the winds, we extracted wind speed
daily maxima at the locations of ISD-Lite stations and aver-
aged these values over all stations in the area. Then, we com-
pared the simulated mean, 95th centile, and 99th centile with
the observed equivalent for each model ensemble (Table 1). For HadGEM3-A and weather@home, as daily maxima were
not available, we used daily averages of the wind speeds. For RACMO, HadGEM3-A, and weather@home, model
values are pooled together to compute the distribution statis- Earth Syst. Dynam., 10, 271–286, 2019 R. Vautard et al.: Human influence on European winter wind storms 278 EURO-CORDEX for Friederike (Eleanor), and 1.12 (1.22)
for HadGEM3-A for Friederike (Eleanor). EURO-CORDEX for Friederike (Eleanor), and 1.12 (1.22)
for HadGEM3-A for Friederike (Eleanor). a factor of 4 (95 % CI: 1 to 100) (see Table 2). Using the
global mean temperature as a covariate instead of time gives
slightly higher trends. The shape parameter ξ of the GEV is
most likely negative, so the distribution has a tail that is thin-
ner than an exponential distribution. This implies that the ra-
tio of probabilities is larger for the same difference in wind
speed for a higher baseline, i.e. stronger storms. In each case, the attribution of the event consists of com-
paring probabilities of the exceedance of an indicator, e.g. 5.2
RACMO ensemble The storm indicator is scaled to have the same 99th per-
centile as the observed indicator in the historical period. In-
dicator statistics are then obtained for four climate periods:
1951–1980, simulating the “past” period; the “current” pe-
riod taken as 2001–2030; and two future periods assuming
the RCP8.5 scenario (2021–2050 and 2041–2070). The ob-
served indicator value for storm Friederike (16.0 m s−1) has
a return period of about 13 years for the current climate
(95 % CI 10–19 years, see Fig. 4 and Table 1), which is close
to observed values. The probability of witnessing higher in-
dicator values is no different or marginally significantly dif-
ferent between past and current periods (Fig. 5a and d). How-
ever, the change of probability becomes larger in future peri-
ods, with a probability ratio (PR) of about 1.5 (95 % CI: 1–2)
in the near term (Fig. 5d and Table 2). For this particular
case, the increase is also stronger for stronger storms due to
an increase in the variability relative to the mean. 4
Observations, model ensembles, and evaluation The result is confirmed in a different dataset from KNMI
observations (not shown), with most stations showing a clear
downward trend over the whole period (1971–2017 for two
stations, 1982–2017 for most others). A simultaneous fit to
all stations scaled to the same mean show a decrease in in-
tensity of −15 % (95 % CI: −7 % to −17 %), the same as
the ISD-Lite data show. The trends are much less clear when
starting in 1990 (using stations with at least 25 years of data). The trends in potential wind are much smaller (around −5 %)
and not significantly different from zero, even when pooled
over all stations. 5.2
RACMO ensemble 5.1
Observations (d–f) Probability ratio of exceeding the return value of the indicator as compared
with the counterfactual period as a function of the return value, with 5 %–95 % significance intervals as dashed lines with corresponding
colour, calculated from a nonparametric bootstrap and shown for RACMO (d), EURO-CORDEX (e) and HadGEM3-A (f) ensembles. Figure 5. (a–c) Return values as a function of return periods for the storm Friederike indicator, for differen Table 2. Event return periods and probability ratios summarized for all model ensembles and for storm Friederike. Probability ratios (PRs)
are calculated with respect to a past or counterfactual period. PI is pre-industrial and n/a is not applicable. Table 2. Event return periods and probability ratios summarized for all model ensembles and for storm Friederike. Probability ratios (PRs)
are calculated with respect to a past or counterfactual period. PI is pre-industrial and n/a is not applicable. ble 2. Event return periods and probability ratios summarized for all model ensembles and for storm Friederi
e calculated with respect to a past or counterfactual period. PI is pre-industrial and n/a is not applicable. Table 2. Event return periods and probability ratios summarized for all model ensembles and for storm Friederike. Probability ratios (PRs)
are calculated with respect to a past or counterfactual period. PI is pre-industrial and n/a is not applicable. Table 2. Event return periods and probability ratios summarized for all model ensembles and for storm Friederike. Probability ratios (PRs)
are calculated with respect to a past or counterfactual period. PI is pre-industrial and n/a is not applicable. Ensemble
Ret. period (year)
PR for current
PR for period
PR for period
PR for period
PR for period
climate
2021–2050
2041–2070
PI +1.5 ◦C
PI +2.0 ◦C
Obs. ISD-Lite
12.5 [8–400]
1/4 [1/100–1]
n/a
n/a
n/a
n/a
Models using wind speed daily maximum (over three-hourly data)
RACMO
15 [11–20]
1.1 [0.8–1.7]
1.5 [1.0–2.2]
1.5 [1.1–2.3]
–
–
EURO-CORDEX
40 [25–80]
0.9 [0.4–2.0]
1.4 [0.7–3.0]
1.6 [0.8–4.1]
–
–
Models using wind speed daily mean
HadGEM3-A
1.2 [1.15–1.27]
1.02 [0.98–1.06]
–
–
–
–
weather@home
1.3 [1.29–1.35]
1.03 [0.97–1.2]
–
–
1.039 [0.98–1.16]
1.04 [0.98–1.17] 5.1
Observations As mentioned before, we compute the daily maximum of
three-hourly wind speed (taken at 00:00, 03:00, 06:00, 09:00,
12:00, 15:00, 18:00, and 21:00 UTC) at each station and av-
eraged it over the ISD-Lite stations available in the box. The
winter maximum of this quantity is shown in Fig. 4a as a
function of time (labelled with the year of the second half of
winter). The data have been fitted by a GEV distribution in
which the location parameter µ and scale parameter σ vary
exponentially with time, such that their ratio remains con-
stant. The shape parameter ξ is not time-dependent. This fit
shows a significant decrease (p <0.05 two-sided) in wind
speed over 1976–2017, in agreement with earlier analyses
(Smits et al., 2005; Vautard et al., 2010). The decrease in in-
tensity of about 12 % with a 95 % confidence interval (CI) of
0 % to 30 %, which we will denote thereafter (95 % CI: 0 %
to 30 %), corresponds to a decrease in probability of about Therefore, according to this model’s representation, we do
not identify a climate change impact currently, but the in-
crease in probability of storms like Friederike emerges in the
coming decades. A fit with a GEV that scales with the global
mean temperature of the driving EC-Earth ensemble gives
no change (PR between 0.95 and 1.16), overlapping with the
30-year time window analysis (not shown). www.earth-syst-dynam.net/10/271/2019/ Earth Syst. Dynam., 10, 271–286, 2019 R. Vautard et al.: Human influence on European winter wind storms 279 Figure 5. (a–c) Return values as a function of return periods for the storm Friederike indicator, for different time periods and the RACMO (a),
EURO-CORDEX (b), and HadGEM3-A (c) ensembles. (d–f) Probability ratio of exceeding the return value of the indicator as compared
with the counterfactual period as a function of the return value, with 5 %–95 % significance intervals as dashed lines with corresponding
colour, calculated from a nonparametric bootstrap and shown for RACMO (d), EURO-CORDEX (e) and HadGEM3-A (f) ensembles. Figure 5. (a–c) Return values as a function of return periods for the storm Friederike indicator, for different time periods and the RACMO (a),
EURO-CORDEX (b), and HadGEM3-A (c) ensembles. 6.2
RACMO ensemble Storm Eleanor is now investigated through the wind daily
maximum indicator with an average over the large region as
defined above. Due to the southern boundary that is exclud-
ing a small band of the large region, we used a boundary at
43.5◦N instead of 42◦N for this model. This makes the indi-
cator return value for stations slightly lower than when calcu-
lated over the full region (11.9 m s−1 instead of 12.3 m s−1). The corresponding RACMO return period is in the range of
3 to 5 years. The climate change is not significant for the cur-
rent period and marginally significant for future periods, as
for storm Friederike (Fig. 8a, d). The estimated PR is 1.1 and
slightly higher for future periods. Interestingly, for stronger
storms, the PR increases. The same results hold for a GEV
fit with covariate of all data in 1971–2070, with a PR signifi-
cantly different from 1 (95 % CI 1.0 to 1.2, not shown). Figure 6. Return values as a function of return periods for the storm
Friederike indicator, for the weather@home ensemble with 5 %–
95 % significance intervals, calculated from a nonparametric boot-
strap, under the assumptions of actual, counterfactual, 1.5 ◦C, and
2 ◦C warming. 6.3
EURO-CORDEX ensemble Using the EURO-CORDEX ensemble, the return period of
the large-scale storm Eleanor, characterized by the chosen in-
dicator, is estimated to about 7–10 years (Fig. 8b). A climate
change signal is absent in the simulations when comparing
periods 1971–2000 and 2001–2030. For the indicator value,
the PR is in the range [0.5–1]. Only for later periods and for
larger indicator values, a marginally significant increase in
the PR in the range [1–2] can be seen (Fig. 8b, e). A GEV
with a modelled global mean temperature (from EC-Earth)
as covariate also gives a nonsignificant increase with a PR
between 0.99 and 1.15 (95 % CI). 6.4
HadGEM3-A ensemble For HadGEM3-A, using the daily mean wind, we find no
climate change signal in the estimation of the probabilities
of high winds of any magnitude; but for the very extreme
winds, we find marginally significant changes in the direc-
tion of more frequent high winds under current conditions
than under natural conditions (Fig. 8c, f). The estimated re-
turn period for the indicator value corresponding to Eleanor,
which does not fall in the extreme tail, also lies between 3
and 5 years. R. Vautard et al.: Human influence on European winter wind storms R. Vautard et al.: Human influence on European winter wind storms 280 Figure 6. Return values as a function of return periods for the storm
Friederike indicator, for the weather@home ensemble with 5 %–
95 % significance intervals, calculated from a nonparametric boot-
strap, under the assumptions of actual, counterfactual, 1.5 ◦C, and
2 ◦C warming. 5.5
weather@home For weather@home, using the suboptimal definition of
maximum of daily mean wind to define storm Friederike
(8.7 m s−1), we find no significant change in the likelihood
of storms like Friederike to occur (Fig. 6). In contrast to the
EURO-CORDEX assessment, this also holds for rarer events
(not shown). 5.4
HadGEM3-A ensemble The HadGEM3-A ensemble exhibits a significant difference
between actual and counterfactual periods, with a current in-
crease in strong daily mean winds in the area struck by storm
Friederike (Fig. 5c). However, due to the use of the mean
wind speed instead of the maximum wind speed, the indi-
cator does not disentangle extreme winds over a short time
period from less strong winds over an extended time pe-
riod (Fig. 5c). Accordingly, the observed value is not excep-
tional, due to the fast travelling nature of the extremely high
winds in the area: for the value corresponding to Friederike
(8.7 m s−1), such events occur almost every year in both
types of simulations. 6.1
Observations The same observational analysis on the Eleanor index as in
Sect. 4.1 gives a more significant downward trend for this
storm (p <0.01 two-sided), with a decrease of about 20 %
(3 %–35 %) (Fig. 7). This corresponds to an increase in return
period of a factor of 8 (95 % CI 1.5–100). The return period
is also about 20 years in the current climate according to the
fit of Fig. 7. 5.3
EURO-CORDEX ensemble dency for more storms like Friederike in the future with an
anthropogenic signal emergence not yet achieved. The GEV
with smoothed EC-Earth global mean temperature as covari-
ate confirms this conclusion, with an increase in probability
of 1.0 to 1.2 (p ∼0.1); this corresponds to the assumption
that the percentage increase is a constant 0.0 % to 1.4 % per
degree global warming over the whole range of Fig. 5a–b. This assumption holds well over the four 30-year time peri-
ods considered before. In the EURO-CORDEX simulations, the return period cor-
responding to the scaled indicator (25–40 years) is larger,
making it a more extreme event (Fig. 5b). The shape of the
distribution is clearly different from that of the RACMO sim-
ulations and that of the observations (compare with Fig. 5a
and b). However, as for RACMO, the PR (Fig. 5e) is not sig-
nificantly different from 1 (see Fig. 5e and the low boundary
of the 95 % confidence interval), despite systematic values
of PR above 1. Such an increase becomes marginally signifi-
cant in the middle of the century with PR values in the range
1 to 3 for lower wind thresholds. Again, this indicates a ten- Earth Syst. Dynam., 10, 271–286, 2019 www.earth-syst-dynam.net/10/271/2019/ 6
Storm Eleanor Results for storm Eleanor confirm the findings obtained for
storm Friederike. All numerical results are presented in Ta-
ble 3. Earth Syst. Dynam., 10, 271–286, 2019 www.earth-syst-dynam.net/10/271/2019/ R. Vautard et al.: Human influence on European winter wind storms 281 p
Figure 7. Same as Fig. 4 but for the Eleanor index. Figure 8. Same as Fig. 5 for storm Eleanor. Fi
7 S
Fi
4 b
f
h El
i d Figure 7. Same as Fig. 4 but for the Eleanor index. Figure 8. Same as Fig. 5 for storm Eleanor. Figure 8. Same as Fig. 5 for storm Eleanor. 6.5
weather@home sults to changes in the domain definition, we have recalcu-
lated the changes in return periods of the same indicator cal-
culated over a smaller domain and a larger domain for each
storm (see Table 4 for the domain coordinates). This calcula-
tion is made only with the RACMO simulations. Results for
each domain are reported in Table 4. Each row indicates the
return period of an event with the same current-climate re-
turn period as the storm for each domain. Changing domain
induces a change in return value for a given return period. Confidence intervals are indicated. As shown in the table,
the return period almost systematically decreases with time,
but 95 % significance is generally not reached for the differ-
ence between current period and past period. Our result is
therefore robust to a change in the domain definition. For weather@home, using the maximum of daily mean wind
to define storm Eleanor (8.3 m s−1), we find no significant
change in the likelihood of storms like Eleanor to occur
(Fig. 9). In contrast to the EURO-CORDEX assessment, this
also holds for rarer events where the weather@home model
shows a nonsignificant decrease in high wind speeds. www.earth-syst-dynam.net/10/271/2019/ Earth Syst. Dynam., 10, 271–286, 2019 7
Sensitivity to the domain definition Return periods are all given for return value characteristics
of the storms (15 years for Friederike and 4.2 years for Eleanor); F0 is storm Friederike, domain as in Table 2; F1 is the same with a smaller
domain focused more on the Netherlands; F2 is larger domain including parts of UK; E0 is storm Eleanor, as in Table 3; E1 is the smaller
domain; E2 is the larger domain. The coordinates (Lat, Long) of domains are indicated in the first data row. Bold numbers are significantly
different from the value in 1951–1980. Table 4. Sensitivity experiment results. Return periods and their confidence intervals (in square brackets) for the different time periods and the
RACMO simulations. Each column contains results for one domain experiment. Return periods are all given for return value characteristics
of the storms (15 years for Friederike and 4.2 years for Eleanor); F0 is storm Friederike, domain as in Table 2; F1 is the same with a smaller
domain focused more on the Netherlands; F2 is larger domain including parts of UK; E0 is storm Eleanor, as in Table 3; E1 is the smaller
domain; E2 is the larger domain. The coordinates (Lat, Long) of domains are indicated in the first data row. Bold numbers are significantly
different from the value in 1951–1980. Domain
F0
F1
F2
E0
E1
E2
Coord. (Lat, Long)
50–53, 2–15
50.5–52.5, 3–7
49–54, 0–18
43.5–52, 0–10
44–48, 3–9
43.5–53, −5–10
RP 1951–1980
16.0 [12–22]
11.2 [9–15]
20.0 [15–28]
4.8 [4.2–5.6]
5.1 [4.5–5.9]
4.6 [4.0–5.3]
RP 2001–2030
15.0 [11–20]
15.5 [12–21]
15.0 [11–20]
4.2 [3.7–4.8]
4.2 [3.7–4.8]
4.2 [3.7–4.8]
RP 2021–2050
11.2 [9–14]
9.4 [8–12]
12.3 [10–16]
3.9 [3.4–4.4]
4.1 [3.6–4.8]
3.9 [3.4–4.4]
RP 2041–2070
10.2 [8–13]
9.6 [8-12]
10.4 [8–13]
3.6 [3.2–4.1]
3.6 [3.2–4.1]
3.8 [3.3–4.3] Figure 9. Return values as a function of return periods for the storm
Eleanor indicator for the weather@home ensemble with 5 %–95 %
significance intervals, calculated from a nonparametric bootstrap,
under the assumptions of actual, counterfactual, 1.5 ◦C, and 2 ◦C
warming. (on 18 January 2018) and human-induced climate change
have been studied through this attribution analysis involving
several simulation ensembles and observations from tens of
weather stations. 7
Sensitivity to the domain definition From an analysis of two sets of observations, we conclude
that near-surface storms in the areas of the two storms have
a decreasing trend in wind speed and, hence, in frequency
over the past 40 years, consistent with previous observation-
based studies on storminess in these areas (Smits et al., 2005;
Soubeyroux et al., 2017) and with global land wind stilling
(Vautard et al., 2010; McVicar et al., 2012). This trend was
shown to be close to zero over the Netherlands area when us-
ing the potential wind, indicating a strong influence of rough-
ness changes there, as also demonstrated by Wever (2012). Other processes, such as aerosol increase, could also induce
a wind decrease (Bichet et al., 2012), and decadal-scale long-
term variability has been shown to have a significant role as
well (e.g. Matulla et al., 2008). Figure 9. Return values as a function of return periods for the storm
Eleanor indicator for the weather@home ensemble with 5 %–95 %
significance intervals, calculated from a nonparametric bootstrap,
under the assumptions of actual, counterfactual, 1.5 ◦C, and 2 ◦C
warming. We next turn to the model results. Due to the differing ex-
periments that we used, the probability ratios have been com-
puted over different intervals. To compare those we need to
convert them to a common interval. We do this by assum-
ing the probability ratio is an exponential function of some 7
Sensitivity to the domain definition Domain definitions have been taken from a visual inspection
of areas of maximal wind speeds. However, the choice bears
some arbitrariness. In order to test the robustness of our re- www.earth-syst-dynam.net/10/271/2019/ Earth Syst. Dynam., 10, 271–286, 2019 R. Vautard et al.: Human influence on European winter wind storms 282 Table 3. Event return periods and probability ratios summarized for all model ensembles and for storm Eleanor. PI is pre-industrial and n/a
is not applicable. Ensemble
Ret. period (year)
PR for current
PR for period
PR for period
PR for period
PR for period
climate
2021–2050
2041–2070
PI +1.5 ◦C
PI +2.0 ◦C
Obs. ISD-Lite
20 [6–200]
1/8 [1/100–1/1.5]
n/a
n/a
n/a
n/a
RACMO
4.2 [3.7–4.8]
1.2 [1.0–1.4]
1.3 [1.0–1.5]
1.3 [1.1–1.6]
–
–
HadGEM3-A
3.9 [3.4–4.5]
1.0 [0.8–1.2]
–
–
–
–
EURO-CORDEX
6.6 [5.6–6.9]
0.8 [0.6–1.0]
1.2 [1.0–1.4]
1.2 [0.9–1.6]
–
–
weather@home
13.9 [13.6–15]
1.01 [0.62–2.35]
–
–
0.94 [0.6–2.35]
0.94 [0.59–2.36] Table 3. Event return periods and probability ratios summarized for all model ensembles and for storm Eleanor. PI is pre-industrial and n/a
is not applicable. nd probability ratios summarized for all model ensembles and for storm Eleanor. PI is pre-industrial and n/a Table 4. Sensitivity experiment results. Return periods and their confidence intervals (in square brackets) for the different time periods and the
RACMO simulations. Each column contains results for one domain experiment. Return periods are all given for return value characteristics
of the storms (15 years for Friederike and 4.2 years for Eleanor); F0 is storm Friederike, domain as in Table 2; F1 is the same with a smaller
domain focused more on the Netherlands; F2 is larger domain including parts of UK; E0 is storm Eleanor, as in Table 3; E1 is the smaller
domain; E2 is the larger domain. The coordinates (Lat, Long) of domains are indicated in the first data row. Bold numbers are significantly
different from the value in 1951–1980. Table 4. Sensitivity experiment results. Return periods and their confidence intervals (in square brackets) for the different time periods and the
RACMO simulations. Each column contains results for one domain experiment. R. Vautard et al.: Human influence on European winter wind storms 283 R. Vautard et al.: Human influence on European winter wind storms
283
Figure 10. Synthesis of the probability ratios (PRs) compared for storms Friederike and Eleanor. Panels (a) and (b) compare PRs for the
daily maximum of three-hourly instantaneous wind speeds (blue for observations and red for models). The PRs have been converted to apply
to the common interval 1975–2055 assuming the logarithm scales with the CO2 concentration. Panels (c) and (d) compare PRs for the two
models with daily mean wind speeds. The PRs have been converted to apply to the common interval 1860–2055 to show the change that
occurs between pre-industrial and 1.5 ◦C conditions. Figure 10. Synthesis of the probability ratios (PRs) compared for storms Friederike and Eleanor. Panels (a) and (b) compare PRs for the
daily maximum of three-hourly instantaneous wind speeds (blue for observations and red for models). The PRs have been converted to apply
to the common interval 1975–2055 assuming the logarithm scales with the CO2 concentration. Panels (c) and (d) compare PRs for the two
models with daily mean wind speeds. The PRs have been converted to apply to the common interval 1860–2055 to show the change that
occurs between pre-industrial and 1.5 ◦C conditions. indicator of global warming f (yr): indicator of global warming f (yr): HadGEM3-A). This explains at least partially the conflict
with the observed trends, as the potential wind results for the
Netherlands showed that roughness plays a large role in the
observed decrease in storminess. By contrast, these model
ensembles mainly reflect changes due to greenhouse gases. PR(y1,yend) = PR(y2,yrend)PR(yr1,yr2)
= PR(yr2,yrend)exp[f (yr1) −f (yr2)]. (1) (1) We prefer not to use time for f (yr), as the warming trend
is not linear in time. The global mean temperature as a proxy
for the trend in the local temperature is often used (e.g. van
der Wiel, 2017), but this would necessitate using a model
result for the future, which would be model-dependent. We
therefore use the observed/RCP4.5 CO2 concentration for
f (yr). This measure correlates very well with the global
mean temperature on the observed record (r = 0.94) and per-
mits extrapolation of the probability ratios into the future. R. Vautard et al.: Human influence on European winter wind storms We conclude that storms like Friederike and Eleanor have
not become significantly more or less frequent due to climate
change, but our model results indicate that global warming
due to greenhouse gases could make storms like them some-
what more frequent in the future, with a frequency increase
up to at most a factor of 2, or equivalently a few percent
higher wind speeds. However, this may seem contradictory
with the observations showing a clear and significant de-
cline in high wind speeds in accordance with earlier studies. This is equivalent to declining probabilities of these kinds
of storms, but our analysis and previous studies find expla-
nations for these changes in factors other than greenhouse
gases. The increase in surface roughness due to forest growth
and urbanization potentially explains a major part of this de-
crease (Vautard et al., 2010; Wever et al., 2012), and does not
exclude other factors, such as decadal variability and aerosol
effects. Until a quantitative attribution of past observed de-
creases is established – and with that an understanding of
the interplay between greenhouse gas forcing and those other
factors, and scenarios for them – the confidence on future
evolutions of wind storms will remain low, based on sim-
ulations reflecting mainly the effects of greenhouse gas in-
creases. This comes in addition to the poor understanding of
how atmospheric circulation variability changes (Shepherd,
2014). In contrast to the observations, global and regional climate
models do not simulate a decrease over the past decades. In-
stead, simulations of the daily maximum of three-hourly in-
stantaneous wind, of the same spatial and temporal character-
istics of these storms and, hence, the observational analysis,
indicate increases in probability between 1975 and 2055, cor-
responding to increases in wind speed for this return period
(Fig. 10). These are not all significantly different from 1, but
model consensus and future trends support the presence of
such a positive tendency. The change is small though: a prob-
ability ratio of 1.5 for Friederike with an uncertainty range of
1 to 2, corresponding with an increase in intensity of the wind
of only about 5 % (0 % to 10 %). For Eleanor the numbers are
even smaller: an increase in PR of about 1.25 (1.0 to 1.6) or
an increase in intensity of 2 % (0 % to 5 %). 8
Synthesis and conclusions Western European countries have been struck by high-impact
wind storms during the month of January 2018. The link be-
tween storms like Eleanor (on 3 January 2018) and Friederike www.earth-syst-dynam.net/10/271/2019/ Earth Syst. Dynam., 10, 271–286, 2019 www.earth-syst-dynam.net/10/271/2019/ R. Vautard et al.: Human influence on European winter wind storms The changes in daily mean wind are smaller still and in-
distinguishable from no change. However, as these do not
correspond directly to the impact of these storms, we do not
take them into account in the synthesis. We finally note that changes in extreme probabilities, in
general, can be due to both dynamical changes (changes
in atmospheric circulation types) and thermodynamical
changes (changes in temperature, its gradient, humidity, etc.)
due to anthropogenic factors. Our analyses do not attempt The climate model simulations do not always include
changes in aerosols and either have no roughness changes
(e.g. regional models) or capture these only partially (e.g. www.earth-syst-dynam.net/10/271/2019/ Earth Syst. Dynam., 10, 271–286, 2019 Earth Syst. Dynam., 10, 271–286, 2019 R. Vautard et al.: Human influence on European winter wind storms R. Vautard et al.: Human influence on European winter wind storms R. Vautard et al.: Human influence on European winter wind storms 284 to discern the exact origin and processes of changes as it
would involve a completely different set of analyses such as
done in Vautard et al. (2016) or Yiou et al. (2017). While
for temperature and precipitation extremes such a distinc-
tion between dynamical and thermodynamical processes is
relatively straightforward, we do not think such a separation
would be as simple to interpret here, precisely because the
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UNIVERSIDADE DE BRASILIA
FACULDADE DE EDUCAÇÃO
Projeto Monografia.
Aluno: kelison Aguiar da Silva
Matricula: 0585998
ORIENTADOR: TADEU QUEIROZ MAIA
Título: Experiência acadêmica UNIVERSIDADE DE BRASILIA FACULDADE DE EDUCAÇÃO Projeto Monografia. Aluno: kelison Aguiar da Silva
Matricula: 0585998 Aluno: kelison Aguiar da Silva
Matricula: 0585998 ORIENTADOR: TADEU QUEIROZ MAIA 1 1 RESUMO ............................................................................. 3
1. INTRODUÇÃO ............................................................... 4
2. PROBLEMA .................................................................... 4
3. OBJETIVO ....................................................................... 4
3.1. OBJETIVO GERAL ...................................................... 5
3.2. OBJETIVO ESPECÍFICO ............................................. 5
4. HIPÓTESE ....................................................................... 5
5. PROCEDIMENTOS METODOLÓGICOS ...................... 6
6. FUNDAMENTAÇÃO TEÓRICA .................................... 6
6.1. POLÍTICA E EDUCAÇÃO, UM BREVE RELATO ..... 7
6.2. A TECNOLOGIA ALIADA A EDUÇÃO ...................... 8
6.3. O PAPEL DA ARTE NA EDUCAÇÃO ......................... 9
6.4. O PAPEL SOCIAL DA CRIANÇA ............................. 11
6.5. CIDADANIA E GARANTIA DE DIREITOS – ÉTICA11
6.6. ENTENDENDO O ESTATUTO DA CRIANÇA E DO
ADOLESCENTE ................................................................ 11
7. APRESENTAÇÃO DA PESQUISA – CEM 03. ............. 12
8. Breve Histórico ............................................................... 14
8. ÚLTIMAS CONSIDERAÇÕES .................................... 15
9. REFERENCIAL BIBLIOGRÁFICO .............................. 17 2 2 RESUMO A Corrupção em todas as áreas que produzem as leis da educação está diretamente
relacionado à vontade política de grupos privados, e não enfrenta com unhas e dentes este
problema poderá causa um caos na educação, temos que mudar isso, especialmente no
contexto, onde está presente uma realidade desigual, mais uma vez tentamos e nada. A pes
maioria não tem feito nada, dividir a responsabilidade que temos, lutar juntos, porque a
cultura corrupta ta crescendo e nós estamos ficando mais pobres, esse compromisso com os
despossuídos em Escola privada é muito importante, a luta deve ser mostrado, o problema
ta sendo grande, um exemplo é a educação que existe na Finlândia, onde o
desenvolvimento é praticamente perfeito nesse sentido que o Estado pode investir em
verdade. Não há intenção de desenvolver um plano estratégico ou no Brasil com relação a
educação. o Estado tem de intervir diretamente na educação, O que temos é produzir um
sistema coerente com a nossa realidade. 3 3 Tragetória Acadêmica A experiência que tive nesses 5 anos de vida academica foi muito importante, pois
em diversos momentos que aconteceram na ocasião principalmente na univer-
sidade
de tiveram relação não só com a pesquisa, mas com a experiencia e pratica
acade
mica, as interações por sorte foi estabelecida de maneira tão eficases que todo
os meus
atos foram superado com o gosto que tive de estudar com professores capacita-
dos em
ajudar a conserta o bem social. Por sorte, coube a mim perceber as variantes da educação na perspectiva do
conhe-
cimento, nesta grande divisão de ideias e experiencia dentro de uma universi-
dade im-
plicou na soma de todas as experiencias desgustadas por mim e toco esse corpo
mem
talizado no universo. Consegui escreve e viajar nas ideias que se tranformaram e tomaram conta da
minha RESUMEN La corrupción en todas las áreas que producen las leyes de la educación está
directamente relacionada con la voluntad política de los grupos privados, y no se enfrentan
con uñas y dientes este problema puede causar un caos en la educación, tenemos que
cambiar eso, sobre todo en el contexto en el que está presente una realidad desigual, una
vez más intentó y nada. El personal de la mayoría no está haciendo nada, que comparten la
responsabilidad, de luchar juntos, porque la cultura corrupta ta creciendo y estamos cada
vez más pobres, este compromiso con los desposeídos en la escuela privada es muy
importante, la lucha debe ser demostrado, el problema ta siendo grandes, un ejemplo es la
educación que existe en Finlandia, donde el desarrollo es prácticamente perfecto en este
sentido que el Estado puede invertir en la verdad. No hay intención de desarrollar un plan
estratégico o en el Brasil en relación a la educación. el Estado tiene que intervenir
directamente en la educación, lo que tenemos es producir un sistema coherente con nuestra
realidad. 4 4 Tragetória Acadêmica 1. INTRODUÇÃO No presente trabalho desdobrei alguns conceitos de filosofia direcionando o da
seguinte maneira: posto em questão diversos assuntos relacionados com o direcionamento
das atividades voltadas para assuntos que são demandas com relação ao grupo, as questões
das imagens e o seu uso. Inicialmente foi retificado alguns temas abordados enfatizando a
filosofia, a maneira como ela se desdobra no contexto da sociedade, deixando para trás, os 5 aspectos positivistas e anacrônicos, contudo, o desenvolvimento e a minha produção no
contexto do trabalho foi sem sombra de dúvida bastante relevante. Outro ponto chave a ser compreendido foi o foco das imagens e a sua relação
positiva com o trabalho, onde a minha contribuição foi um fator determinante e sem dúvida
bastante eficaz no sentido de descoberta de uma filosofia que relaciona fatos e esquemas
das imagens com o social, nesse sentido, a minha contribuição foi sem dúvida excelente,
principalmente, quando temos uma equipe direcionada e focada no trabalho em questão. A minha luta começa na escola que foi inaugurada em 17 de setembro de 2007, o
Centro de Ensino do Recanto das Emas 113 (CEF 113), então poderá perceber que o
trabalho com a filosofia na escola trás uma mínima relação com a política de
financiamento dessa escola, ou seja, a prática aliada à boa coordenação é a chave, é a saída
sem depender muito de programas e financiamento público, pois os recursos repassados
nessa escola não chegam a garantir uma melhor oferta de ensino de qualidade por sorte há
na escola uma gama de ações que criam oportunidades para aqueles alunos. O sistema de avaliação conforme os requisitos ainda não é cumprido, pois a escola
é muito recente, daí o único sistema de avaliação feito foi o SIAD (sistema de avaliação do
desempenho das instituições educacionais do sistema de ensino do DF.), ou seja, como a
escola tem a modalidade de ensino fundamental, ainda não houve a aplicação do SAEB (O
Sistema Nacional de Avaliação da Educação Básica), é o sistema usado como indicador do
desempenho do IDEB (Índice de Desenvolvimento da Educação Básica). 1. INTRODUÇÃO Na turma da 4ª série tive uma atuação não distanciada e sim reflexiva e com
dialogo em vários momentos em sala, elaborei temas e dividi esses em categorias, daí
então, dividi a sala em 8 grupos e cada grupo era responsável por cada tema, foram vários
temas ou categorias como a política, esporte, namoro e etc., nesse sentido o momento de
cada um, era respeitado, e cada componente de cada grupo refletia o tema escolhido, foi
muito importante, pois, a reflexão aliada ao dialogo foi um ato reflexivo, não houve
distancia, as categorias trabalhadas foram se tornando mais evidente no intervalo de tempo,
a formula seria M=D/t, onde o M seria o momento ou ato a ser produzido, D seria o ato ou
diálogo-coletivo e T seria o tempo presente, essa formula não é absoluta, mas com certeza
seria muito útil em grupos diferenciados com a finalidade de um dialogo e uma maior ação
comunicativa de reflexão filosófica. O xadrez aliado com a filosofia desse modo poderia ser trabalhado com o momento
ou ato produzido em questão e isto não é absoluto, outras possibilidades são levadas em
consideração e abarcadas nessa forma de saber. O quanto à escola pública é fundamental para isso, principalmente, quando a nossa
realidade está inteiramente controlada por a desigualdade e as disparidades no contexto 6 social tanto com relação aos recursos, quanto em relação à distribuição deles, uma escola
de qualidade naquele local no Recanto da Emas é fundamental para levar aquelas pessoas
que não tem a mínima condição de pagar uma escola particular, vendo esta questão percebi
o quanto o Estado é fundamental para aplicação de uma política pública Estatal na
educação que não deixe se levar por interesses privados dominar a nossa educação é nesse
sentido que o lucro é muito relevante para eles, e as noções sociais são mascaradas com
objetivos irrelevantes para nossa educação. 2. PROBLEMA O trabalho vai analisar a estratégica das leis e os direitos e garantias perante a
educação pública e o que trata a Constituição Brasileira. O presente trabalho busca visar uma educação de jovens e adultos direcionadas para
uma formação complementar e articulada com as políticas de governo, escola e
universidade, quanto à profissionalização dos jovens e adulto que não tem nenhuma opção
nessa modalidade depois de ter conseguido se formar e também como tecnologias
inovadoras ou mesmo a arte podem contribuir na formação de um cidadão melhor
qualificado. As fontes e documentos pretendem mostrar que isso já é uma realidade
presente no DF, Isto de forma articulada é de suma importância para a formação do aluno e
seu itinerário formativo. O estudo será feitos de forma investigatória com analise e fontes documentais
produzindo uma pesquisa e uma contribuição do ponto de vista da educação de jovens e
adultos (EJA). 3.1. OBJETIVO GERAL Analisar os principais aspectos decorrentes da política voltada à educação e os
impactos dos recursos na educação. Com isso verificar a interferência dessas ações no
sistema de ensino público é ou não um entrave para educação. Outro aspecto do ponto de
vista filosófico é a educação e os seus processos estratégicos de financiamento. 3. OBJETIVO 7 7 O presente trabalho tem como objetivo analisar a educação brasileira no contexto
da Constituição Brasileira, especificamente o artigo 205 que trata sobre educação no
contexto político, estratégico e social, possibilitando por meio de estudos filosóficos,
delimitar e traçar um panorama da lei e em que medida, as políticas públicas de educação
estão realmente coerente com as demandas e efetivadas claramente nas escolas e perceber
se estão coerentes com a nossa realidade. Com isso, abrindo e possibilitando formas DE SABER construídas na comunidade
e direcionadas ao diálogo e a filosofia. Art. 205. A educação, direito de todos e dever do Estado e da família, será
promovida e incentivada com a colaboração da sociedade, visando ao pleno
desenvolvimento da pessoa, seu preparo para o exercício da cidadania e sua qualificação
para o trabalho. O processo tem como abordagem uma construção do saber na perspectiva da
educação. A educação é um dever do Estado, é direito de todos, porém, a sua omissão se faz
presente, obstando um direito e disseminado a estratégia que se tem, não é garantidora de
um desenvolvimento eficaz. A principal meta e efetivamente descobrir o que se tem de distorções e contradições
na educação. 3.2. OBJETIVO ESPECÍFICO
Identificar as principais omissões da educação no Brasil perante o ensino
público.
Relacionar os estudos filosóficos com a educação de maneira a fazer uma
reflexão critica da educação no Brasil, principalmente a distribuição dos recursos.
Mostrar os principais problemas que efetivamente enfrentados pela
educação para uma escola pública verdadeira e de qualidade. 8 5. PROCEDIMENTOS METODOLÓGICOS Para elaboração do seguinte trabalho são usados os seguintes procedimentos:
Levantamento bibliográfico, esta etapa será abordados textos e materiais
bibliográficos, para fornecer subsídios para o trabalho,
Pesquisa por sites de educação para desdobramento do trabalho,
Pesquisa em escola: Centro de Ensino Fundamental 113 do Recanto das
Pesquisa em escola: Centro de Ensino Fundamental 113 do Recanto das
Emas. Emas. 4. HIPÓTESE Demonstrar que as deficiências de políticas governamentais são um entrave para
um sistema de ensino público de qualidade ocasionando um caos na estrutura social do país
tendo como aumento a desigualdade social. O presente trabalho pretende demonstrar que as ações estatais se sobrepõem as
ações governamentais e a promoção de medidas direcionadas para um sistema de ensino
público, e verdadeiramente de qualidade são medidas essenciais para ordem social do país. Tais, implementações diagnosticadas de forma reflexiva e crítica, reduziriam esse
transtorno no sistema de ensino público e reduziria em grande parte a desigualdade no
Brasil. Atualmente podemos ver uma serie de distorções tanto na constituição brasileira
como na LDB, Lei de Diretrizes e Bases da Educação, com a crescente falta de substancia
enquadras como lógica nas normas e o que se diz dos processos de educação, o brasileiro
se ver longe de uma escola publica e verdadeira de qualidade, pois os impactos resultantes
do capitalismo e os processos de comunicações distorcidas geraram um caos no
enquadramento jurídico brasileiro e na educação. O que se tem a fazer e uma gama de
estudos reflexivos direcionantes filosóficos por meio da comunicação de todas as infra
estruturas e super estruturas brasileira para modificação da escola publica. 9 9 6. FUNDAMENTAÇÃO TEÓRICA Atualmente podemos ver uma série de distorções tanto na constituição brasileira
como na LDB, Lei de Diretrizes e Bases da Educação, com a crescente falta de substância
enquadras como lógica nas normas e o que se diz dos processos de educação. O brasileiro
se vê longe de uma escola pública e verdadeira de qualidade, pois os impactos resultantes
do capitalismo e os processos de comunicações distorcidas geraram um caos no
enquadramento jurídico brasileiro e na educação. O que se tem a fazer e uma gama de
estudos reflexivo onde a ação social comunicativa de todas as instituições do ponto de vista
filosófico brasileiro para modificação da escola pública. No projeto no Centro de Ensino Fundamental do Recanto das Emas – CEF 113
pôde perceber o quanto a filosofia se faz presente em determinados processos e contextos
escolares, principalmente quando falamos em ensino fundamental, pois essa educação
sempre teve mascarada e sem dúvida distanciada dela. O ensino de filosofia é um próprio
reflexo do atual momento em questão, que vivemos, principalmente quando percebemos
que nossas ações estão direcionadas para uma reflexão do nosso próprio ato, do nosso
movimento e tudo que é processado nesse imenso coletivo diferenciado, a nossa ação
comunicativa se faz presente em determinados contextos sociais. A produção desse
conhecimento pode ser um ato voltado para o dialogo e a sabedoria não somente nessa
escola e sim todas as redes escolares públicas. 10 A história local remete a pensar sobre uma história que usualmente é escrita por
seus próprios membros evidenciando problemas e várias críticas na perspectiva da história,
com isso consigo ver essas críticas e pensamentos deturpados, pois a construção de nossa
história é feita por questões que estão abarcadas até mesmos nessas localidades
evidenciando, contudo, categorias que possam construir e distribuir estas fontes, de forma
a alcançar um conhecimento histórico inteiramente construído com essas pequenas
localidades. É nesse sentido que podemos perceber não somente algo diferenciado abarcada até
mesmo nos PCNs (Planos Curriculares Nacionais), pois a questão dos diferentes modos de
repensar a história junto aos parâmetros é sem sombra de dúvida outra questão que entra
em choque com pontos e evidências de um ensino concebido e organizado positivista, em
contra partida um ensino voltado realmente para um conhecimento verídico. 6. FUNDAMENTAÇÃO TEÓRICA Tais questões
trás uma maneira de pensar e fazer a história em termos de uma aprendizagem e
concepções, bastante problemática, a qual podem ser acrescidas questões como
anacronismo, desenvolvimento de perspectivas etnocêntricas ao contrario do que podemos
refazer uma nova direção das construções dessas fontes conseguindo estabelecer relações e
produção de uma história realmente construídas dessas localidades. Com isso, podemos perceber claramente que essas fontes são produzidas por
homens e suas relações sócias naquele período, por azar, argumentos que deixam pra trás
estas questões evidenciam realmente o que podemos chamar de reducionismo na
construção de fontes históricas claras e precisas. É nesse sentido que hoje essas categorias podem, sim, ser trabalhadas e
direcionadas na perspectiva da nossa realidade, cai por terra este pensamento que as teorias
e as construções dessa história têm que ser distanciadas de indivíduos que fazem desta
história local uma construção social. 6.1. POLÍTICA E EDUCAÇÃO, UM BREVE RELATO A República Federativa do Brasil constitui se em Estado Democrático de Direito, e
está fundamentada pela cidadania conforme inciso II, art. 1º da CF/88. assim, todos os
políticos assumem a educação como prioridade em seus discursos de campanha. Não há
uma pessoa sensata que seja capaz de dissociar a idéia de progresso de um país de
consistentes investimentos para a educação. No entanto, uma grande diferença entre o que
é apregoado na política e em relação ao que vemos em nossas escolas e redes públicas de
ensino. Ninguém quer mudar a educação de uma hora para a outra, isso vai com o tempo,
lógico, mas é preciso ação, apenas promessas em comícios de nada adiantam. A escola real é aquela na qual é possível construir, é aquela na qual esta ao alcance do governo, e está
em nosso alcance, mas que é dependente do Governo, do Estado E PRINCIPALMENTE
DE NOS MESMOS O POVO BRASILEIRO. De acordo com Machado (2010), quando pensamos em Estado como formulador de
políticas públicas, logo nos vêm à cabeça as várias funções sociais possíveis de serem
exercidas pelo Estado, tais como saúde, educação, previdência, moradia, saneamento
básico, entre outras. Na prática se trata disso, entretanto para que sejam implementadas as diversas
políticas em cada área social é necessário definir e compreender a estrutura institucional do
Estado que contempla tais funções, ou seja, seu conjunto de órgãos, autarquias, ministérios
competentes em cada setor, além do processo de financiamento e gestão. Demerval Saviani afirma não existir uma identidade entre educação e política,
embora ambas se constituam em fenômenos inseparáveis e prevaleça a distinção entre a
dimensão política na educação e a dimensão da prática educativa. Para ele a captação da
intervenção seguiria o seguinte. Saviani assegura que a interferência da política na educação e vice-versa só pode
ser captada quando as concebemos como distintas entre si, o que torna necessário
especificá-las: a)
A educação, alicerçada na persuasão (consenso, compreensão), acaba sendo uma
"relação de hegemonia" e sua especificidade se define pelo caráter de uma relação travada
entre contrários não-antagônicos; b)
A política, alicerçada na dissuasão (dissenso, repressão), por outro lado, é uma
"relação de dominação" e sua especificidade se define pelo caráter de uma relação travada
entre contrários antagônicos. 6.1. POLÍTICA E EDUCAÇÃO, UM BREVE RELATO De acordo com Schwartzman (1990) nós evoluímos de um tempo em que políticas
públicas eram formuladas e acompanhadas de forma explícita, e havia otimismo sobre o
que governos podiam fazer para a situação atual, em que as políticas são muito mais
difusas ou mesmo inexistentes. Hoje sabemos muito mais sobre a natureza e os limites da
administração pública, em termos gerais e no Brasil em particular, assim como sobre suas
relações complexas com o sistema político e a cultura do país. O resultado desta maior
sabedoria, no entanto, não tem sido melhorar a capacidade governamental em formular
políticas e levá-las à cabo, mas um grande ceticismo a respeito do que o possa ser feito;
ceticismo reforçado pelas experiências frustrantes do governo Sarney, às quais se somam
os ataques generalizados ao Estado que fazem parte da ideologia dominante destes últimos
anos. 12 Podemos concluir que a educação que se tem hoje é o futuro de amanha, Os
políticos que elegemos hoje são o futuro de amanha. Com boa educação hoje, quem sabe,
uma melhor política no país de amanha. A premissa constitucional diz: somente pela participação popular, tem se um Estado
Democrático de Direito, isto é, governo do povo, para o povo, pelo povo quando desta não
participação, não raro se formam governos tiranos, formando oligarquias que usurpam o
poder daquele natural, o povo. O presente trabalho tem como objetivo analizar a educação
brasileira no contexto da constituição Brasileira, especificamente o artigo 205 que trata
sobre educação no contexto político estratégico possibilitando por meio de estudos
filosóficos descobrir se a estratégica de desenvolvimento faz presente em uma realidade
inteiramente pobre, em que medida a oferta de vagas nas escolas PUBLICA estão coerente
com a nossa realidade no quadro onde desigualdade social é inteiramente real, isso tudo
POSSIBILITANDO UMA forma que possa por meio de estudos filosóficos descobrir se a
estratégica de desenvolvimento COM RELAÇÃO A ESCOLA PRIVADA CONDIZ COM
A NOSSA REALIDADE MESMO. 6.2. A TECNOLOGIA ALIADA A EDUÇÃO De acordo com Caminha (2010) na educação a distância, entende-se que o avanço
das tecnologias possibilitando a integração de mídias, proporcionam variadas possibilida-
des de estudo ao aluno, atendendo às diferentes formas de aprender. No entanto, é claro
que as tecnologias por elas próprias não dão conta de garantir a aprendizagem. Assim a
preocupação com a abordagem educacional é fundamental. A Internet, as redes, o celular, a multimídia estão
revolucionando nossa vida no cotidiano. Cada vez
resolvemos mais problemas conectados, à distância. Na
educação, porém, sempre colocamos dificuldades para a
mudança, sempre achamos justificativas para a inércia ou
vamos mudando mais os equipamentos do que os
procedimentos. A educação de milhões de pessoas não pode
ser mantida na prisão, na asfixia e na monotonia em que se
encontra. Está muito engessada, previsível, cansativa. (M
2009 ) 13 As tecnologias são apenas apoios, meios. Mas elas nos permitem realizar atividades
de aprendizagem de formas diferentes às de antes. Podemos aprender estando em juntos
em lugares distantes, sem precisamos estar sempre juntos numa sala para que isso
aconteça. Muitos expressam seu receio de que o virtual e as
atividades à distância sejam um pretexto para baixar o nível
de ensino, para aligeirar a aprendizagem. A qualidade não
acontece apenas por estarmos juntos num mesmo lugar,
mas
por
estabelecermos
ações
que
facilitem
a
aprendizagem. A escola continua sendo uma referência
importante. Ir até ela ajuda a definir uma situação oficial de
aprendiz, a conhecer outros colegas, a aprender a conviver. Mas, pela inércia diante de tantas mudanças sociais, ela está
se convertendo em um lugar de confinamento, retrógrado e
pouco estimulante. M
2009 Moran, 2009 O viver virtual vai tornar-se quase tão importante como o coexistir presencial. A
escola precisa de uma renovação. Isso se consegue com uma gestão administrativa e
pedagógica mais flexível, com tempos e espaços menos predeterminados, com modos de
acesso a pesquisa e de desenvolvimento de atividades mais dinâmicas. Passando pelos corredores das salas das universidades, o que se vê é quase sempre
uma pessoa falando e uma classe cheia de alunos semi-atentos. A infra-estrutura é
decadente. Salas barulhentas, a voz do professor mal chega aos que estão mais distantes
como nos anfiteatros da UnB. Moran (2009) nos mostra que é hora de partir para soluções mais adequadas para o
aluno de hoje. 6.2. A TECNOLOGIA ALIADA A EDUÇÃO Os adultos mantemos o status quo, em nome da qualidade, mas na verdade
nos apavoramos diante da mudança, do risco do fracasso. A escola pode ser um espaço de inovação, de experimentação saudável de novos
caminhos. Não precisamos nos desfazer com tudo, mas implementar alteração e
supervisioná-las com comedimento e maturidade. Manter o currículo e as normas, tal como estão, na prática é insustentável. As
secretarias de educação precisam ser mais proativas e incentivar mudanças, flexibilização,
criatividade. Professores, alunos e administradores podem avançar muito mais em organizar
currículos mais flexíveis, aulas diferentes. A rotina, a repetição, a previsibilidade é uma 14 arma letal para a aprendizagem. A monotonia da repetição esteriliza a motivação dos
alunos. São muitos os recursos a nossa disposição para aprender e para ensinar. A chegada
da Internet, dos programas que gerenciam grupos e possibilitam a publicação de materiais
estão trazendo possibilidades inimagináveis vinte anos atrás. A resposta dada até agora
ainda é muito tímida, deixada a critério de cada professor, sem uma política institucional
mais ousada, corajosa, incentivadora de mudanças. Está mais do que na hora de evoluir,
modificar nossas propostas, aprender fazendo. Moran (2009) ainda nos mostra que hoje obrigamos os alunos a ir a um local para
aprender. Em determinados momentos isso é um contra-senso. O importante é que gostem
de aprendem de várias formas, motivados, utilizando as potencialidades de estar juntos e
de estar em rede. Os alunos gostam da comunicação online, da pesquisa instantânea, de
tudo o que acontece just in time, naquele momento. As salas de aula precisam estar
equipadas com acesso a Internet para mostrar rapidamente o resultado de uma pesquisa em
tempo real na sala. Os alunos necessitam de mais laboratórios conectados, principalmente
os mais carentes, sem esse acesso em casa. Para alunos com acesso a Internet é possível
realizar uma parte do processo de aprendizagem a distância/conectados. E os alunos sem
esse acesso poderiam fazer essas mesmas atividades nos laboratórios. “Não basta tentar remendos com as atuais tecnologias. Temos quer fazer muitas
coisas diferentemente. É hora de mudar de verdade e vale a pena fazê-lo logo, chamando
os que estão dispostos, incentivando-os de todas as formas – entre elas a financeira – dando
tempo para que as experiências se consolidem e avaliando com equilíbrio o que está dando
certo. 6.2. A TECNOLOGIA ALIADA A EDUÇÃO Precisamos trocar experiências, propostas, resultados.”(Moran, 2009) Tentemos
fazer, cada qual, a sua parte, mesmo parecendo pouco ou que não dará resultados, mas
juntos, cada inovador em seu ambiente de trabalho, em suas ações e discursos, será
possível alcançar a mudança que fará a diferença. A escola deve acompanhar as mudanças, especialmente no campo tecnológico, pois
os alunos já dominam as novas tecnologias e isso exige que a escola, o professor também
conheça e saiba aproveitar da melhor forma estas ferramentas para acrescentar nas aulas e
nos seus estudos. As tecnologias que num primeiro momento são utilizadas de forma separada –
computador, celular, Internet, mp3, câmera digital – e caminham na direção da
convergência, da integração, dos equipamentos multifuncionais que agregam valor. O computador continua, mas ligado à internet, à câmera digital, ao celular, ao mp3,
principalmente nos pockets ou computadores de mão. O telefone celular é a tecnologia que 15 atualmente mais agrega valor: é wireless (sem fio) e rapidamente incorporou o acesso à
Internet, à foto digital, aos programas de comunicação (voz, TV), ao entretenimento (jogos,
música-mp3) e outros serviços. Estas tecnologias começam a afetar profundamente a educação. Esta sempre
esteve e continua presa a lugares e tempos determinados: escola, salas de aula, calendário
escolar, grade curricular. Moran (2009) mostra que apesar da resistência institucional, as pressões pelas
mudanças são cada vez mais fortes. As empresas estão muito ativas na educação on-line e
buscam nas universidades mais agilidade, flexibilização e rapidez na oferta de educação
continuada. Os avanços na educação a distância com a LDB e a Internet estão sendo
notáveis. A LDB legalizou a educação a distância e a Internet lhe tirou o ar de
isolamento, de atraso, de ensino de segunda classe. A interconectividade que a Internet e
as redes desenvolveram nestes últimos anos está começando a revolucionar a forma de
ensinar e aprender. A educação presencial está incorporando tecnologias, funções, atividades que
eram típicas da educação a distância, e a EAD está descobrindo que pode ensinar de
forma menos individualista, mantendo um equilíbrio entre a flexibilidade e a interação. 6.3. O PAPEL DA ARTE NA EDUCAÇÃO De acordo com Ana Mae Barbosa, a educação em arte, assim como a educação
geral e plena do indivíduo, acontece na sociedade de duas formas: Assistematicamente através dos meios de comunicação de massa e das manifes-
tações não institucionalizadas da cultura, como as relacionadas ao folclore (entendido
como manifestação viva e em mutação, não limitado apenas à preservação de tradi-
ções); e sistematicamente na escola ou em outras instituições de ensino. Com isso a arte
esta em vários espaços dentro da escola e outros mil espaços que direcionam os desdo-
bramento da magia e o folclore em varias Culturas sociais. A arte-educação tem um objetivo maior que a formação de profissionais dedica-
dos a esta área de conhecimento, no âmbito da escola regular busca oferecer aos indiví-
duos condições para que compreenda o que ocorre no plano da expressão e no plano do 16 significado ao interagir com as Artes, permitindo sua inserção social de maneira mais
ampla. Arte é um extraordinário trabalho educacional, pois procura, de lado a lado das
tendências individuais, orientar a formação do gosto, estimula a inteligência e contribui
para a formação da originalidade do indivíduo, sem ter como preocupação única e mais
importante à formação de artistas. No seu trabalho criador, o indivíduo utiliza e aperfeiçoa processos que desen-
volvem a percepção, a imaginação, a observação, o raciocínio, o controle gestual. Capa-
cidade psíquica que influem na aprendizagem. No processo de criação ele pesquisa a
própria emoção, liberta-se da tensão, ajusta-se, organiza pensamentos, sentimentos, sen-
sações e forma hábitos de trabalho. Educa-se. Ana Mae Barbosa (1991) Raimundo Matos de Leão (1996) nos mostra que sendo a escola o primeiro espaço
formal onde se dá o desenvolvimento de cidadãos, nada melhor que por aí se dê o contato
sistematizado com o universo artístico e suas linguagens: artes visuais, teatro, dança,
música e literatura. Contudo, o que se percebe é que o ensino da arte está relegado ao
segundo plano, ou é encarado como mera atividade de lazer e recreação. Ao longo dos anos, muito se tem falado e escrito sobre a necessidade da inclusão da
arte na escola de forma mais efetiva. Desde 1971, pela Lei 5692, a disciplina Educação
Artística torna-se parte dos currículos escolares. Ao longo dos anos, muito se tem falado e escrito sobre a necessidade da inclusão da
arte na escola de forma mais efetiva. 6.3. O PAPEL DA ARTE NA EDUCAÇÃO Desde 1971, pela Lei 5692, a disciplina Educação
Artística torna-se parte dos currículos escolares. No Brasil, a Lei de Diretrizes e Bases da Educação Nacional (LDB nº 9.394/96)
estabeleceu em seu artigo 26 parágrafo 2º que: No Brasil, a Lei de Diretrizes e Bases da Educação Nacional (LDB nº 9.394/96)
estabeleceu em seu artigo 26, parágrafo 2º que:
"O ensino da arte constituirá componente curricular obrigatório, nos diversos níveis
da educação básica, de forma a promover o desenvolvimento cultural dos alunos".
"A arte é um patrimônio cultural da humanidade, e todo ser humano tem direito ao
acesso a esse saber" Leão continua nos mostrando que o ensino da arte deve estar em consonância com a
contemporaneidade. A sala de aula deve ser um espelho do atelier do artista ou do
laboratório do cientista. Neles são desenvolvidas pesquisas, técnicas são criadas e
recriadas, e o processo criador toma forma de maneira viva, dinâmica. A pesquisa e a
construção do conhecimento é um valor tanto para o educador quanto para o educando, 17 rompendo com a relação sujeito/objeto do ensino tradicional. Este processo poderá ser
desafiador. Delimite-se o ponto de partida e o ponto de chegada será resultante da
experimentação. Dessa forma, o ensino da arte estará intimamente ligado ao interesse de
quem aprende. A dinâmica entre o sentir, o pensar e o agir. Promove a interação entre saber e
prática relacionados à história, às sociedades e às culturas, possibilitando uma relação
ensino/aprendizagem de forma efetiva, a partir de experiências vividas, múltiplas e
diversas. Considera-se também nesta proposta a vertente lúdica como processo e resultado,
como conteúdo e forma. É necessário que se pense o lúdico na sua essencialidade,
conforme nos fala o professor Edmir Perroti: "...gostaria de chamar a atenção para o conceito de
lúdico. Sim, porque no mundo atual as diferentes dimensões
do lúdico vêm sendo reduzidas a praticamente uma, a do
lúdico instrumental. Esta que é, por exemplo utilizada pela
publicidade, vem sendo tomada enquanto dimensão que dá
conta das possibilidades todas do lúdico, como se este se
esgotasse em tal perspectiva. Gostaria,assim, de lembrar aqui
que o lúdico compreende pelo menos outra dimensão, que
além de instrumental, o lúdico pode e deve ser essencial. No primeiro caso, o do lúdico instrumental, o jogo é
compreendido
enquanto
recurso
motivador,
simples
instrumento, meio para a realização de objetivos que podem
ser educativos, publicitários ou de inúmeras naturezas. seguinte forma: não existe a necessidade de nomear filosofia-educação, biologia-educação,
português-educação. A esse respeito, Ana Mae Barbosa faz a seguinte consideração: "Como a matemática, a história e as ciências, a arte
tem domínio, uma linguagem e uma história. Se constitui
portanto, num campo de estudos específicos e não apenas em
meia atividade [...] A arte-educação é epistemologia da arte e,
portanto, é a investigação dos modos como se aprende arte na
escola de 1° grau, 2° grau, na universidade e na intimidade
dos ateliers. Talvez seja necessário para vencer o preconceito,
sacrificarmos a própria expressão arte-educação que serviu
para identificar uma posição e vanguarda do ensino da arte
contra o oficialismo da educação artística dos anos setenta e
oitenta. Eliminemos a designação arte-educação e passemos a
falar diretamente de ensino da arte e aprendizagem da arte
sem eufemismos, ensino que tem de ser conceitualmente
revisto na escola fundamental, nas universidades, nas escolas
profissionalizantes, nos museus, nos centros culturais a ser
previsto nos projetos de politécnica que se anunciam. (1991) Para Selma de Asssis Moura a arte é cultura: Para Selma de Asssis Moura a arte é cultura: Para Selma de Asssis Moura a arte é cultura:
É fruto de sujeitos que expressam sua visão de mundo,
visão esta que está atrelada a concepções, princípios,
espaços, tempos, vivências. O contato com a arte de
diversos períodos históricos e de outros lugares e regiões
amplia a visão de mundo, enriquece o repertório estético,
favorece a criação de vínculos com realidades diversas e
assim propicia uma cultura de tolerância, de valorização da
diversidade, de respeito mútuo, podendo contribuir para
uma cultura de paz. (2008) Para Selma de Asssis Moura a arte é cultura:
É fruto de sujeitos que expressam sua visão de mundo,
visão esta que está atrelada a concepções, princípios,
espaços, tempos, vivências. O contato com a arte de
diversos períodos históricos e de outros lugares e regiões
amplia a visão de mundo, enriquece o repertório estético,
favorece a criação de vínculos com realidades diversas e
assim propicia uma cultura de tolerância, de valorização da
diversidade, de respeito mútuo, podendo contribuir para
uma cultura de paz. (2008) É fruto de sujeitos que expressam sua visão de mundo,
visão esta que está atrelada a concepções, princípios,
espaços, tempos, vivências. 6.3. O PAPEL DA ARTE NA EDUCAÇÃO No
segundo caso, brincar sob todas as formas físicas e/ou
intelectuais, é visto como atitude essencial, como categoria
que não necessita de uma justificativa externa, alheia a ela
mesma para se validar. No primeiro caso, o que conta é a
produtividade. No segundo, a produtividade é o próprio
processo de brincar, uma vez nessa concepção jogar é
intrinsecamente educativo, é essencial enquanto forma de
humanização." (1995) A interação entre a concepção de arte e a concepção de educação encaminha-se na
confluência do que conhecemos como arte-educação, conceito este que aponta para o
entendimento de uma questão mais ampla que é a arte no espaço educativo: um projeto
pedagógico com uma prática em arte. Destacamos a questão, tendo em vista que nenhuma
outra disciplina tem necessidade de uma ênfase na sua nomenclatura quando da inclusão
numa proposta pedagógica. Para melhor compreensão da afirmativa, exemplificamos da 18 seguinte forma: não existe a necessidade de nomear filosofia-educação, biologia-educação,
português-educação. A esse respeito, Ana Mae Barbosa faz a seguinte consideração: 6.4. O PAPEL SOCIAL DA CRIANÇA O ponto de partida é que devemos pensar, sentir e agir em relação à criança e ao
adolescente enquanto pessoas humanas. E, além disso, como pessoas em condições
peculiares de desenvolvimento pessoal e social. A primeira manifestação de cidadania se dá quando a pessoa exerce o direito de não
ser violada em seus direitos fundamentais. Então, o que deve ser observado é o interesse de
cada um tanto da criança como do adolescente. É importante entendermos o tipo de comunicação que se estabelece entre crianças e
o meio onde se dá suas relações para um maior conhecimento do processo de
desenvolvimento. A criança em si não toma atitudes e sim desenvolve atitude (crenças,
valores, opiniões), em relação aos objetos do meio social. Essas atitudes são importantes,
pois, muitas vezes, são elas que norteiam o comportamento. seguinte forma: não existe a necessidade de nomear filosofia-educação, biologia-educação,
português-educação. A esse respeito, Ana Mae Barbosa faz a seguinte consideração: O contato com a arte de
diversos períodos históricos e de outros lugares e regiões
amplia a visão de mundo, enriquece o repertório estético,
favorece a criação de vínculos com realidades diversas e
assim propicia uma cultura de tolerância, de valorização da
diversidade, de respeito mútuo, podendo contribuir para
uma cultura de paz. (2008) (2008) Ela conclui nos mostrando que o conhecimento da arte produzida em sua própria
cultura permite ao sujeito conhecer-se a si mesmo, percebendo-se como ser histórico que
mantém conexões com o passado, que é capaz de intervir modificando o futuro, que toma
consciência de suas concepções e idéias, podendo escolher criticamente seus princípios,
superar preconceitos e agir socialmente para transformar a sociedade da qual faz parte. Diante de tal importância que a arte assume na educação, pode-se fazer uma revisão
crítica do que a escola tem alcançado em termos de ensino da arte. Estamos vivenciando um momento histórico de grande importância na educação
como um todo e na arte-educação especificamente: o desafio de superar concepções
tecnicistas e utilitaristas, mas também de ir além do “deixar fazer” e da livre expressão 19 apenas, para reconhecer que a arte tem características próprias que devem ser melhor
conhecidas pelos educadores, que tem objetivos próprios e seus próprios métodos. Como o ensino da arte é um universo amplo, uma vez que diz respeito ao que é
humano e envolve o fazer e o pensar, o ensino da arte não poderia deixar de interagir com
outras áreas do conhecimento. Dessa forma, o trabalho de produção e ensino da arte a ser
desenvolvido pela escola deverá configurar-se numa concepção onde arte e educação sejam
práticas que se relacionam com outras, pretendendo a criação de novas práticas na arte e na
vida. De acordo com os Parâmetros Curriculares Nacionais, A educação em arte propicia
o desenvolvimento do pensamento artístico e da percepção estética, que caracterizam um
modo próprio de ordenar e dar sentido à experiência humana:o aluno desenvolve sua
sensibilidade, percepção e imaginação, tanto ao realizar formas artísticas quanto na ação de
apreciar e conhecer as formas produzidas por ele e pelos colegas,pela natureza e nas
diferentes culturas.(PCN- Arte-1997). 6.5. CIDADANIA E GARANTIA DE DIREITOS – ÉTICA Para viver em sociedade é preciso possuir valores, critérios, e, mais ainda,
estabelecer relações e hierarquias entre esses valores. Escolher implica comparar e valorar. Assim, torna-se necessário a elaboração de critérios que classifiquem as ações como boas
ou más, corretas ou inadequadas, e que orientem e justifiquem a escolha, que se configura 20 com a resposta diante das prescrições da sociedade. A moral sofre transformações,
principalmente quando submetida À reflexão critica realizada pela ética. A ética não tem um caráter normativo, pois, ao fazer uma reflexão ética, pergunta-
se sobre a consciência e a coerência dos valores que norteiam as ações. A ética serve,
portanto, para verificar a coerência entre praticas e princípios, e questionar, reformular ou
fundamentar os valores e as normas componentes de uma moral, sem ser em si mesma
normativa. Ela deve possibilitar aos adolescentes o desenvolvimento de atitude critica, de
reconhecimento dos limites e possibilidades dos sujeitos e das circunstancia, de
problematização das ações e das relações entre indivíduos. Para tanto, requer por parte dos
adultos o reconhecimento das características psicológicas e sociais dessa fase. Nessa etapa
de vida, os adolescentes ampliam a sua capacidade de analisar situações complexas, de
considerar diferentes fatores envolvidos e de construir critérios de justiça. 6.6. ENTENDENDO O ESTATUTO DA CRIANÇA E DO ADOLESCENTE O estatuto é um marco e um divisor de águas na história recente da cidadania de
meninos e meninas no Brasil. Ele vem substituir o código de menores criado em 1979 que
ditou regra e normas durante 10 anos. Redigido por uma comissão composta por
representantes do setor jurídico, das políticas publicas e dos movimentos sociais, o estatuto
agrega o mérito de resultar de um trabalho conjunto, no qual os mais diversos segmentos
da sociedade puderam ser ouvidos. O estatuto garante os direitos e deveres de cidadania a
crianças e adolescentes, determinando à família, à sociedade, à comunidade e ao Estado a
co-responsabilidade pela proteção integral desse meninos e meninas, que antigamente era
tarefa exclusiva do juiz de menores. O estatuto não se resume a um conjunto de leis isoladas. Sua proposta é muito mais
ampla, pois prevê a criação de uma rede de atendimento, caracterizada por ações integras. Para garantir a criação dessa rede, o estatuto estipula que cada município criem órgãos para
o cumprimento de políticas voltadas a crianças e ao adolescente. São eles: o conselho
Municipal de Direitos da criança e do adolescente, os conselhos tutelares e as delegacias
especializadas. Com atribuições diferentes, todos devem atuar de acordo com sua
competência e em consonância com o juiz da infância e da juventude, personagem que
ganhou um novo papel a partir do estatuto, e com ministério público. A questão dos direitos e deveres da criança e do adolescente, pela primeira vez na
historia brasileira, tem prioridade absoluta, e sua proteção é dever da família o artigo trás:
O principio da prioridade absoluta garante à criança e ao adolescente a inclusão em 21 programas de orientação e promoção em todas as áreas: saúde, educação, assistência
social, esporte, cultura, lazer e outras. Em outro artigo podemos compreender que é dever da família, da comunidade, da
sociedade em geral e do poder publico assegurar, com absoluta prioridade, a efetivação dos
direitos referentes à vida, à saúde, à alimentação, à educação, ao esporte, ao lazer, à
profissionalização, à cultura, à dignidade, ao respeito, à liberdade e a]à convivência
familiar e comunitária. O ECA trás inovações, amplia e divide a responsabilidade da
família, do estado, da sociedade e da comunidade na proteção integral de crianças e
adolescentes, e estabelece um sistema participativo de formulação, controle e fiscalização
das políticas publicas de atendimento entre o Estado, a sociedade civil e o município. 6.6. ENTENDENDO O ESTATUTO DA CRIANÇA E DO ADOLESCENTE Ele
também distingue a criança como de 0 a 12 anos incompleto e adolescente de 12 a 18 anos
completo. Determina a obrigatoriedade de pais e responsáveis matricularem seus filhos e
acompanharem sua freqüência e seu aproveitamento escolar. 7. APRESENTAÇÃO DA PESQUISA – CEM 03. Essa parte do trabalho buscou analisar e compreender as concepções da área de
tecnologia e da arte na educação de jovens e adultos (EJA), de modo investigatório
permitindo uma maior compreensão do ponto de vista profissional. A pesquisa investigou com fontes e dados diversos, com análises e debates junto
aos órgãos governamentais, compreendendo do ponto de vista coletivo a profissionalização
e as elaborações das ações do governo e seu papel quanto às convicções da arte voltada
para a para a tecnologia e trabalho nas escolas com educação de jovens e adultos (EJA). No Centro de Ensino Fundamental 113, do Recanto das Emas, os professores da
faculdade de educação da Universidade de Brasília estão desenvolvendo um projeto com a
participação de 04 professores doutores e 02 alunos de mestrado e 02 alunos de graduação,
constituindo um grupo com o objetivo de promoverem pesquisas voltadas para os três
campos do conhecimento da educação de jovens e adultos, ciência e artes e educação
profissional. 22 Todo o grupo articulado estrategicamente reuniu, na construção de um foco que
permitirá o acesso aos alunos da educação de jovens e adultos (EJA), à tecnologia voltada
à arte e educação com o objetivo de assegurar a cada, conhecimentos e atualizações
tecnológicas na área profissional. Os debates e reflexões na rodada e as produções por
parte de alunos e professores da escola junto aos docentes, mestres e graduados da
Universidade de Brasília construirá um círculo de construção coletiva. A referida pesquisa buscou identificar em que medida a estratégia governamental
de resgate da educação, a população de jovens e adultos atinge aqueles que são objeto de
sua política, isso permite compreender se a educação está ou não articulada ou não com as
políticas governamentais e se suas contribuições no campo da educação de jovens e adultos
e educação profissional estão permitindo um maior acesso a tecnologia. Com as Duas modalidades: ensino regular e EJA, esta escola vem contribuindo que
a participação dos alunos da educação de jovens e adultos se envolva com o projeto de
pesquisa da Universidade de Brasília direcionando a uma pesquisa do ponto de vista da
escola e a Universidade de Brasília. A diretoria corrobora com essa perspectiva de pesquisa
junto aos professores e alunos e para o desenvolvimento dela. A formação profissional fica evidente acerca do desenvolvimento do ensino voltado
para tecnologia, aperfeiçoamento e atualização. 7. APRESENTAÇÃO DA PESQUISA – CEM 03. Nesse sentido a educação não é deixada
para trás e as produções ficam mais evidentes quando a arte e a tecnologia são voltadas
para a formação profissional do aluno. A história desses alunos e os papéis de cada um nas transformações ocorridas
dentro da escola e na comunidade vão desdobrar um maior entendimento sobre o nível e as
capacidades desenroladas no presente trabalho, remetendo a um aprimoramento dando
possibilidade para uma formação permanente e continuada do ponto de vista da educação
profissional. Desse modo, a educação é concebida de maneira significativa quanto ao
direcionamento da formação e desenvolvimento dos alunos quanto as perspectiva
profissional. Nesse sentido o que é esperado no presente trabalho com a implementação da
pesquisa é compreender na perspectiva histórica de um trabalho coletivo se os processos
tanto do ponto de vista das ações do atual governo estão confluentes e se coadunam com as
transformações não somente na escola, onde será desenvolvida a pesquisa, porém por outro
lado, se essas ações estão mesmo ocorrendo no Distrito Federal (DF). O trabalho nos remete a compreender os papeis e os processos das tecnologias
direcionadas à aprendizagem e a formação profissional de cada aluno tendo como partida
grupos formados por professores, alunos, doutores, mestres e graduados no centro de
ensino 113 do Recanto das Emas. 23 Outra pesquisa, foi na escola CEM 113 inaugurada em 17 de setembro de 2007, no
recanto das emas, onde pode vivenciar como o papel da filosofia com as crianças é muito
importante para a construção de conhecimentos em sala de aula foi bastante legal este
projeto, então, pode perceber que não há um sistema de avaliação ainda conforme os
requisitos nesta escola pois a escola não participa, daí o único sistema de avaliação feito foi
o CIAD(sistema de avaliação do desempenho das instituições educacionais do sistema de
ensino do DF.), ou seja, como a escola tem a modalidade de ensino fundamental, ainda não
houve a aplicação do SAEB (O Sistema Nacional de Avaliação da Educação Básica),
é o sistema usado como indicador do desempenho do IDEB (Índice de
Desenvolvimento da Educação Básica). Introdução: No centro de ensino Médio 03, da CEILÂNDIA, os professores da faculdade de
educação da Universidade de Brasília estão desenvolvendo um projeto com a participação
de 04 professores doutores e 02 alunos de mestrado e 02 alunos de graduação, constituindo
um grupo com o objetivo de promoverem pesquisas voltadas para os três campos do
conhecimento da educação de jovens e adultos, ciência e artes e educação profissional. Todo o grupo articulado estrategicamente reunirá na construção de um foco que
permitirá o acesso aos alunos da educação de jovens e adultos (EJA) à tecnologia voltada a
arte e educação com o objetivo de assegurar a cada, conhecimentos e atualizações
tecnológicas na área profissional. Os debates e reflexões na rodada e as produções por
parte de alunos e professores da escola junto aos docentes, mestres e graduados da
Universidade de Brasília construirá um circulo de construção coletiva. A referida pesquisa busca identificar em que medida a estratégia governamental de
resgate da educação, a população de jovens e adultos atinge aqueles que são objeto de sua
política, isso permite compreender se a educação está ou não articulada e coadunam ou não
com as políticas governamentais e se suas contribuições no campo da educação de jovens e
adultos e educação profissional estão permitindo um maior acesso a tecnologia. Com as Duas modalidades: ensino regular e EJA está escola vem contribuindo que a
participação dos alunos da educação de jovens e adultos se envolva com o projeto de
pesquisa da Universidade de Brasília direcionando a uma pesquisação do ponto de vista da
escola e a Universidade de Brasília. A diretoria corrobora com essa perspectiva de pesquisa
junto aos professores e alunos e para o desenvolvimento dela. A referida pesquisa busca identificar em que medida a estratégia governamental de
resgate da educação, a população de jovens e adultos atinge aqueles que são objeto de sua
política, isso permite compreender se a educação está ou não articulada e coadunam ou não
com as políticas governamentais e se suas contribuições no campo da educação de jovens e
adultos e educação profissional estão permitindo um maior acesso a tecnologia. Com as Duas modalidades: ensino regular e EJA está escola vem contribuindo que a
participação dos alunos da educação de jovens e adultos se envolva com o projeto de
pesquisa da Universidade de Brasília direcionando a uma pesquisação do ponto de vista da
escola e a Universidade de Brasília. 7. APRESENTAÇÃO DA PESQUISA – CEM 03. Como a escola ainda não aderiu ao sistema de avaliação nacional o IDEB e é uma
escola muito recente, o que foi constatado é que o único sistema de avaliação propriamente
ocorrido na escola foi o CIAD (sistema de avaliação do desempenho das instituições
educacionais do sistema de ensino do DF.) O SIADE - Sistema de Avaliação do
Desempenho das Instituições Educacionais do Sistema de Ensino do Distrito Federal foi
criado por meio do Decreto nº 29.244, de 2 de julho de 2008. É um instrumento
permanente de planejamento, destinado a aferir as condições da oferta do ensino nas
escolas públicas e privadas do DF, de forma a garantir o pleno desenvolvimento do
educando, seu preparo para o exercício da cidadania e sua qualificação para o trabalho. este por sua vez é usado aqui no DF, Esta media é calculada por disciplina, A escola
teve uma média muito boa em português com relação as escolas do DF. Nesse sentido, a filosofia da educação aliada as praticas escolares na escola publica deixa
claramente evidente o papel do compromisso que tive eu e meus colegas de pesquisa com a
escola. Do ponto de vista filosófico a educação é abarcada com valores nítidos e revestidos
com pleno compromisso. Nesse sentido, O educador tem que propiciar condições para que o individuo
construa sua cidadania, possibilitando mudanças que ocorrem através do exercício da
cidadania participativa, que vai se construindo de muitas formas, sendo uma delas o
desenvolvimento de iniciativas comunitárias, gerando projetos de transformações sociais,
com isso, o projeto de filosofia com as crianças é muito importante para compreendemos
essas relações entre aluno e professor. 24 Então, Para viver em sociedade é preciso possuir valores, critérios, e , mais ainda,
estabelecer relações e hierarquias entre esses valores. Escolher implica comparar e valorar. Assim, torna-se necessário uma filosofia com a elaboração de critérios que classifiquem as
ações como boas ou más, corretas ou inadequadas, e que orientem e justifiquem a escolha,
que se configura com a resposta diante das prescrições da sociedade. A moral sofre
transformações, principalmente quando submetida À reflexão critica realizada pela ética. 1. Introdução ao plano de trabalho Este projeto de pesquisa identificou em que medida a estratégia governamental de
resgate da educação à população de jovens e adultos atinge aqueles que são objeto da sua
formulação. No caso particular, pretende-se com essa ação, acompanhar um grupo de
beneficiários da Educação de Jovens e Adultos integrada à Educação Profissional na
localidade do Recanto das Emas – DF, mais precisamente os alunos do EJA do CEF-113. Buscar-se-á acompanhar os alunos desses cursos e em que medida a sua trajetória de
sucesso no mercado de trabalho é explicada pelo sucesso das ações a que foram
beneficiados. Plano de trabalho 1. Introdução ao plano de trabalho Introdução: A diretoria corrobora com essa perspectiva de pesquisa
junto aos professores e alunos e para o desenvolvimento dela. A formação profissional fica evidente acerca do desenvolvimento do ensino voltado
para tecnologia, aperfeiçoamento e atualização. Nesse sentido a educação não é deixada
para trás e as produções ficam mais evidentes quando a arte e a tecnologia são voltadas
para a formação profissional do aluno. 25 A historia desses alunos e os papeis de cada um nas transformações ocorridas
dentro da escola e na comunidade vão desdobrar um maior entendimento sobre o nível e as
capacidades desenroladas no presente trabalho, remetendo a um aprimoramento dando
possibilidade para uma formação permanente e continuada do ponto de vista da educação
profissional. Desse modo, a educação é concebida de maneira significativa quanto ao
direcionamento da formação e desenvolmento dos alunos quanto as perspectiva
profissional. Nesse sentido o que é esperado no presente trabalho com a implementação da
pesquisa é compreender na perspectiva histórica de um trabalho coletivo se os processos
tanto do ponto de vista das ações do atual governo estão confluentes e se coadunam com as
transformações não somente na escola, onde será desenvolvida a pesquisa, porém por outro
lado, se essas ações estão mesmo ocorrendo no Distrito Federal (DF). O trabalho nos
remete a compreender os papeis e os processos das tecnologias direcionadas à
aprendizagem e a formação profissional de cada aluno tendo como partida grupos
formados por professores, alunos, doutores, mestres e graduados no centro de ensino 03 da
CEILÂNDIA. 3. Resultados Esperados na Execução do Plano de Trabalho 3. Resultados Esperados na Execução do Plano de Trabalho A partir dessas pesquisas com a comunidade escolar do CEF-113, a transiarte foi
explicada ao grupo como uma linguagem. Linguagem tecnológica que está associada a
valores, culturas e criações artísticas capazes de levar a compreensão da realidade do
jovem e adulto dentro das suas possibilidades e limitações, o que leva a uma reflexão de
valores, do que é ser jovem e ser adulto. Criações artísticas de imagens e vídeos
envolvendo temas e outras categorias com a vivência da comunidade, grupo escolar e sua
divulgação no ambiente virtual. 2. Metodologia do Plano de Trabalho Pretende despertar a identidade cultural na produção artística virtual de jovens/
adultos através da criação artística coletiva e individual. A intenção é fortalecer os
conhecimentos epistemológicos da arte e as práticas artísticas, com a implementação da
pesquisa é compreender na perspectiva histórica de um trabalho coletivo se os processos
tanto do ponto de vista das ações do atual governo estão confluentes e se coadunam com as 26 transformações não somente na escola, onde será desenvolvida a pesquisa, porém por outro
lado, se essas ações estão mesmo ocorrendo no Distrito Federal (DF). transformações não somente na escola, onde será desenvolvida a pesquisa, porém por outro
lado, se essas ações estão mesmo ocorrendo no Distrito Federal (DF). Breve Histórico Um fundo próprio para a educação não foi algo pensado somente nestas duas
últimas décadas, desde os anos 30 do século passado ele está sendo cogitado, inicialmente
com o movimento dos pioneiros da educação, principalmente por Anísio Teixeira. Porém,
apenas em menos de quinze anos é que o Brasil passa a ter um fundo para a educação
pensado de forma mais sistemática. “Em 1996 é aprovado uma Emenda Constitucional 14 que obriga Estados, Distrito
Federal e Municípios aplicarem, até 2006, pelo menos 60% do percentual constituído
mínimo de 25% (ou seja 15%) de imposto no Ensino Fundamental” (DAVIES, 2001, p. 15). A EC 14, também cria o Fundo de Manutenção e Desenvolvimento do Ensino
Fundamental e de Valorização do Magistério - Fundef. O Fundef regulamentado pela Lei
9.424/96 foi implantado no governo FHC. A avaliação feita, por Davies, do Fundef, enquanto, ainda estava vigorando, era que
ele pudesse enfraquecer os outros níveis de ensino: o infantil, o médio, o supletivo e o de
jovens e adultos, uma vez que, a distribuição do fundo arrecadado era feita levando-se em
conta a número de matriculas do Ensino Fundamental regular, do ano anterior, isso gerou
uma corrida por matriculas e muito desvio de dinheiro, pois criaram muitos estudantes
fantasmas, com o objetivo de arrecadar mais fundos. (DAVIES, 2001, p. 25) 27 Um outro problema apontado por Davies refere-se a pequena participação do
governo federal em relação aos recursos arrecadados, apesar dele ter sido o responsável
pela iniciativa. O autor, então disponibiliza os dados de outubro de 2000, encontrado na
página do MEC, na internet para comprovar a pequena participação do GF. Ele então diz
que a “receita total do Fundef somava 13, 273 bilhões”, os contribuintes eram os estados e
os Municípios, o DF não estava incluso e, a contribuição do Governo Federal era de “486,6
bilhões o equivale a 3,7% do total”. (DAVIES, 2001, p. 16-17) 8. ÚLTIMAS CONSIDERAÇÕES A omissão presente em todas as esferas que produzem leis sobre educação e ensino
está diretamente relacionada intencionalmente com a política da desorientação e
desorganização burocrática aos moldes da produção capitalista, para enfrenta isso com
unhas e dentes este problema, temos ajustar as limitações presente na própria lei, na cultura
e no ensino e em grande parte na educação. Temos, no contexto social, uma verdadeira instabilidade das instituições políticas,
as reformas não são do ponto de vista democrático geradores de ações táticas na área da
educação, a estratégia que se tem em grande parte não orienta e coordena essas ações
principalmente com relação as políticas publicas de maneira geral. O fator determinante é a
omissão Estatal nesse caos gerado no sistema de ensino, devemos mudar e orientar a
estratégia da Lei e das políticas publicas com relação a cultura e o FUNDEB que não estão
coerente com a realidade, de outra forma, a produção social existente com relação as
políticas públicas propriamente não conseguem de maneira alguma permitir que um
financiamento ou ate um planejamento consistente chegassem a ser concluído
efetivamente. O que se faz presente em uma realidade traçada pela desigualdade social. Não
adianta dançar na mesma música se não podemos nem mesmo modificar nossos passos,
tentamos modificar os nossos passos mais uma vez e dançamos a mesma música sempre,
tentamos modificar os nossos passos e nada mais uma vez, não adiantou temos que
direcionar nossas ações a cerca da educação mobilizando todas as redes sociais frente a
esse problema, dividimos a responsabilidade agora temos que lutar juntos, pois a cultura
corrupta está crescendo e estamos ficando pobres culturalmente, as produções liberalistas
de Estado estão definindo um novo individuo social, a palavra para os miseráveis na escola
é de suma importância, a luta tem que ser traçada, o problema ta tomando grandes
dimensões, um exemplo disso é a educação lá na Finlândia, onde o sonho e a estratégia são
praticamente perfeitos. 8. ÚLTIMAS CONSIDERAÇÕES 28 De outra maneira o Estado pode reverter uma realidade processando leis que criam
oportunidades melhores para alunos em condições de pobreza extrema, se não há uma
intencionalidade ou uma estratégica de desenvolver o Brasil, o Estado tem que intervir
diretamente no privado radicalmente no contexto da educação e da própria escola, a
pobreza ta tomando conta das ruas, há muito marginal e até a cultura da corrupção já ta
traçada em todas as esfera sociais, parece que ser corrupto virou modo até mesmo na
escola, as ruas estão cheio de miseráveis, o que se tem, é produzir um sistema coerente
com a nossa realidade. A escola privada tem que abarca nessa luta o poder da polícia não está mais
adiantado nada, o que podemos fazer não é chamar a policia e sim reverte isso com
medidas legais e com luta e mobilização das pessoas. As omissões e a incoerência traçada
nos meios de comunicação de massa gerara esta incoerência por motivos e intenções de
grupos formadores de um “saber” fantasiado gerando e produzindo indivíduos meta-
individual carente de valores equilibrados e estáveis na sociedade, a produção gerou
mentes desajustadas, desconfiguradas, suprida de poder e dinheiro e sonhos privatista isto
fluindo inteiramente na mente humana e com força determinante configurada na tecnologia
e na produção da imaginação e da imagem, as cadeias para muitos é motivo de vantagem
entre todos os presentes. A condição humana continua em xeque pronto para a violência. Nenhum desses problemas terá solução se continuarmos trotando pelos mesmos
caminhos direitistas, indiferentes à educação popular e ao progresso do país. É
indispensável para o Brasil, como o foi para todos os povos que deram certo realizando
suas potencialidades, empreender um grande esforço nacional no sentido de alcançar
algumas metas mínimas no campo da educação popular. ANEXO: A cultura constitui, por assim dizer, a natureza específica do ser humano,
que está sempre causando surpresas. A motricidade em desordem significa que,
a cada surpresa ambiental, há a possibilidade de constituir uma nova ordem. Sendo assim, precisamos, como propõe Fonseca (1988), pensar o fenômeno da
motricidade como algo extremamente complexo e integrado a outros sistemas,
em que a dinâmica das interações com o meio ambiente determina as habilidades
que serão construídas. Exemplos a respeito disso são fartos, e alguns bastante
banais. Imaginemos um professor que, segurando uma pilha de cadernos tente
sair da sala e para isso precise abrir a porta. Ora, as mãos que seguram os ca- 29 dernos constituem, momentaneamente, uma ordem para realização desse gesto;
portanto, não podem realizar simultaneamente outros gestos. Para tanto, nosso
professor deverá desmanchar, isto é, desordenar a coordenação atual para reali-
zar outra, a de abrir a porta. Portanto, a nova ordem será precedida de uma de-
sordem. Essa capacidade de estar disponível para realizar novas coordenações,
a partir da desordenação das anteriores, é excepcionalmente desenvolvida nos
humanos e constitui uma das características básicas de nossa motricidade, espe-
cialmente quando o gesto requerido refere-se a situações desconhecidas e inédi-
tas. Porém se o professor segurando os cadernos, fosse chamado por um dos
alunos, poderia, sem deixar o material, virar-se e prestar atenção ao que a criança
lhe está perguntando, sem desfazer a ordem das mãos que seguram os cader-
nos. Nesse caso, a constituição de uma nova ordem resultaria na combinação de
duas outras ordens. As atividades motoras fazem parte do cotidiano em quaisquer
instituições dedicadas à educação de crianças. Nelas, o movimento, o brinquedo
e os jogos tradicionais da cultura popular obtiveram, de alguma forma, espaços e
momentos nas rotinas das salas de aula. Em algumas escolas encontramos core-
ografias no início dos trabalhos, parque livre ou dirigido, cantinhos com jogos ou
materiais lúdicos etc. Em um estudo sobre a antropologia dos movimentos, Daólio (1995) afirma
que a motricidade resulta das interações sociais e da relação dos homens com o
ambiente; seu significado constrói-se em função de diferentes necessidades, inte-
resses e possibilidades corporais presentes nas diferentes culturas, em diferentes
épocas da história. Assim, ao brincar, jogar, imitar e criar ritmos, as crianças tam-
bém se apropriam do repertório da cultura corporal na qual estão inseridas. ANEXO: Nesse
sentido, as instituições educativas devem favorecer um ambiente físico e social
onde a criança se sinta estimulada e segura para arriscar-se e vencer desafios. Quanto mais rico e desafiador for esse ambiente (do ponto de vista dos movimen-
tos), maior será a possibilidade de ampliação de conhecimentos acerca de si
mesma, dos outros e do meio em que vive. Como dissemos, a participação em jogos, brincadeiras e atividades rítmi-
cas revelam, por outro lado, a cultura corporal de cada grupo social, constituindo-
se em atividades privilegiadas nas quais o movimento é assimilado de forma sig-
nificativa. Dessa forma, é muito importante que a instituição incorpore esse reper-
tório de forma intencional à rotina diária, permitindo e estimulando a expressivida-
de e a mobilidade das crianças. Compreender tal caráter expressivo poderá aju-
dar o professor a organizar melhor a sua prática. 30 As posturas do professor também exercem influência sobre a motricidade
infantil, uma vez que os corpos dos adultos ao redor são veículos expressivos. A
atenção a esse aspecto valorizará e adequará os próprios gestos, mímicas e mo-
vimentos na comunicação com as crianças, fornecendo-lhes um repertório de ex-
pressões, por exemplo, ao contar histórias pontuando idéias ou se utilizando de
recursos vocais para enfatizar sua dramaticidade. Da mesma forma, conhecer
jogos e brincadeiras e refletir sobre seus movimentos são condições importantes
para estimular adequadamente o desenvolvimento infantil. Competências a serem desenvolvidas ao longo da Educação Infantil: * Participar de diferentes atividades corporais procurando adotar uma atitude coo-
perativa e solidária, sem discriminar os colegas pelo desempenho ou por razões
sociais, físicas sexuais ou culturais. * Participar de diferentes atividades corporais procurando adotar uma atitude coo-
perativa e solidária, sem discriminar os colegas pelo desempenho ou por razões
sociais, físicas sexuais ou culturais. * Organizar autonomamente alguns jogos, brincadeiras ou outras atividades cor-
porais simples. * Organizar autonomamente alguns jogos, brincadeiras ou outras atividades cor-
porais simples. Para desenvolver tais competências podemos às crianças a oportunidade de: * Participar em diversos jogos e brincadeiras, respeitando as regras e não discri-
minando os colegas. * Resolver situações-problema por meio do diálogo e experimentação, com a aju-
da do professor. Discutir regras dos jogos vividos. * Explicar e demonstrar brincadeiras aprendidas em contextos extra-escolares.
Participar e apreciar brincadeiras ensinadas pelos colegas.
Participar e apreciar brincadeiras ensinadas pelos colegas. ANEXO: Essa forma de instrumentalizar o jogo e as brincadeiras, retirando o aspec-
to gratuito, do prazer pelo prazer, parece não estar presente nas brincadeiras. Utilizando os rituais, as músicas, as danças e as apresentações folclóricas como
atividade das crianças no lúdico estava presente juntamente com o artístico. No
entanto, todas essas diferentes formas de conceber o jogo coexistiam. sobre jo-
gos e brincadeiras. Na verdade, como já dizia Caillois (1982), a atividade lúdica é
um continuum com duas extremidades: uma ocupada por jogos que manifestam
criatividade, fantasia, espontaneidade; e outra, com os jogos convencionais, su-
bordinados a regras. Na vida social, sempre existem regras, que variam de socie-
dade para sociedade, portanto, o jogo infantil, naquele continuum, pode tanto en- 31 sinar a obediência a elas, como também pode ensinar a sua arbitrariedade. Atra-
vés das manifestações folclóricas, as crianças estavam conhecendo vários tipos
de "regras", de sociedades diferentes, momentos históricos distintos, podendo,
com isso, ter variadas experiências lúdicas entre as referidas extremidades: da
mera obediência disciplinadora, à pura fantasia ociosa. A escola é o lugar onde a criança tem que ficar sentada, bem quietinha,
quatro horas por dia e nove meses por ano, absorvendo abstrações e sem a prá-
tica da experimentação. Tudo se passa como se o interesse da criança gravitasse
em torno de livros, silêncio, passividade, inatividade. A escola é ainda individualis-
ta, na época em que vivemos, não se propondo a ensinar cooperação, iniciativa,
autodireção ou a arte de fazer amigos e dirigir os outros. Violando as leis do cres-
cimento físico e psíquico, prepara homens sem iniciativa, sem vontade, sem
idéias. Forma pulmões e corações débeis, braços e pernas macilentos e debilita-
dos, gerando na criança uma anemia e uma constituição acanhada e incompleta
(...) Ao invés de respeitar o corpo e deixar a mente cuidar de si mesma, respeita-
mos a mente e descuidamos do corpo. Ambas as atitudes são erradas, mas não
podemos deixar de reconhecer esta verdade tão simples: o físico serve de base
ao mental e este último não deve ser desenvolvido em prejuízo do primeiro. A es-
cola não é, pois, o sistema ideal de cultura infantil. Um outro sistema precisa, não
diremos substituí-la, mas completá-la. Um sistema que tome a criança como ela
é, e a nossa complexa civilização como ela é, harmonizando os dois fatos de uma
maneira científica e ao mesmo tempo humana. ANEXO: O valor da filosofia O valor da filosofia O conhe-
cimento a que aspira é o que unifica e sistematiza o corpo das ciências e o que resulta de um
exame crítico dos fundamentos das nossas convicções, dos nossos preconceitos e das nossas
crenças. Mas não se pode dizer que a filosofia tenha tido grande sucesso ao tentar dar respos-
tas exactas às suas questões. Se perguntarmos a um matemático, a um mineralogista, a um
historiador ou a qualquer outro homem de saber, que corpo exacto de verdades a sua ciência
descobriu, a sua resposta durará o tempo que estivermos dispostos a escutá-lo. Mas se colo-
carmos a mesma questão a um filósofo, se for sincero terá de confessar que o seu estudo não
chegou a resultados positivos como aqueles a que chegaram outras ciências. É verdade que
isto se explica em parte pelo facto de que assim que se torna possível um conhecimento exac-
to acerca de qualquer assunto, este assunto deixa de se chamar filosofia e passa a ser uma
ciência separada. A totalidade do estudo dos céus, que pertence actualmente à astronomia,
esteve em tempos incluído na filosofia; a grande obra de Newton chamava-se "os princípios
matemáticos da filosofia natural". Analogamente, o estudo da mente humana, que fazia parte
da filosofia, foi agora separado da filosofia e deu origem à ciência da psicologia. Assim, a
incerteza da filosofia é em larga medida mais aparente do que real: as questões às quais já é
possível dar uma resposta exacta são colocadas nas ciências, e apenas aquelas às quais não é
possível, no presente, dar uma resposta exacta, formam o resíduo a que se chama filosofia. Contudo, esta é apenas uma parte da verdade sobre a incerteza da filosofia. Há muitas ques-
tões ― entre elas aquelas que são do maior interesse para a nossa vida espiritual ―
que, tanto quanto podemos ver, continuarão sem solução, a menos que as capaci-
dades do intelecto humano se tornem de uma ordem completamente diferente da actual. O
universo tem uma unidade de plano ou de propósito, ou é uma confluência fortuita de áto-
mos? A consciência é um componente permanente do universo, dando a esperança de que a
sabedoria aumente indefinidamente, ou é um acidente transitório num pequeno planeta no
qual a vida tem por fim de se tornar impossível? O bem e o mal são importantes para o uni-
verso ou apenas para o homem? O valor da filosofia Tendo agora chegado ao fim da nossa análise breve e muito incompleta dos problemas da
filosofia, será vantajoso que, para concluir, consideraremos qual é o valor da filosofia e por-
que deve ser estudada. É da maior necessidade que examinemos esta questão, tendo em conta
que muitas pessoas, sob a influência da ciência ou de afazeres práticos, se inclinam a duvidar
de que a filosofia seja algo melhor do que frivolidades inocentes mas inúteis, distinções de-
masiado subtis e controvérsias sobre matérias acerca das quais o conhecimento é impossível. Esta visão da filosofia parece resultar em parte de uma concepção errada dos fins da vida e
em parte de uma concepção errada do género de bens que a filosofia procura alcançar. A físi-
ca, por meio de invenções, é útil a inúmeras pessoas que a ignoram completamente, pelo que
seu o estudo é recomendado, não apenas, ou principalmente, devido ao efeito no estudante,
mas sim devido ao efeito na humanidade em geral. A filosofia não tem esta utilidade. Se o 32 estudo da filosofia tem algum valor para os que não estudam filosofia, tem de ser apenas
indirectamente, por intermédio dos seus efeitos na vida daqueles que a estudam. Portanto, se
o valor da filosofia deve ser procurado em algum lado, é principalmente nestes efeitos. Mas mais, se não queremos que a nossa tentativa para determinar o valor da filosofia fracas-
se, temos de libertar primeiro as nossas mentes dos preconceitos daqueles a que se chama
erradamente homens "práticos". O homem "prático", como se usa frequentemente a palavra, é
aquele que reconhece apenas necessidades materiais, que entende que os homens devem ter
alimento para o corpo, mas esquece-se da necessidade de fornecer alimento à mente. Mesmo
que todos os homens vivessem desafogadamente e que a pobreza e a doença tivessem sido
reduzidas ao ponto mais baixo possível, ainda seria necessário fazer muito para produzir uma
sociedade válida; e mesmo neste mundo os bens da mente são pelo menos tão importantes
como os do corpo. É exclusivamente entre os bens da mente que encontraremos o valor da
filosofia; e somente aqueles que não são indiferentes a estes bens podem ser convencidos de
que o estudo da filosofia não é uma perda de tempo. Como todos os outros estudos, a filosofia, aspira essencialmente ao conhecimento. O valor da filosofia Embora a filosofia seja incapaz de nos dizer com certeza qual é
a resposta verdadeira às dúvidas que levanta, é capaz de sugerir muitas possibilidades que
alargam os nossos pensamentos e os libertam da tirania do costume. Assim, embora diminua
o nosso sentimento de certeza quanto ao que as coisas são, a filosofia aumenta muito o nosso
conhecimento do que podem ser; elimina o dogmatismo um tanto arrogante daqueles que
nunca viajaram na região da dúvida libertadora e, ao mostrar as coisas que são familiares com
um aspecto invulgar, mantém viva a nossa capacidade de admiração. Para além da sua utilidade na revelação de possibilidades insuspeitadas, a filosofia adquire
valor ― talvez o seu principal valor ― por meio da grandeza dos objectos
que contempla e da libertação de objectivos pessoais e limitados que resulta desta
contemplação. A vida do homem instintivo está fechada no círculo dos seus interesses priva-
dos. A família e os amigos podem estar incluídos, mas o mundo exterior não é tido em conta
excepto na medida em que possa auxiliar ou impedir o que entra no círculo dos desejos ins-
tintivos. Numa vida assim há algo de febril e limitado, comparada com a qual a vida filosófi-
ca é calma e livre. O mundo privado dos interesses instintivos é um mundo pequeno no meio
de um mundo grande e poderoso que, mais cedo ou mais tarde, reduzirá o nosso mundo pri-
vado a ruínas. A menos que consigamos alargar os nossos interesses de modo a incluir todo o
mundo exterior, somos como uma guarnição numa fortaleza sitiada, que sabe que o inimigo
impede a sua fuga e que a rendição final é inevitável. Numa vida assim não há paz, mas uma
luta constante entre a persistência do desejo e a incapacidade da vontade. De uma forma ou
doutra, se queremos que a nossa vida seja grande e livre, temos de fugir desta prisão e desta
luta. Uma forma de fugir é por intermédio da contemplação filosófica. Na sua perspectiva mais
ampla, a contemplação filosófica não divide o universo em dois campos hostis ― amigos e
inimigos, prestável e hostil, bom e mau ― vê o todo com imparcialidade. Quando é pura,
a contemplação filosófica não procura provar que o resto do universo é semelhante ao ho-
mem. O valor da filosofia Estas são questões que a filosofia coloca e a que diferentes
filósofos responderam de diferentes maneiras. Mas, quer seja ou não possível descobrir res-
postas de outro modo, parece não ser possível demonstrar que alguma das respostas sugeridas
pela filosofia é verdadeira. No entanto, por muito pequena que seja a esperança de descobrir
uma resposta, a filosofia tem o dever de continuar a examinar estas questões, a conscienciali-
zar-nos da sua importância, a examinar todas as respostas que lhes são dadas e a manter vivo
o interesse especulativo pelo universo, que pode ser destruído se nos limitarmos ao conheci-
mento que podemos verificar com exactidão. É verdade que muitos filósofos defenderam que a filosofia pode estabelecer a verdade de
determinadas respostas a estas questões fundamentais. Eles acreditaram ser possível provar
por demonstrações rigorosas que o mais importante nas crenças religiosas é verdadeiro. Para
que possamos julgar estas tentativas, é necessário examinar o conhecimento humano e formar 33 uma opinião quanto aos seus métodos e às suas limitações. Seria insensato pronunciarmo-nos
dogmaticamente sobre um assunto destes, mas se as investigações dos capítulos anteriores
não nos induziram em erro, somos forçados a renunciar à esperança de encontrar provas filo-
sóficas das crenças religiosas. Não podemos, portanto, incluir como parte do valor da filoso-
fia qualquer conjunto de respostas exactas a essas questões. Por esta razão, mais uma vez, o
valor da filosofia não depende de qualquer pretenso corpo de conhecimentos que podemos
verificar com exactidão e que aqueles que a estudam adquiram. Na verdade, o valor da filosofia tem de ser procurado sobretudo na sua própria incerteza. O
homem que não tem a mais pequena capacidade filosófica, vive preso aos preconceitos deri-
vados do senso comum, das crenças habituais da sua época ou da sua nação, e das convicções
que se formaram na sua mente sem a cooperação ou o consentimento reflectido da sua razão. Para um tal homem o mundo tende a tornar-se definido, finito, óbvio; os objectos vulgares
não levantam quaisquer questões e as possibilidades invulgares são desdenhosamente rejeita-
das. Assim que começamos a filosofar, pelo contrário, verificamos, como vimos nos capítulos
iniciais, que mesmo os objectos mais comuns levam a problemas a que apenas podemos dar
respostas muito incompletas. O valor da filosofia Toda a aquisição de conhecimento é um alargamento do Eu, mas alcança-se melhor
este alargamento quando ele não é directamente procurado. É obtido quando o desejo de co-
nhecimento é apenas operativo, por um estudo que não deseja antecipadamente que os seus
objectos tenham esta ou aquela característica, mas adapta o Eu às características que encontra
nos seus objectos. Este alargamento do Eu não é obtido quando, aceitando o Eu como é, ten-
tamos mostrar que o mundo é de tal modo semelhante a este Eu que é possível conhecê-lo
sem ter de admitir o que parece estranho. O desejo de provar isto é uma forma de auto-
afirmação e, como toda a auto-afirmação, é um obstáculo ao crescimento do Eu que ela dese-
ja e de que o Eu sabe ser capaz. Na especulação filosófica como em tudo o mais, a auto-
afirmação vê o mundo como um meio para os seus próprios fins; considera, assim, o mundo
menos importante do que o Eu e o Eu limita a grandeza dos seus bens. Na contemplação,
pelo contrário, partimos do não-Eu e por intermédio da sua grandeza alargamos os limites do 34 Eu; por intermédio da infinidade do universo a mente que o contempla participa da infinida-
de. Eu; por intermédio da infinidade do universo a mente que o contempla participa da infinida-
de. Por esta razão, as filosofias que adaptam o universo ao Homem não promovem a grandeza de
alma. O conhecimento é uma forma de união do Eu e do não-Eu e, como todas as uniões, é
prejudicado pelo domínio e, portanto, por qualquer tentativa de forçar o universo a confor-
mar-se ao que encontramos em nós. Há uma ampla tendência filosófica para o ponto de vista
que nos diz que o Homem é a medida de todas as coisas, que a verdade é feita pelo homem,
que o espaço, o tempo e o mundo dos universais são propriedades da mente e que, se existir
algo que não tenha sido criado pela mente, é incognoscível e não tem qualquer importância
para nós. Se as nossas discussões anteriores estavam correctas, este ponto de vista é falso;
mas para além de ser falso, tem o efeito de despojar a contemplação filosófica de tudo o que
lhe dá valor, uma vez que a confina ao Eu. O valor da filosofia Aquilo a que chama conhecimento não é uma
união com o não-Eu, mas um conjunto de preconceitos, de hábitos e de desejos, que constitu-
em um véu impenetrável entre nós e o mundo fora de nós. O homem que encontra prazer
numa teoria do conhecimento destas é como o homem que nunca deixa o círculo doméstico
por receio de que a sua palavra possa não ser lei. A verdadeira contemplação filosófica, pelo contrário, encontra satisfação em todo o alarga-
mento do não-Eu, em tudo o que engrandeça os objectos contemplados e, por essa via, o su-
jeito que contempla. Tudo o que na contemplação seja pessoal ou privado, tudo o que depen-
da do hábito, do interesse pessoal ou do desejo, deforma o objecto e, por isso, prejudica a
união que o intelecto procura. Ao criarem desta forma uma barreira entre o sujeito e o objec-
to, estas coisas pessoais e privadas tornam-se uma prisão para o intelecto. O intelecto livre
verá como Deus pode ver, sem um aqui e agora, sem esperanças nem temores, sem o empeci-
lho das crenças vulgares e dos preconceitos tradicionais, calmamente, desapaixonadamente,
no desejo único e exclusivo de conhecimento ― conhecimento tão impessoal e tão pu-
ramente contemplativo quanto o homem possa alcançar. Também por este motivo, o intelecto
livre dará mais valor ao conhecimento abstracto e universal, no qual os acidentes da história
privada não entram, do que ao conhecimento originado pelos sentidos e dependente, como
este conhecimento tem de ser, de um ponto de vista exclusivo e pessoal e de um corpo cujos
órgãos dos sentidos deformam tanto quanto revelam. A mente que se habituou à liberdade e à imparcialidade da contemplação filosófica conserva-
rá alguma desta mesma liberdade e imparcialidade no mundo da acção e da emoção. Encarará
os seus propósitos e desejos como partes do todo, com a falta de persistência que resulta de
os ver como fragmentos minúsculos num mundo no qual nada mais é afectado por qualquer
acção humana. A imparcialidade que, na contemplação, é o desejo puro da verdade, é a mes-
ma qualidade da mente que, na acção, é a justiça e na emoção é o amor universal que pode
ser dado a tudo e não apenas aos que consideramos úteis ou dignos de admiração. O valor da filosofia Com base em observações simples acerca da experiência perceptiva ― o facto de que,
digamos, uma mesa parece ter diferentes cores, formas e texturas, depen-
dendo de variações no observador ou nas condições em que é percepcionada ― podemos
ver que há uma distinção a fazer entre as aparências das coisas e aquilo que elas são em si
mesmas. Como é que podemos ter a certeza de que a aparência representa fielmente a reali-
dade que supomos encontrar-se por trás dela? Pode-se mesmo perguntar, como sugerem os
argumentos cépticos sobre sonhos e ilusões, se podemos ter a certeza de que existem de facto
coisas reais 'por detrás' das nossas experiências sensoriais. Para lidar com estas questões, Russell introduz o termo 'dado dos sentidos' para designar as
coisas que são imediatamente conhecidas na sensação: ocorrências particulares na consciên-
cia perceptiva de cores, sons, gostos, cheiros e texturas, correspondendo cada classe de dados
a um dos cinco sentidos. Os dados dos sentidos distinguem-se dos actos de os sentir: eles são
aquilo de que temos imediatamente consciência nos actos de sentir. Mas eles têm também,
como as considerações do parágrafo anterior mostram, de ser distinguidos das coisas no
mundo fora de nós com que os supomos associados. A questão crucial portanto é: qual a rela-
ção dos dados dos sentidos com os objectos físicos? Russell responde ao céptico que questiona o nosso direito a alegar que conhecemos o que se
encontracos sejam, estritamente falando, irrefutáveis, não há contudo 'a menor razão' para os
supor verdadeiros (PF 44[1]). A sua estratégia é coligir considerações persuasivas para supor-
tar o seu ponto de vista. Primeiro, podemos pegar em que as nossas experiências imediatas do
dado dos sentidos têm uma 'certeza primitiva'. Quando temos experiência de dados dos senti-
dos que vemos naturalmente como estando associados com, digamos, uma mesa, reconhece-
mos que não dissemos tudo o que há a dizer sobre a mesa. Pensamos, por exemplo, que a
mesa continua a existir quando não estamos na sala. Podemos comprá-la, tapá-la com um
pano, movê-la de um lado para o outro. Requeremos que observadores diferentes sejam capa-
zes de percepcionar a mesma mesa. Tudo isto sugere que a mesa é algo além e acima dos
dados dos sentidos que nos aparecem. O valor da filosofia Por conse-
guinte, a contemplação alarga não apenas os objectos dos nossos pensamentos, mas também
os objectos das nossas acções e das nossas afecções; faz-nos cidadãos do universo e não ape-
nas de uma cidade murada em guerra com tudo o resto. A verdadeira liberdade humana e a
sua libertação da sujeição a esperanças e temores mesquinhos consiste nesta cidadania do
universo. Assim, resumindo a nossa discussão sobre o valor da filosofia, a filosofia deve ser estudada,
não por causa de quaisquer respostas exactas às suas questões, uma vez que, em regra, não é
possível saber que alguma resposta exacta é verdadeira, mas antes por causa das próprias
questões; porque estas questões alargam a nossa concepção do que é possível, enriquecem a
nossa imaginação intelectual e diminuem a certeza dogmática que fecha a mente à especula-
ção; mas acima de tudo porque, devido à grandeza do universo que a filosofia contempla, a
mente também se eleva e se torna capaz da união com o universo que constitui o seu mais
alto bem. 5
Uma das questões centrais da filosofia é: o que é o conhecimento e como se obtém? John
Locke e os seus sucessores na tradição empirista defenderam que o fundamento do conheci- 35 mento contingente sobre o mundo se encontra na experiência sensorial ― o uso dos cin-
co sentidos, ajudados quando necessário por telescópios e outros instrumentos
semelhantes. Russell está de acordo com isto. Mas o empirismo enfrenta o desafio dos argu-
mentos cépticos que visam mostrar que as nossas pretensões ao conhecimento podem com
frequência ― talvez sempre ― ser injustificadas. Há várias razões para isto. Às
vezes cometemos erros quando percepcionamos ou raciocinamos, às vezes sonhamos sem
saber que estamos a sonhar, às vezes somos enganados devido aos efeitos da febre ou do
álcool. Quando afirmamos conhecer algo, como podemos estar certos de que esta afirmação
não é posta em causa de nenhum destes modos? Em Os Problemas da Filosofia (PF) em 1912 Russell fez a sua primeira tentativa sistemática
de tratar estas questões. 'Há algum conhecimento', pergunta, 'que seja tão certo que nenhum
homem razoável possa dele duvidar?' Ele responde afirmativamente, mas a certeza, como se
constata, está longe da certeza absoluta da prova. O valor da filosofia Ao conhecimento imediato de verdades Russell chama 'conhecimento intuitivo' e descreve as
verdades que são assim conhecidas como auto-evidentes. Estas são proposições simplesmen-
te 'luminosamente evidentes e não podem ser deduzidas de nada mais evidente'. Por exemplo,
simplesmente vemos que '1 + 1 = 2' é verdade. Entre os itens do conhecimento intuitivo en-
contram-se os relatos da experiência imediata; se afirmo simplesmente de que dados dos sen-
tidos estou agora consciente, não posso (à excepção de lapsos verbais triviais) estar engana-
do. O conhecimento derivado de verdades consiste em tudo o que possa ser inferido de verdades
auto-evidentes por intermédio de princípios de dedução auto-evidentes. Apesar da aparência de rigor que a nossa posse de conhecimento a priori introduz, diz Rus-
sell, temos de aceitar que o nosso conhecimento quotidiano geral é apenas tão bom quanto o
seu fundamento na justificação pela 'melhor explicação' e nos instintos que o tornam plausí-
vel. O conhecimento comum, portanto, equivale na melhor das hipóteses a 'opinião mais ou
menos provável'. Mas quando observamos que as nossas opiniões prováveis formam um sis-
tema que é coerente e se suporta mutuamente ― quanto mais coerente e estável for o
sistema, maior a probabilidade das opiniões que o formam ― vemos por que razão po-
demos confiar nelas. Uma característica importante da teoria de Russell diz respeito ao espaço e, em particular, à
distinção entre o espaço público que tudo contém, assumido pela ciência, e os espaços priva-
dos em que os dados dos sentidos dos observadores individuais existem. O espaço privado é
construído a partir de várias experiências visuais, tácteis e outras que um observador coorde-
na numa matriz com ele mesmo no centro. Mas porque não temos contacto com o espaço
público da ciência, a sua existência e natureza é inteiramente uma questão de inferência. Esta é a primeira versão de uma teoria do conhecimento e da percepção de Russell, tal como
é exposta nos PF. No primeiro encontro ficamos com uma refrescante sensação de senso
comum, mas está longe de não ser problemática. Por exemplo, Russell fala de conhecimento
'primitivo' e descreve-o como intuitivo, mas não diz em que consiste esse conhecimento, para
além de dizer que não exige o suporte de nada mais auto-evidente do que ele próprio. O valor da filosofia Mas, se não existisse qualquer mesa no mundo tería-
mos de formular uma hipótese complicada acerca de existirem tantas mesas-aparentes dife-
rentes quantos os observadores existentes e explicar como, apesar disso, falamos todos como
se estivéssemos a percepcionar o mesmo objecto. Mas repara que do ponto de vista céptico, como Russell indica, nem devemos sequer pensar
que existem outros observadores. Afinal de contas, se não podemos refutar o cepticismo
acerca dos objectos, como é que refutamos o cepticismo acerca da existência de outras men-
tes? Russell ultrapassa esta dificuldade aceitando uma versão daquilo a que se chama 'o argumen-
to da melhor explicação'. É certamente muito mais simples e mais poderoso, argumenta ele,
adoptar a hipótese de que, primeiro, há realmente objectos físicos que existem independen-
temente da nossa experiência sensorial e, segundo, que eles causam as nossas percepções e,
portanto, lhes 'correspondem' de uma forma fiável. Seguindo Hume, Russell vê a crença nesta 36 hipótese como 'instintiva'. hipótese como 'instintiva'. A este, argumenta ele, podemos juntar outro género de conhecimento, a saber, o conhecimen-
to a priori das verdades da lógica e das matemáticas puras (e até talvez das proposições fun-
damentais da ética). Tal conhecimento é totalmente independente da experiência e depende
completamente da auto-evidência das verdades conhecidas, como '1 + 1 = 2' e 'A = A'. Quan-
do o conhecimento perceptivo e o conhecimento a priori são unidos permitem-nos adquirir
conhecimento geral do mundo para além da nossa experiência imediata, porque o primeiro
género de conhecimento dá-nos os dados empíricos e o segundo género permite-nos extrair
deles inferências. Cada um destes dois géneros de conhecimento pode ser ainda dividido em subgéneros, des-
critos por Russell respectivamente como conhecimento imediato e conhecimento derivado. Ele chama 'contacto' ao conhecimento imediato de coisas. Os objectos do contacto são eles
próprios de duas espécies: particulares, isto é, dados dos sentidos individuais e ― talvez ―
nós próprios; e universais. Os universais são de vários tipos. Eles incluem qualidades sensí-
veis como vermelhidão e lisura, relações espaciais e temporais como 'à direita de' e 'antes' e
certas abstracções lógicas. Ao conhecimento derivado de coisas Russell chama 'conhecimento por descrição', que é co-
nhecimento geral de factos tornado possível pela combinação de e a inferência de aquilo com
que temos contacto. O nosso conhecimento de que o Everest é a montanha mais alta do mun-
do é um exemplo de conhecimento descritivo. O valor da filosofia Mas
esta definição é pouco adequada e ainda é mais obscurecida quando ele acrescenta que há
dois géneros de auto-evidência, dos quais apenas um é básico. Faz esta distinção sentido? Em
todo o caso o que significa 'auto-evidência'? Ele também não tem em conta a possibilidade de 37 duas proposições se poderem contradizer mutuamente apesar de parecerem auto-evidentes
quando consideradas separadamente. Se isto acontecesse, qual escolher e com base em que
princípios de auto-evidência adicionais? Outra crítica dirigida ao ponto de vista de Russell é a de que faz uma assunção importante
mas questionável acerca da natureza básica da experiência sensorial. É que os dados dos sen-
tidos, enquanto sensoriais mínimos como cores particulares, cheiros ou sons, são simples-
mente dados na experiência e são os seus elementos mais primitivos. Mas a experiência sen-
sorial não é de todo desta forma 'diminuta' e imediata. É antes uma experiência rica e com-
plexa de casas, árvores, pessoas, gatos e nuvens ― é fenomenologicamente 'cheia' e chega-se
apenas aos dados dos sentidos por um complicado processo de esvaziamento da experiência
perceptiva comum de tudo o que normalmente significa para nós. Assim, não vemos um re-
ctângulo e inferimos que é uma mesa; vemos uma mesa e, quando nos concentramos na sua
forma, vemos que é um rectângulo. Esta crítica é, até onde alcança, de certeza correcta, mas há formas de a ter em conta que nos
permitem ainda assim descrever os aspectos sensoriais da experiência independentemente da
carga usual de crenças e teorias que carrega. Uma vez que o objectivo é tentar justificar a
posse daquelas crenças, mostrando que a experiência perceptiva nos dá o direito a tê-las,
precisamos obviamente de uma descrição da experiência perceptiva considerada puramente
como tal, de modo a podermos avaliar a sua adequação para a tarefa. O objectivo de Russell
ao falar de dados dos sentidos é fazer precisamente isso. Além disso, Russell reconheceu que
os dados dos sentidos não são o que é imediatamente dado na percepção; em escritos da dé-
cada após os PF ele refere repetidamente que as especificações dos dados dos sentidos são
obtidas em último lugar na análise e não primeiro na experiência. Outra crítica é que Russell assume que a experiência imediata pode ser expressa em proposi-
ções que, apesar de descreverem apenas o que é subjectivamente 'dado', podem ser usadas
como base para o conhecimento do mundo. O valor da filosofia Mas como pode o que parece aplicar-se apenas à
experiência privada e não conter qualquer referência ao que está para além da experiência ser
a base para a teoria do conhecimento? Não serve de nada dizer que Russell também admite
conhecimento a priori dos princípios lógicos que permitem fazer inferências a partir destas
proposições, porque não haveria qualquer motivação para as fazer a menos que, além dele, o
sujeito possua algumas crenças empíricas gerais para servir como premissas maiores em tais
inferências e algumas hipóteses empíricas que as inferências efectivamente testam ou supor-
tam. Mas quer umas quer outras não estão disponíveis a um experimentador dotado apenas,
como Russell o apresenta, com dados dos sentidos e verdades auto-evidentes da lógica. Este problema era importante para o próprio Russell e muito mais tarde (em Human Knowle-
dge) tratou-o aceitando uma versão de algo que anteriormente tinha rejeitado na filosofia de
Kant, a saber, que têm de existir coisas (diferentes das verdades da lógica) que conhecemos a
priori para que o conhecimento seja de todo possível. Este ponto altamente importante é dis-
cutido no lugar apropriado mais a baixo. Outro problema avançado pelos críticos é que as considerações em que Russell se baseia para
mostrar que há uma distinção aparência-realidade, como ele as enuncia, não persuadem. O
facto de que um objecto parecer de uma cor ou forma para um observador e de outra cor ou
forma para outro observador, ou de diferentes cores ou formas para o mesmo observador sob
diferentes condições ― por exemplo, consoante o veja à luz do dia ou no escuro, ou
de um ponto de vista ou outro ― diz-nos que a questão de como os objectos aparecem à
percepção é uma questão complicada, mas por si só não nos diz que estamos a perceber algo
diferente do objecto em questão. Esta crítica é válida, mas acontece que, como mostram trabalhos mais recentes na filosofia da
percepção, há outras formas perfeitamente adequadas de traçar uma distinção aparência-
realidade; assim, os argumentos de Russell podem aqui ser vistos ― como ele próprio os
via ― como heurísticos, isto é, como ilustrando apenas o ponto a fim de começar a dis-
cussão. 38 Mas esta crítica sugere uma outra mais importante. É que Russell, como todos os seus prede-
cessores desde Descartes e como alguns dos seus sucessores como H. H. Price e A. J. O valor da filosofia Ayer,
aceitava uma suposição extremamente importante de Descartes. Esta suposição é que o ponto
de partida correcto para uma investigação sobre o conhecimento é a experiência individual. O
indivíduo começa com os dados privados da consciência e descobre razões entre eles para
apoiar as suas inferências para ― ou, de forma mais geral, crenças sobre ― um
mundo fora da sua cabeça. Uma das maiores mudanças na filosofia do século XX foi a
rejeição desta suposição cartesiana. Entre as sérias dificuldades desta suposição encontra-se
que se a aceitarmos o cepticismo torna-se impossível de ignorar ou de refutar. Outra é que
com uma base tão pequena não estamos simplesmente autorizados a pensar no solipsístico
pretenso conhecedor, sozinho dentro da sua cabeça, como sendo capaz de nomear e pensar
sobre as suas sensações e experiências e ainda menos como sendo capaz de raciocinar a partir
delas para um mundo exterior. Ambos os pensamentos nos empurram firmemente para o pen-
samento de que o lugar adequado para começar a epistemologia é, de certo modo, no domínio
público. Tradução de Álvaro Nunes Tradução de Álvaro Nunes [1] Da tradução portuguesa publicada pela editora Arménio Amado. [1] Da tradução portuguesa publicada pela editora Arménio Amado. A. C. Grayling, Russell, Oxford University Press, Oxford, 1996, pp. 39-44. 9. REFERENCIAL BIBLIOGRÁFICO SUPLICY, Eduardo Matarazzo. RENDA DE CIDADANIA. A saída pela porta. SP:
Cortez/ Fundação Perseu Abreu, 2002. 39 ANGELIM, Maria L.P. Educar é descobrir - um estudo observacional exploratório. Brasília: Universidade de Brasília (dissertação de mestrado), 1988. 2v. ANGELIM, Maria L.P. Educar é descobrir - um estudo observacional exploratório. Brasília: Universidade de Brasília (dissertação de mestrado), 1988. 2v. BARBIER, René. A pesquisa-ação. Tradução Lucie Didio. Brasília: E.Plano, 2002 FREIRE, Paulo. Pedagogia da autonomia - saberes necessários à prática educativa. São Paulo: Paz e Terra, 1997. FREIRE, Paulo. Pedagogia da autonomia - saberes necessários à prática educativa. São Paulo: Paz e Terra, 1997. _. Pedagogia do Oprimido. 20Ed. Rio de Janeiro: Paz e Terra,1987. ________. Pedagogia do Oprimido. 20Ed. Rio de Janeiro: Paz e Terra,1987. ARAÚJO, Raimundo Luiz Silva. Financiamento da Educação Básica no Governo
Lula: elementos de ruptura e de continuidade. 2007. 182f. Dissertação (Mestrado em
Políticas Públicas e Gestão da Educação), Programa de Pós-Graduação em Educação da
Universidade de Brasília. Brasília, 2007. Sítios: EJA- http//www.forumeja.org.br
http://scholar.google.com.br/ EJA- http//www.forumeja.org.br http://scholar.google.com.br/ http://www.mds.gov.br DAVIES, Nicholas. O Fundef e as verbas da educação. São Paulo: Xamã, 2001. ç
Ministério da Educação. Fundo de Manutenção e Desenvolvimento da Educação
Básica e de Valorização dos Profissionais da Educação – FUNDEB. Disponível em:
http://www.fnde.gov.br/home/index.jsp?arquivo=fundeb.html 40 Tradução de Álvaro Nunes
[1] Da tradução portuguesa publicada pela editora Arménio Amado.
A. C. Grayling, Russell, Oxford University Press, Oxford, 1996, pp. 39-
44 Ministério
da
Educação.
FUNDEB.
Disponível
em:
http://portal.mec.gov.br/index.php?option=com_content&view=article&id=12407&Itemid
=725 em: http://portal.mec.gov.br/index.php?option=com_content&view=article&id=12407&Itemid
=725 http://portal.mec.gov.br/index.php?option=com_content&view=article&id=12407&Itemid
=725 FERNANDES, Francisco das Chagas. Por que o Fundeb? Disponível em:
<http://mecsrv04.mec.gov.br/news/ArtigosDiaImp.asp?Id=513 FERNANDES, Francisco das Chagas. Por que o Fundeb? Disponível em:
<http://mecsrv04.mec.gov.br/news/ArtigosDiaImp.asp?Id=513 Anexo: Tradução de Álvaro Nunes
[1] Da tradução portuguesa publicada pela editora Arménio Amado. A. C. Grayling, Russell, Oxford University Press, Oxford, 1996, pp. 39-
44 41 41
|
https://openalex.org/W3113903879
|
https://projecteuclid.org/journals/electronic-communications-in-probability/volume-25/issue-none/On-the-boundary-behavior-of-multi-type-continuous-state-branching/10.1214/20-ECP364.pdf
|
English
| null |
On the boundary behavior of multi-type continuous-state branching processes with immigration
|
Electronic communications in probability
| 2,020
|
cc-by
| 9,506
|
*Peng
Jin
is
supported
by
the
Guangdong
Basic
and
Applied
Basic
Research
Foundation
(No.
2020A1515010436) and the STU Scientific Research Foundation for Talents (No. NTF18023).
†School of Mathematical Sciences, Dublin City University, Glasnevin Campus, Dublin 9, Ireland.
E-mail: martin.friesen@dcu.ie
‡Division of Science and Technology, BNU-HKBU United International College, Zhuhai, China.
E-mail: pengjin@uic.edu.cn
§Bergische Universität Wuppertal, Gaußstraße 20, 42119 Wuppertal, Germany.
E-mail: ruediger@uni-wuppertal.de Electron. Commun. Probab. 25 (2020), article no. 84, 1–14.
https://doi.org/10.1214/20-ECP364
ISSN: 1083-589X Electron. Commun. Probab. 25 (2020), article no. 84, 1–14. https://doi.org/10.1214/20-ECP364
ISSN: 1083-589X Electron. Commun. Probab. 25 (2020), article no. 84, 1–14. https://doi.org/10.1214/20-ECP364
ISSN: 1083-589X ELECTRONIC
COMMUNICATIONS
in PROBABILITY Abstract In this article, we provide a sufficient condition for a continuous-state branching
process with immigration (CBI process) to not hit its boundary, i.e. for non-extinction. Our result applies to arbitrary dimension d ≥1 and is formulated in terms of an
integrability condition for its immigration and branching mechanisms F and R. The
proof is based on a comparison principle for multi-type CBI processes being compared
to one-dimensional CBI processes, and then an application of an existing result for
one-dimensional CBI processes. The same technique is also used to provide a sufficient
condition for the transience of multi-type CBI processes. Keywords: multi-type continuous-state branching process with immigration; extinction; tran-
sience; comparison principle. p
p
p
MSC2020 subject classifications: 60G17; 60J25; 60J80. MSC2020 subject classifications: 60G17; 60J25; 60J80. Submitted to ECP on February 4, 2019, final version accep Submitted to ECP on February 4, 2019, final version accepted on November 18, 2020. On the boundary behavior of multi-type continuous-state
branching processes with immigration* Martin Friesen†
Peng Jin‡
Barbara Rüdiger§ Barbara Rüdiger§ 1
Introduction Continuous-state branching processes with immigration (abbreviated as CBI pro-
cesses) form a class of time-homogeneous Markov processes with state space Rd
+ = {x ∈Rd | x1, . . . , xd ≥0}, d ∈N, whose Laplace transform is an exponentially affine function of the initial state variable. In particular, CBI processes are affine processes in the sense of [7, Definition 2.6]. For
x = (x1, . . . , xd) ∈Rd
+ each component xi denotes the (continuous) number of individuals
of type i ∈{1, . . . , d}, while d is the number of types. Such processes have been first
studied in [10] for single-type models (d = 1) in the diffusion case without immigration
and in [23] for multi-types (d ≥1) without immigration including models with jumps, see
also [34] for a pioneering work in this direction. CBI processes arise as large population
limits of Galton-Watson branching processes. Indeed, the single-type case was studied in
[28, 20] without immigration and in [26, 29, 3] with immigration. Analogous results for whose Laplace transform is an exponentially affine function of the initial state variable. In particular, CBI processes are affine processes in the sense of [7, Definition 2.6]. For
x = (x1, . . . , xd) ∈Rd
+ each component xi denotes the (continuous) number of individuals
of type i ∈{1, . . . , d}, while d is the number of types. Such processes have been first
studied in [10] for single-type models (d = 1) in the diffusion case without immigration
and in [23] for multi-types (d ≥1) without immigration including models with jumps, see
also [34] for a pioneering work in this direction. CBI processes arise as large population
limits of Galton-Watson branching processes. Indeed, the single-type case was studied in
[28, 20] without immigration and in [26, 29, 3] with immigration. Analogous results for On the boundary behavior of multi-type CBI processes with immigration multi-type CBI processes have been then obtained in [24, 5]. An introduction, additional
references and results for single-type CBI processes are given in [27, 33] and [30,
Chapter 3], while a construction of multi-type CBI processes from a system of stochastic
equations was studied in [2]. 1
Introduction Let us mention that the CIR process, as well as several
multi-factor extensions of it, form a particular class of examples closely related with
applications in mathematical finance, see, e.g., [1] and [7] and the references therein. Here and below ⟨·, ·⟩denotes the euclidean scalar product on Rd and | · | the induced
norm. Below we describe the parameters of the multi-type CBI process. Definition 1.1. The tuple (c, β, B, ν, µ) is called admissible if (i) c = (c1, . . . , cd) ∈Rd
+. (ii) β = (β1, . . . , βd) ∈Rd
+. (
)
+
(ii) β = (β1, . . . , βd) ∈Rd
+. (ii) β = (β1, . . . , βd) ∈Rd
+. +
(iii) B = (bkj)k,j∈{1,...,d} is such that bkj ≥0 for k, j ∈{1, . . . , d} with k ̸= j. (iii) B = (bkj)k,j∈{1,...,d} is such that bkj ≥0 for k, j ∈{1, . . . , d} with k ̸= j. (iii) B = (bkj)k,j∈{1,...,d} is such that bkj ≥0 for k, j ∈{1, . . . , d} with k ̸= j. {
}
(iv) ν is a Borel measure on Rd
+ satisfying
R
Rd
+(1 ∧|z|)ν(dz) < ∞and ν({0}) = 0. (iv) ν is a Borel measure on Rd
+ satisfying
R
Rd
+(1 ∧|z|)ν(dz) < ∞and ν({0}) = 0. +
(vi) µ = (µ1, . . . , µd), where, for each j ∈{1, . . . , d}, µj is a Borel measure on Rd
+
satisfying +
(vi) µ = (µ1, . . . , µd), where, for each j ∈{1, . . . , d}, µj is a Borel measure on Rd
+
satisfying Z
Rd
+
zj ∧z2
j +
X
k∈{1,...,d}\{j}
zk
µj(dz) < ∞, µj({0}) = 0. (1.1) (1.1) Note that this definition is a special case of [7, Definition 2.6] in the sense that we
consider here the state space Rd
+, exclude killing of the process, and assume that
the measures µ1, . . . , µd satisfy the additional moment condition for the big jumps
R
|z|>1 |z|µj(dz) < ∞for j = 1, . . . , d. Let (c, β, B, ν, µ) be admissible parameters in
the sense of Definition 1.1. ECP 25 (2020), paper 84.
Page 2/14
https://www.imstat.org/ecp 1
Introduction For given admissible parameters (c, β, B, ν, µ) there exists a unique con-
servative Feller transition semigroup (Pt)t≥0 acting on the Banach space of continuous
functions vanishing at infinity with state space Rd
+ such that its generator has core
C∞
c (Rd
+) and is, for f ∈C2
c (Rd
+), given by (Lf)(x) =
d
X
j=1
cjxj
∂2f(x)
∂x2
j
+ ⟨β + Bx, (∇f)(x)⟩+
Z
Rd
+
(f(x + z) −f(x))ν(dz)
(1.4)
+
d
X
j=1
xj
Z
Rd
+
f(x + z) −f(x) −zj
∂f(x)
∂xj
µj(dz). (1.5) (1.4) (1.5) The corresponding transition semigroup coincides with the one given by (1.2) and hence
describes a multi-type CBI process with admissible parameters (c, β, B, ν, µ). The smoothness of transition probabilities for one-dimensional CBI processes was
recently studied in [6], where very precise results have been obtained. In [14] (see also
[11] for related results) we have studied existence of transition densities for multi-type
CBI processes. It was shown that, under appropriate conditions, such a density exists on
the interior of its state space, i.e. on Γ = {x ∈Rd
+ | x1, . . . , xd > 0}. In this work provide
conditions under which the corresponding multi-type CBI process is supported on Γ, i.e. P[X(t) ∈Γ,
t ≥0] = 1. Such property simply states that the population described by X
does not get extinct. As a consequence, it has, under the conditions of [14] and those
presented in this work, a density on the whole state space Rd
+. p
y
p
+
The boundary behavior for one-dimensional CBI processes was studied in [19], for
two-dimensional diffusion processes in [34], and more recently in [4, 8, 12] where
also recurrence and transience was studied. Let us also mention the work [35] where
recurrence and transience for general Lévy driven OU-processes was characterized. Note that the class of CBI processes satisfying c = 0 and µ = 0 form a particular class
of Lévy driven OU-processes whose state-space is Rd
+. In order to study the multi-
dimensional case, we establish first in Section 2 a general comparison principle for
multi-type CBI processes. Such comparison allows us to relate two CBI processes with
different admissible parameters with respect to the classical order on R+. 1
Introduction It was shown in [7, Theorem 2.7] (see also [2, Theorem 2.4])
that there exists a unique Markov transition kernel Pt(x, dy) with representation Z
Rd
+
e−⟨ξ,y⟩Pt(x, dy) = exp
−⟨x, v(t, ξ)⟩−
Z t
0
F(v(s, ξ))ds
, x, ξ ∈Rd
+, t ≥0,
(1.2) (1.2) where, for any ξ ∈Rd
+, the continuously differentiable function t 7−→v(t, ξ) ∈Rd
+ is the
unique locally bounded solution to the system of differential equations ∂v(t, ξ)
∂t
= −R(v(t, ξ)), v(0, ξ) = ξ. (1.3) (1.3) Here F and R are of Lévy-Khinchine form Here F and R are of Lévy-Khinchine form F(ξ) = ⟨β, ξ⟩+
Z
Rd
+
1 −e−⟨ξ,z⟩
ν(dz),
Rj(ξ) = cjξ2
j −⟨Bej, ξ⟩+
Z
Rd
+
e−⟨ξ,z⟩−1 + ξjzj
µj(dz),
j ∈{1, . . . , d}, and e1, . . . , ed denote the canonical basis vectors in Rd. The corresponding Markov
process with transition kernel Pt(x, dy) is called multi-type CBI process. Here, the
function F is the so-called immigration mechanism and describes the immigration of
individuals into the system from outside. Such immigration may be continuous described
by a drift parameter β, but also discontinuous described by a Lévy subordinator with
Lévy measure ν. The function R describes the branching mechanism, i.e., Rj is the
branching mechanism where each individual of type j may produce new individuals of
types k ∈{1, . . . , d} independently of all other individuals. Here c = (c1, . . . , cd) denotes ECP 25 (2020), paper 84. https://www.imstat.org/ecp Page 2/14 Page 2/14 On the boundary behavior of multi-type CBI processes with immigration the classical local branching associated with a diffusion process, Bej = (bjk)k∈{1,...,d}
describes the continuous branching rates in term of a drift, and finally the Lévy process
associated with the Lévy measure µj describes the branching of individuals produced by
an individual of type j. The following is a particular case of [7], see also [2, Theorem
2.4]. Remark 1.2. ECP 25 (2020), paper 84. ECP 25 (2020), paper 84.
Page 3/14
https://www.imstat.org/ecp 2
Comparison principle for multi-type CBI processes The possibility to describe a multi-type CBI process as a unique Rd
+-valued strong
solution to a stochastic differential equation was studied in [2]. The main technical tool
there was a comparison principle (see [2, Lemma 4.1]) for multi-type CBI processes
with respect to the initial condition and the drift parameter β from the immigration
mechanism. Below we provide an extension of this principle where the comparison can
also be made with respect to jump measures µ and ν, respectively, from the branching and
immigration mechanisms. Let (c, β, B, ν, µ) and (c, eβ, eB, eν, eµ) be admissible parameters
satisfying (A1) βk ≥eβk and bkk = ebkk for all k = 1, . . . , d, and bkj ≥ebkj for all k, j = 1, . . . , d with
k ̸= j, k ̸= j,
(A2) ν ≥eν, µk ≥eµk, and µk ◦pr−1
k
= eµk ◦pr−1
k
holds for all k = 1, . . . , d, where prk(z) = zk
denotes the projection onto the k-th coordinate. (A2) ν ≥eν, µk ≥eµk, and µk ◦pr−1
k
= eµk ◦pr−1
k
holds for all k = 1, . . . , d, where prk(z) = zk
denotes the projection onto the k-th coordinate. Condition (A1) imposes a comparison on the drift parameters while condition (A2)
imposes a comparison on the state-dependent and state-independent jump measures. Since we can only compare terms with finite variation, the drift coefficients B, eB and
jump measures µj, eµj, j = 1, . . . , d, are supposed to coincide on the diagonal. Condition (A1) imposes a comparison on the drift parameters while condition (A2)
imposes a comparison on the state-dependent and state-independent jump measures. Since we can only compare terms with finite variation, the drift coefficients B, eB and
jump measures µj, eµj, j = 1, . . . , d, are supposed to coincide on the diagonal. Below we construct multi-type CBI processes X and Y with admissible parameters
(c, β, B, ν, µ) and (c, eβ, eB, eν, eµ), respectively. These processes should be defined on the
same filtered probability space (Ω, F, (Ft)t≥0, P) with the usual conditions and are
obtained as the unique strong Rd
+-valued solutions to a system of stochastic differential
equations. Write µk = eµk + (µk −eµk), k = 1, . . . 1
Introduction Based on this
comparison result we show that each component of the multi-type CBI process dominates
a one-dimensional CBI process obtained from the original multi-type CBI process by
ignoring all possibilities that an individual of type k ∈{1, . . . , d} can create individuals of
different types j ̸= k. By assuming conditions sufficient for the smaller one-dimensional
CBI processes to not hit zero, or converge to infinity, we obtain a similar result also for
the original multi-type CBI process. At this point we implicitly use the results obtained
in [12] and [8] for one-dimensional CBI processes. At this point, we would like to mention that the property P[X(t) ∈Γ,
t ≥0] = 1
proved in this work can be combined with the Besov regularity of the law of X(t) (see
[14]) to show that the multi-type CBI process has the strong Feller property. Assuming
in addition mild additional conditions such that this CBI process has a unique invariant
measure (see [22]), this method can be combined with the coupling argument from
[16] to show that its transition probabilities converge in the total variation distance to
the unique invariant measure. Such an approach was first established in [13] for the
anisotropic stable JCIR process, see Example 3.8 for its definition. It is clear from the https://www.imstat.org/ecp https://www.imstat.org/ecp Page 3/14 On the boundary behavior of multi-type CBI processes with immigration proofs given in [13] that they can also be adapted to general multi-type CBI processes
satisfying the conditions imposed in this work and those given in [14]. Finally, let us
mention that convergence in total variation for extensions of CBI processes have been
studied by different techniques in [32, 18]. 2
Comparison principle for multi-type CBI processes , d, and note that µk −eµk ≥0 is a
Le´vy measure satisfying (1.1). Hence we may consider the following objects defined on
(Ω, F, (Ft)t≥0, P): • A d-dimensional (Ft)t≥0-Brownian motion W = (W(t))t≥0. • (Ft)t≥0-Poisson random measures Neµ1(ds, dz, dr), . . . , Neµd(ds, dz, dr) on R+ × Rd
+ ×
R+ with compensators b
Neµj(ds, dz, dr) = dseµj(dz)dr, j = 1, . . . , d, and (Ft)t≥0-
Poisson random measures Nµ1−eµ1(ds, dz, dr), . . . , Nµd−eµd(ds, dz, dr) on R+×Rd
+×R+
with compensators b
Nµj−eµj(ds, dz, dr) = ds (µj(dz) −eµj(dz)) dr, j = 1, . . . , d. • (Ft)t≥0-Poisson random measures Neµ1(ds, dz, dr), . . . , Neµd(ds, dz, dr) on R+ × Rd
+ ×
R+ with compensators b
Neµj(ds, dz, dr) = dseµj(dz)dr, j = 1, . . . , d, and (Ft)t≥0-
Poisson random measures Nµ1−eµ1(ds, dz, dr), . . . , Nµd−eµd(ds, dz, dr) on R+×Rd
+×R+
with compensators b
Nµj−eµj(ds, dz, dr) = ds (µj(dz) −eµj(dz)) dr, j = 1, . . . , d. • (Ft)t≥0-Poisson random measures Neν and Nν−eν on R+ × Rd
+ with compensators
b
Neν(ds, dz) = dseν(dz) and b
Nν−eν(ds, dz) = ds(ν(dz) −eν(dz)). • (Ft)t≥0-Poisson random measures Neν and Nν−eν on R+ × Rd
+ with compensators
b
Neν(ds, dz) = dseν(dz) and b
Nν−eν(ds, dz) = ds(ν(dz) −eν(dz)). The random objects W, Neν, Nν−eν, Neµ1, . . . , Neµd, Nµ1−eµ1, . . . , Nµd−eµd are supposed to be
independent. Denote by e
Nν = Nν −b
Nν, etc., the corresponding compensated Poisson
random measures. For given x, y ∈Rd
+ we consider the following system of stochastic
equations on the state space Rd
+: Xk(t) = xk +
Z t
0
βk +
d
X
j=1
bkjXj(s)
ds +
√
2ck
Z t
0
p
Xk(s)dWk(s)
(2.1)
+
Z t
0
Z
Rd
+
zkNeν(ds, dz) +
Z t
0
Z
Rd
+
zkNν−eν(ds, dz) (2.1) ECP 25 (2020), paper 84. Page 4/14
https://www.imstat.org/ecp ECP 25 (2020), paper 84. Page 4/14
https://www.imstat.org/ecp ECP 25 (2020), paper 84. 2
Comparison principle for multi-type CBI processes Page 4/14 On the boundary behavior of multi-type CBI processes with immigration +
Z t
0
Z
Rd
+
Z
R+
zk1{r≤Xk(s−)}
e
Neµk(ds, dz, dr) + e
Nµk−eµk(ds, dz, dr)
+
X
j̸=k
Z t
0
Z
Rd
+
Z
R+
zk1{r≤Xj(s−)}
Neµj(ds, dz, dr) + Nµj−eµj(ds, dz, dr)
and Yk(t) = yk +
Z t
0
eβk +
d
X
j=1
ebkjYj(s)
ds +
√
2ck
Z t
0
p
Yk(s)dWk(s)
(2.2)
+
Z t
0
Z
Rd
+
zkNeν(ds, dz) +
Z t
0
Z
Rd
+
Z
R+
zk1{r≤Yk(s−)} e
Neµk(ds, dz, dr)
+
X
j̸=k
Z t
0
Z
Rd
+
Z
R+
zk1{r≤Yj(s−)}Neµj(ds, dz, dr) (2.2) It is not difficult to verify that X and Y are multi-type CBI processes with the corre-
sponding admissible parameters. It is not difficult to verify that X and Y are multi-type CBI processes with the corre-
sponding admissible parameters. Proposition 2.1. Let (c, β, B, ν, µ) and (c, eβ, eB, eν, eµ) be admissible parameters satisfying
(A1) and (A2). Then there exist unique Rd
+-valued strong solutions to (2.1) and (2.2). Moreover, (2.1) determines a CBI process with admissible parameters (c, β, B, ν, µ),
and (2.2) determines a CBI process with admissible parameters (c, eβ, eB, eν, eµ). Proof. Using the Itô formula and a simple computation shows that any solution to (2.1)
is also a solution to the martingale problem (L, C∞
c (Rd
+), δx) and any solution to (2.2) is
a solution to the martingale problem (eL, C∞
c (Rd
+), δy), see [9, Chapter 4] for the general
theory of martingale problems. Here L denotes the generator of the conservative multi-
type CBI process with admissible parameters (c, β, B, ν, µ) and eL denotes the generator
with admissible parameters (c, eβ, eB, eν, eµ), respectively. Since C∞
c (Rd
+) is a core for the
generators, it follows that the martingale problems are well-posed, see [9, Chapter
4, Proposition 1.7, Theorem 2.7, Theorem 4.4]. Hence (2.1) and (2.2) determine the
corresponding CBI processes. Note that existence of uniqueness Rd
+-valued strong solutions to (2.1) and (2.2) was
essentially shown in [2, Theorem 4.6], provided that the immigration measures ν, eν
satisfy the additional moment condition Z
|z|>1
|z| (ν(dz) + eν(dz)) < ∞. 2
Comparison principle for multi-type CBI processes (2.3) (2.3) However, since ν({|z| > 1}), eν({|z| > 1}) < ∞, a standard interlacing argument similar
to [21, Proof of Propostion 9.1] or [17, Section 2] shows that existence and uniqueness
to (2.1) and (2.2) for general immigration measures ν, eν is equivalent for those satisfying
this additional moment condition. However, since ν({|z| > 1}), eν({|z| > 1}) < ∞, a standard interlacing argument similar
to [21, Proof of Propostion 9.1] or [17, Section 2] shows that existence and uniqueness
to (2.1) and (2.2) for general immigration measures ν, eν is equivalent for those satisfying
this additional moment condition. The following is our main result for this section. Theorem 2.2. Let (c, β, B, ν, µ) and (c, eβ, eB, eν, eµ) be admissible parameters satisfying
(A1) and (A2). Let X and Y be the unique strong solutions to (2.1) and (2.2), respectively. If xk ≥yk holds for all k = 1, . . . , d, then P[Xk(t) ≥Yk(t),
∀t ≥0,
k = 1, . . . , d] = 1
holds. ECP 25 (2020), paper 84. Page 5/14
https://www.imstat.org/ecp ECP 25 (2020), paper 84. Page 5/14 On the boundary behavior of multi-type CBI processes with immigration Proof. Define ∆k(t) := Yk(t) −Xk(t) and δk(r, s−) = 1{r≤Yk(s−)} −1{r≤Xk(s−)}. Then
∆k(0) = 0 and we obtain, for each k ∈{1, . . . , d}, Proof. Define ∆k(t) := Yk(t) −Xk(t) and δk(r, s−) = 1{r≤Yk(s−)} −1{r≤Xk(s−)}. Then
∆k(0) = 0 and we obtain, for each k ∈{1, . . . , d}, ∆k(t) = yk −xk +
Z t
0
eβk −βk +
d
X
j=1
ebkjYj(s) −bkjXj(s)
ds
+
√
2ck
Z t
0
p
Yk(s) −
p
Xk(s)
dWk(s) −
Z t
0
Z
Rd
+
zkdNν−eν
+
Z t
0
Z
Rd
+
Z
R+
zkδk(r, s−)d e
Neµk −
Z t
0
Z
Rd
+
Z
R+
zk1{r≤Xk(s−)}d e
Nµk−eµk
+
X
j̸=k
Z t
0
Z
Rd
+
Z
R+
zkδk(r, s−)dNeµj −
X
j̸=k
Z t
0
Z
Rd
+
Z
R+
zk1{r≤Xj(s−)}dNµj−eµj. Let φm : R −→R+ be twice continuously differentiable functions with the properties: Let φm : R −→R+ be twice continuously differentiable functions with the properties: (i) φm(z) ↗z+ := max{0, z}, as m →∞for all z ∈R. (ii) φ′
m(z) ∈[0, 1] for all m ∈N and z ≥0. 2
Comparison principle for multi-type CBI processes , R7
k,m are given by R1
k,m(s) = φ′
m(∆k(s))
eβk −βk +
d
X
j=1
ebkjYj(s) −bkjXj(s)
,
R2
k,m(s) = ckφ′′
m(∆k(s))
p
Yk(s) −
p
Xk(s)
2
,
R3
k,m(s) =
Z
Rd
+
[φm(∆k(s) −zk) −φm(∆k(s))] (ν(dz) −eν(dz)),
R4
k,m(s) =
Z
Rd
+
Z
R+
φm(∆k(s) + zkδk(r, s)) −φm(∆k(s)) −zkδk(
R5
k,m(s) =
Z
Rd
+
Z
R+
φm(∆k(s) −zk1{r≤Xk(s−)}) −φm(∆k(s)) R1
k,m(s) = φ′
m(∆k(s))
eβk −βk +
d
X
j=1
ebkjYj(s) −bkjXj(s)
,
R2
k,m(s) = ckφ′′
m(∆k(s))
p
Yk(s) −
p
Xk(s)
2
, R1
k,m(s) = φ′
m(∆k(s))
eβk −βk +
d
X
j=1
ebkjYj(s) −bkjXj(s)
,
R2
k,m(s) = ckφ′′
m(∆k(s))
p
Yk(s) −
p
Xk(s)
2
,
R3
k,m(s) =
Z
Rd
+
[φm(∆k(s) −zk) −φm(∆k(s))] (ν(dz) −eν(dz)),
R4
k,m(s) =
Z
Rd
+
Z
R+
φm(∆k(s) + zkδk(r, s)) −φm(∆k(s)) −zkδk(r, s)φ′
m(∆k(s))
dreµk(dz),
R5
k,m(s) =
Z
Rd
+
Z
R+
φm(∆k(s) −zk1{r≤Xk(s−)}) −φm(∆k(s)) R3
k,m(s) =
Z
Rd
+
[φm(∆k(s) −zk) −φm(∆k(s))] (ν(dz) −eν(dz)), +
R4
k,m(s) =
Z
Rd
+
Z
R+
φm(∆k(s) + zkδk(r, s)) −φm(∆k(s)) −zkδk(r, s)φ′
m(∆k(s))
dreµk(dz),
R5
k,m(s) =
Z
Rd
Z
R
φm(∆k(s) −zk1{r≤Xk(s−)}) −φm(∆k(s)) R4
k,m(s) =
Z
Rd
+
Z
R+
φm(∆k(s) + zkδk(r, s)) −φm(∆k(s)) −zkδk(r, s)φ′
m(∆k(s))
dreµk(dz), R5
k,m(s) =
Z
Rd
+
Z
R+
φm(∆k(s) −zk1{r≤Xk(s−)}) −φm(∆k(s)) Z
R+
φm(∆k(s) −zk1{r≤Xk(s−)}) −φm(∆k(s)) + zk1{r≤Xk(s−)}φ′
m(∆k(s))
dr(µk(dz) −eµk(dz)), { ≤
k(
)}φm(
( ))
(µ (
)
µ (
))
R6
k,m(s) =
X
j̸=k
Z
Rd
+
Z
R+
[φm(∆k(s) + zkδk(r, s)) −φm(∆k(s))] dreµj(dz),
R7
k,m(s) =
X
j̸=k
Z
Rd
+
Z
R+
φm(∆k(s) −zk1{r≤Xj(s)}) −φm(∆k(s))
dr(µj(dz) R6
k,m(s) =
X
j̸
k
Z
Rd
+
Z
R+
[φm(∆k(s) + zkδk(r, s)) −φm(∆k(s))] dreµj(dz), R6
k,m(s) =
X
j̸=k
Z
Rd
+
Z
R+
[φm(∆k(s) + zkδk(r, s)) −φm(∆k(s))] dreµj(dz),
R7
k,m(s) =
X
j̸=k
Z
Rd
+
Z
R+
φm(∆k(s) −zk1{r≤Xj(s)}) −φm(∆k(s))
dr(µj(dz) −eµj(dz)), j̸=k
+
R7
k,m(s) =
X
j̸=k
Z
Rd
+
Z
R+
φm(∆k(s) −zk1{r≤Xj(s)}) −φm(∆k(s))
dr(µj(dz) −eµj(dz)), (Mk,m(t))t≥0 is a local martingale and δk(r, s) = 1{r≤Yk(s)} −1{r≤Xk(s)}. 2
Comparison principle for multi-type CBI processes (iii) φ′
m(z) = φm(z) = 0 for all m ∈N and z ≤0. (vi) φ′′
m(x −y)|x −y| ≤2/m for all m ∈N and x, y ≥0. (vi) φ′′
m(x −y)|x −y| ≤2/m for all m ∈N and x, y ≥0. (vi) φ′′
m(x −y)|x −y| ≤2/m for all m ∈N and x, y ≥0. The existence of such a sequence was shown in the proof of [31, Theorem 3.1]. Applying
the Itô formula to φm(∆k(t)) gives The existence of such a sequence was shown in the proof of [31, Theorem 3.1]. Applying
the Itô formula to φm(∆k(t)) gives φm(∆k(t)) = φm(yk −xk) +
7
X
n=1
Z t
0
Rn
k,m(s)ds + Mk,m(t),
(2.4) (2.4) where R1
k,m, . . . , R7
k,m are given by where R1
k,m, . . . 2
Comparison principle for multi-type CBI processes For l ∈N, define
the stopping time τl = inf{t > 0 |
max
i∈{1,...,d} max{Xi(t), Yi(t)} > l}. Using the precise form (Mk,m(t))t≥0 is a local martingale and δk(r, s) = 1{r≤Yk(s)} −1{r≤Xk(s)}. For l ∈N, define
the stopping time τl = inf{t > 0 |
max
i∈{1,...,d} max{Xi(t), Yi(t)} > l}. Using the precise form ECP 25 (2020), paper 84. Page 6/14
https://www.imstat.org/ecp ECP 25 (2020), paper 84. Page 6/14 ECP 25 (2020), paper 84. Page 6/14 Page 6/14 On the boundary behavior of multi-type CBI processes with immigration of Mk,m given by Itô’s formula combined with similar estimates to [2, Lemma 4.1], one
can show that (Mk,m(t ∧τl))t≥0 is a martingale for any l ∈N. Next we will prove that
there exists a constant C > 0 such that 7
X
n=1
Rn
k,m(s) ≤C
d
X
j=1
∆j(s)+ + C
m. (2.5) (2.5) Taking then expectations in (2.4), using that (Mk,m(t ∧τl))t≥0 is a martingale and
estimating as in (2.5) combined with φm(yk −xk) = 0 by (iii), gives E[φm(∆k(t ∧τl))] ≤C
Z t
0
E
d
X
j=1
∆j(s ∧τl)+
ds + Ct
m . Letting m →∞, using property (i), and finally summing over k = 1, . . . , d gives E
d
X
j=1
∆j(t ∧τl)+
≤C
Z t
0
E
d
X
j=1
∆j(s ∧τl)+
ds. Applying Gronwall lemma shows that, for any l ∈N, one has E
hPd
j=1 ∆j(t ∧τl)+
i
= 0. Letting now l →∞and using τl →∞a.s. (since X, Y have cádlág paths) yields
Pd
j=1 ∆j(t)+ = 0 a.s. which proves the assertion. j
1
j
Hence it remains to prove (2.5). In order to estimate R1
k,m we use properties (ii), (iii),
(A1), eβk −βk ≤0, ebkj −bkj ≤0 and ebkj ≥0 for k ̸= j, and Xj(s) ≥0 to obtain R1
k,m(s) ≤φ′
m(∆k(s))
bkk∆k(s) +
X
j̸=k
ebkj∆j(s) +
X
j̸=k
(ebkj −bkj)Xj(s)
≤|bkk|∆k(s)+ +
sup
j̸=k
ebkj
! X
j̸=k
∆j(s)+. R1
k,m(s) ≤φ′
m(∆k(s))
bkk∆k(s) +
X
j̸=k
ebkj∆j(s) +
X
j̸=k
(ebkj −bkj)Xj(s)
≤|bkk|∆k(s)+ +
sup
j̸=k
ebkj
! X
j̸=k
∆j(s)+. For R2
k,m we obtain from (iv) the estimate R2
k,m(s) ≤2ck
m . 2
Comparison principle for multi-type CBI processes For the last term
we use again (ii) and similar arguments as above to find that R4,3
k,m(s) ≤0. For R5
k,m(s)
we use the fact that µk ◦pr−1
k
= eµk ◦pr−1
k
to conclude that R5
k,m(s) = 0. For R6
k,m(s) we
use property (ii) to find that R6
k,m(s) ≤1{∆k(s)>0}
X
j̸=k
Z
Rd
+
Z
R+
zkδk(r, s)dreµj(dz) ≤
X
j̸=k
Z
Rd
+
zkeµj(dz)∆k(s)+, where we have used
R
R+ δk(r, s)dr = ∆k(s) a.s. on {∆k(s) > 0}. To estimate the last term
R7
k,m(s) we use property (ii) so that φm(∆k(s) −zk1{r≤Xj(s)}) −φm(∆k(s)) ≤0 and hence
R7
k,m(s) ≤0 since µj(dz) ≥eµj(dz). Combining all estimates proves (2.5) and hence the
assertion. where we have used
R
R+ δk(r, s)dr = ∆k(s) a.s. on {∆k(s) > 0}. To estimate the last term
R7
k,m(s) we use property (ii) so that φm(∆k(s) −zk1{r≤Xj(s)}) −φm(∆k(s)) ≤0 and hence
R7
k,m(s) ≤0 since µj(dz) ≥eµj(dz). Combining all estimates proves (2.5) and hence the
assertion. The next theorem shows that the additional restriction µk ◦pr−1
k
= eµk ◦pr−1
k , k =
1, . . . , d, can be omitted if these jump measures have finite first moment also for the small
jumps. Theorem 2.3. Let (c, β, B, ν, µ) and (c, eβ, eB, eν, eµ) be admissible parameters satisfying
(A1), ν(dz) ≥eν(dz), and µk(dz) ≥eµk(dz) for each k = 1, . . . , d. Let X and Y be con-
structed as above. Suppose that Z
|z|≤1
|z|µk(dz) < ∞,
k = 1, . . . , d,
(2.6) (2.6) and that xk ≥yk for all k = 1, . . . , d. Then P[Xk(t) ≥Yk(t),
∀t ≥0] = 1 for each
k = 1, . . . , d. and that xk ≥yk for all k = 1, . . . , d. Then P[Xk(t) ≥Yk(t),
∀t ≥0] = 1 for each
k = 1, . . . , d. and that xk ≥yk for all k = 1, . . . , d. Then P[Xk(t) ≥Yk(t),
∀t ≥0] = 1 for each
k = 1, . . . , d. Proof. 2
Comparison principle for multi-type CBI processes Using (ii) we easily find that
φm(∆k(s) −zk) −φm(∆k(s)) ≤0, for zk ≥0, and hence R3
k,m ≤0 since ν(dz) ≥eν(dz). In
order to estimate R4
k,m(s) we first write R4
k,m(s) = R4,1
k,m(s) + R4,2
k,m(s) + R4,3
k,m(s) with R4,1
k,m(s) =
Z
|z|≤1
Z
R+
[φm(∆k(s) + zkδk(r, s)) −φm(∆k(s)) −zkδk(r, s)φ′
m(∆k(s))] dreµk(dz),
R4,2
k,m(s) =
Z
|z|>1
Z
R+
[φm(∆k(s) + zkδk(r, s)) −φm(∆k(s))] dreµk(dz),
R4,3
k,m(s) = −
Z
|z|>1
Z
R+
zkδk(r, s)φ′
m(∆k(s))dreµk(dz). R4,1
k,m(s) =
Z
|z|≤1
Z
R+
[φm(∆k(s) + zkδk(r, s)) −φm(∆k(s)) −zkδk(r, s)φ′
m(∆k(s))] dreµk(dz), R4,1
k,m(s) =
Z
|z|≤1
Z
R+
[φm(∆k(s) + zkδk(r, s)) −φm(∆k(s)) −zkδk(r, s)φ′
m(∆k(s))] dreµk(dz),
R4,2
k,m(s) =
Z
|z|>1
Z
R+
[φm(∆k(s) + zkδk(r, s)) −φm(∆k(s))] dreµk(dz), R4,2
k,m(s) =
Z
|z|>1
Z
R+
[φm(∆k(s) + zkδk(r, s)) −φm(∆k(s))] dreµk(dz), R4,3
k,m(s) = −
Z
|z|>1
Z
R+
zkδk(r, s)φ′
m(∆k(s))dreµk(dz). R4,3
k,m(s) = −
Z
|z|>1
Z
R+
zkδk(r, s)φ′
m(∆k(s))dreµk(dz). R4,3
k,m(s) = −
Z
|z|>1
Z
R+
zkδk(r, s)φ′
m(∆k(s))dreµk(dz). For the first term we use property (iv) so that, for each y > 0, z ≥0 and m ∈N, there
exists ϑ = ϑ(y, z) ∈[0, 1] such that For the first term we use property (iv) so that, for each y > 0, z ≥0 and m ∈N, there
exists ϑ = ϑ(y, z) ∈[0, 1] such that φm(y + z) −φm(y) −φ′
m(y)z = φ′′
m(y + ϑz)z2
2 ≤
2z2
2m(y + ϑz) ≤z2
my . Next observe that δk(r, s) > 0 if and only if ∆k(s) > 0 and r ∈(Xk(s), Yk(s)]. Applying
both observations to R4,1
k,m(s) gives Next observe that δk(r, s) > 0 if and only if ∆k(s) > 0 and r ∈(Xk(s), Yk(s)]. Applying
both observations to R4,1
k,m(s) gives R4,1
k,m(s) ≤1{∆k(s)>0}
m∆k(s)
Z
|z|≤1
Z
R+
z2
kδk(r, s)2dreµk(dz) ≤1
m
Z
|z|≤1
z2
keµk(dz), ECP 25 (2020), paper 84. Page 7/14
https://www.imstat.org/ecp ECP 25 (2020), paper 84. Page 7/14 Page 7/14 On the boundary behavior of multi-type CBI processes with immigration where we have used
R
R+ δk(r, s)2dr = ∆k(s) a.s. on {∆k(s) > 0}. For R4,2
k,m we use
property (ii), so that R4,2
k,m(s) ≤1{∆k(s)>0}∆k(s)
Z
|z|>1
Z
R+
zkδk(r, s)eµk(dz)dr ≤∆k(s)+
Z
|z|>1
zkeµk(dz), where we have also used
R
R+ δk(r, s)dr = ∆k(s) a.s. on {∆k(s) > 0}. 2
Comparison principle for multi-type CBI processes Due to condition (2.6) we can absorb the compensation in the stochastic integrals
against Neµk and Nµk−eµk which readily gives ∆k(t) = yk −xk +
Z t
0
eβk −βk +
d
X
j=1
(egkjYj(s) −gkjXj(s))
ds
+
√
2ck
Z t
0
p
Yk(s) −
p
Xk(s)
dWk(s) −
Z t
0
Z
Rd
+
zkdNν−eν
+
d
X
j=1
Z t
0
Z
Rd
+
Z
R+
zkδk(r, s−)dNeµj −
d
X
j=1
Z t
0
Z
Rd
+
Z
R+
zk1{r≤Xj(s−)}dNµj−eµj, where gkj = bkj, egkj = ebkj for k ̸= j, and gkk = bkk −
R
Rd
+ zkµk(dz) = egkk. The assertion
can now be literarly shown in the same way as in Theorem 2.2. where gkj = bkj, egkj = ebkj for k ̸= j, and gkk = bkk −
R
Rd
+ zkµk(dz) = egkk. The assertion
can now be literarly shown in the same way as in Theorem 2.2. ECP 25 (2020), paper 84. 3
Application to multi-type CBI processes Here and below we denote by X a multi-type CBI process with admissible parameters
(c, β, B, ν, µ). We start with the simple case where one component of the multi-type CBI
process has bounded variation. ECP 25 (2020), paper 84. https://www.imstat.org/ecp Page 8/14 On the boundary behavior of multi-type CBI processes with immigration On the boundary behavior of multi-type CBI processes with immigration On the boundary behavior of multi-type CBI processes with immigration Proposition 3.1. Suppose that there exists k ∈{1, . . . , d} such that ck = 0
and
Z
|z|≤1
zkµk(dz) < ∞. (3.1) (3.1) Then Xk has bounded variation and Then Xk has bounded variation and Xk(t) ≥
(
eθktxk + βk eθkt−1
θk
,
if θk ̸= 0
xk + βkt,
if θk = 0
,
t ≥0,
(3.2) (3.2) where θk = bkk −
R
Rd
+ zkµk(dz) ∈R. Proof. Let (c, eβ, eB, eν, eµ) be admissible parameters given by eβ = β, ebkj = 0 for k ̸= j
and ebkk = bkk, eν = 0, and eµ1 = · · · = eµd = 0. In view of condition (3.1) the process Xk
obtained from (2.1) also satisfies Xk(t) = xk +
Z t
0
βk +
d
X
j=1
gkjXj(s)
ds +
Z t
0
Z
Rd
+
zkNν(ds, dz)
d +
d
X
j=1
Z t
0
Z
Rd
+
Z
R+
zk1{r≤Xj(s−)}Nµj(ds, dz, dr), where gkj = bkj for k ̸= j and gkk = θk. Hence Xk has finite variation. The process Y
given by (2.2) satisfies Yk(t) = xk +
R t
0 (βk + θkY (s)) ds, i.e., where gkj = bkj for k ̸= j and gkk = θk. Hence Xk has finite variation. The process Y
given by (2.2) satisfies Yk(t) = xk +
R t
0 (βk + θkY (s)) ds, i.e., Yk(t) =
(
xkeθkt + βk eθkt−1
θk
,
if θk ̸= 0
xk + βkt,
if θk = 0
,
t ≥0. Using Theorem 2.3 yields P[Xk(t) ≥Yk(t)] = 1 for all t ≥0 and this fixed choice of k. This proves the assertion. Using Theorem 2.3 yields P[Xk(t) ≥Yk(t)] = 1 for all t ≥0 and this fixed choice of k. This proves the assertion. From this we easily obtain the following corollary. Corollary 3.2. Let k ∈{1, . . . 3
Application to multi-type CBI processes Theorem 2.3 yields P[Xk(t) ≥Yk(t)] = 1 for all t ≥0 and k ∈{1, . . . , d}. This proves the
assertion. for each k = 1, . . . , d. Let Y (t) be the unique solution to (2.2) with parameters given
as in (3.5), i.e., Y (t) = x +
R t
0 (β + GY (s)) ds, which is given by Y (t) = etGx +
R t
0 esGβds. Theorem 2.3 yields P[Xk(t) ≥Yk(t)] = 1 for all t ≥0 and k ∈{1, . . . , d}. This proves the
assertion. In view of this estimate we restrict our further analysis to the case where (3.1)
does not hold, i.e., the process has unbounded variation. In this case we define, for
k ∈{1, . . . , d}, the projected immigration and branching mechanisms by F (k)(ξ) = βkξ +
Z
Rd
+
1 −e−ξzk
ν(dz),
(3.6)
R(k)(ξ) = −bkkξ + ckξ2 +
Z
Rd
+
e−ξzk −1 + ξzk
µk(dz). (3.7) (3.6) (3.7) Each of these immigration and branching mechanisms describes a one-dimensional CBI
process which is obtained from a multi-type CBI process with admissible parameters
(c, β, B, ν, µ) by ignoring all possibilities that a particle of type k may create another
particle of type j ̸= k. Theorem 3.4. Suppose that there exists k ∈{1, . . . , d} and κ > 0 such that R(k)(ξ) > 0
for ξ ≥κ. If ck > 0 or
R
|z|≤1 zkµk(dz) = ∞, and it holds that Theorem 3.4. Suppose that there exists k ∈{1, . . . , d} and κ > 0 such that R(k)(ξ) > 0
for ξ ≥κ. If ck > 0 or
R
|z|≤1 zkµk(dz) = ∞, and it holds that Z ∞
κ
exp
Z ξ
κ
F (k)(u)
R(k)(u)du
! 1
R(k)(ξ)dξ = ∞,
(3.8) (3.8) then P[Xk(t) > 0, t ≥0] = 1, provided xk > 0. then P[Xk(t) > 0, t ≥0] = 1, provided xk > 0. then P[Xk(t) > 0, t ≥0] = 1, provided xk > 0. Proof. Let (c, eβ, eB, eν, eµ) be admissible parameters with eβ = β, eB = diag(b11, . . . , bdd),
eν = ν, eµk = µk, and eµj = 0 for j ̸= k. 3
Application to multi-type CBI processes , d} and suppose that (3.1) holds. If either xk > 0 or βk > 0,
then P[Xk(t) > 0, t ≥0] = 1. From this we easily obtain the following corollary. Corollary 3.2. Let k ∈{1, . . . , d} and suppose that (3.1) holds. If either xk > 0 or βk > 0,
then P[Xk(t) > 0, t ≥0] = 1. orollary 3.2. Let k ∈{1, . . . , d} and suppose that (3.1) holds. If either xk > 0 or βk > 0,
en P[Xk(t) > 0, t ≥0] = 1. The next proposition gives a multi-dimensional analogue of this result. For x, y ∈Rd
we will write x ≤y to mean that xi ≤yi for all i = 1, . . . , d. Proposition 3.3. Suppose that (3.1) holds for all k ∈{1, . . . , d}. Then X has bounded
variation and it holds that X(t) ≥etGx +
Z t
0
esGβds,
(3.3) (3.3) where G = (gkj)k,j∈{1,...,d} is given by where G = (gkj)k,j∈{1,...,d} is given by gkj =
(
bkj,
k ̸= j
bkk −
R
Rd
+ zkµk(dz),
k = j. (3.4) (3.4) Proof. Let (c, eβ, eB, eν, eµ) be admissible parameters given by Proof. Let (c, eβ, eB, eν, eµ) be admissible parameters given by eβ = β, eB = B, eν = 0, and eµ1 = · · · = eµd = 0. (3.5) (3.5) ECP 25 (2020), paper 84. Page 9/14
https://www.imstat.org/ecp ECP 25 (2020), paper 84. Page 9/14 ECP 25 (2020), paper 84. Page 9/14 On the boundary behavior of multi-type CBI processes with immigration On the boundary behavior of multi-type CBI processes with immigration On the boundary behavior of multi-type CBI processes with immigration Observe that under (3.1) the process X obtained from (2.1) also satisfies Xk(t) = xk +
Z t
0
βk +
d
X
j=1
gkjXj(s)
ds +
Z t
0
Z
Rd
+
zkNν(ds, dz) +
d
X
j=1
Z t
0
Z
Rd
+
Z
R+
zk1{r≤Xj(s−)}Nµj(ds, dz, dr) for each k = 1, . . . , d. Let Y (t) be the unique solution to (2.2) with parameters given
as in (3.5), i.e., Y (t) = x +
R t
0 (β + GY (s)) ds, which is given by Y (t) = etGx +
R t
0 esGβds. Then (3.8) is satisfied, provided one of the following conditions holds: Then (3.8) is satisfied, provided one of the following conditions holds: (a) αk ∈(0, 1 + γk). (b) αk = 1 + γk and γk ≤C1
C2 . (a) αk ∈(0, 1 + γk). (b) αk = 1 + γk and γk ≤C1
C2 . (b) αk = 1 + γk and γk ≤C1
C2 . Note that, if βk > 0, then F (k)(ξ) ≥βkξ and hence γk = 1. However, Corollary 3.5
also applies in the particular case where β1 = · · · = βd = 0. Proof of Remark 3.6. Set κ = max{M0, M1, M2}. If αk < 1 + γk, then F (k)(u)
R(k)(u) ≥C1
C2 uγk−αk,
for u ∈[κ, ξ], and hence exp
Z ξ
κ
F (k)(u)
R(k)(u)du
! ≥exp
C1
C2
Z ξ
κ
uγk−αkdu
! = exp
−C1
C2
κ1+γk−αk
1 + γk −αk
exp
C1
C2
ξ1+γk−αk
1 + γk −αk
and Z ∞
κ
exp
Z ξ
κ
F (k)(u)
R(k)(u)du
! dξ
R(k)(ξ)
≥
exp
−C1
C2
κ1+γk−αk
1+γk−αk
C2
Z ∞
κ
exp
C1
C2
ξ1+γk−αk
1 + γk −αk
dξ
ξαk = ∞. This proves (3.8) under (a). If αk = 1 + γk, then we obtain for ξ ≥κ and u ∈[κ, ξ], exp
Z ξ
κ
F (k)(u)
R(k)(u)du
! ≥exp
C1
C2
Z ξ
κ
uγk−αkdu
! = κ−C1
C2 ξ
C1
C2 . Using αk ≤1 + C1
C2 gives Z ∞
κ
exp
Z ξ
κ
F (k)(u)
R(k)(u)du
! dξ
R(k)(ξ) ≥κ−C1
C2
C2
Z ξ
κ
ξ
C1
C2
ξαk dξ = ∞, and hence proves (3.8) under (b). and hence proves (3.8) under (b). and hence proves (3.8) under (b). Our next statement provides a sufficient condition for one component of a multi-type
CBI process to converge to infinity. Theorem 3.7. Let k ∈{1, . . . , d} and suppose that R(k)(ξ) > 0 holds for all ξ > 0. Then
P[limt→∞Xk(t) = ∞] = 1, provided one of the following conditions is satisfied: (a) bkk > 0. (b) b
≤0
d (a) bkk > 0. (b) bkk ≤0 and (b) bkk ≤0 and (b) bkk ≤0 and Z 1
0
exp
−
Z 1
ξ
F (k)(u)
R(k)(u)du
dξ
R(k)(ξ) < ∞. (3.10) (3.10) Proof. 3
Application to multi-type CBI processes https://www.imstat.org/ecp Page 10/14 Page 10/14 On the boundary behavior of multi-type CBI processes with immigration On the boundary behavior of multi-type CBI processes with immigration On the boundary behavior of multi-type CBI processes with immigration 3
Application to multi-type CBI processes Applying Theorem 2.2 gives P[Yk(t) > 0,
t ≥0] = 1 ⇒P[Xk(t) > 0,
t ≥0] = 1,
(3.9) (3.9) where X and Y are the unique solutions to (2.1) and (2.2), respectively. It is easy to
see that Yk is a CBI process with immigration and branching mechanisms given by (3.6)
and (3.7), respectively. In view of (3.8) Yk satisfies the conditions of [12, Corollary 6]
which proves the assertion. where X and Y are the unique solutions to (2.1) and (2.2), respectively. It is easy to
see that Yk is a CBI process with immigration and branching mechanisms given by (3.6)
and (3.7), respectively. In view of (3.8) Yk satisfies the conditions of [12, Corollary 6]
which proves the assertion. From this we directly deduce the following corollary. Corollary 3.5. If for each k ∈{1, . . . , d} the conditions of Theorem 3.4 are satisfied, then
P[X(t) ∈Γ,
t ≥0] = 1, provided x ∈Γ = {x ∈Rd
+ | x1, . . . , xd > 0}. Corollary 3.5. If for each k ∈{1, . . . , d} the conditions of Theorem 3.4 are satisfied, then
P[X(t) ∈Γ,
t ≥0] = 1, provided x ∈Γ = {x ∈Rd
+ | x1, . . . , xd > 0}. The following remark provides a sufficient condition for (3.8). Lemma 3.6. Suppose that for some k ∈{1, . . . , d} the following conditions are satisfied: (i) There exists M0 > 0 such that R(k)(ξ) > 0 for ξ ≥M0. (i) There exists M0 > 0 such that R(k)(ξ) > 0 for ξ ≥M0. (i) There exists M0 > 0 such that R(k)(ξ) > 0 for ξ ≥M0. (ii) There exists γk ∈(0, 1] and M1, C1 > 0 such that F (k)(ξ) ≥C1ξγk for ξ ≥M1. (
]
(k)( ) (ii) There exists γk ∈(0, 1] and M1, C1 > 0 such that F (k)(ξ) ≥C1ξγk for ξ ≥M1. (iii) There exists αk ∈(1, 2] and M2, C2 > 0 such that R(k)(ξ) ≤C2ξαk for ξ ≥M2. (iii) There exists αk ∈(1, 2] and M2, C2 > 0 such that R(k)(ξ) ≤C2ξαk for ξ ≥M2. ECP 25 (2020), paper 84. Then (3.8) is satisfied, provided one of the following conditions holds: Let (c, eβ, eB, eν, eµ) and Yk be the same as in the proof of Theorem 3.4. Applying
Theorem 2.2 gives (
Theorem 2.2 gives P[ lim
t→∞Yk(t) = ∞] = 1 =⇒P[ lim
t→∞Xk(t) = ∞] = 1. In view of Proposition 2.1, Yk satisfies the conditions of [8, Theorem 3] which proves the
assertion. ECP 25 (2020), paper 84. Page 11/14
https://www.imstat.org/ecp ECP 25 (2020), paper 84. ECP 25 (2020), paper 84. On the boundary behavior of multi-type CBI processes with immigration Let us close this section with the example of an anisotropic stable JCIR process,
i.e., the multi-type CBI process X with admissible parameters (c = 0, β, B, ν, µ), where
µ = (µ1, . . . , µd) are, for α1, . . . , αd ∈(1, 2), given by µj(dz) = 1R+(zj) dzj
z1+αj
j
⊗
Y
k̸=j
δ0(dzk). (3.11) (3.11) Example 3.8. Let X be the anisotropic stable JCIR process starting from x ∈Rd
+. Fix
k ∈{1, . . . , d}. (a) Suppose that there exist C, M > 0 and γk ∈(0, 1] such that (a) Suppose that there exist C, M > 0 and γk ∈(0, 1] such that βkξ +
Z
Rd
+
1 −e−ξzk
ν(dz) ≥Cξγk,
ξ ≥M. (3.12) (3.12) If xk > 0 and αk ∈(1, 1 + γk), then P[Xk(t) > 0,
t ≥0] = 1. (b) If bkk > 0, then P[limt→∞Xk(t) = ∞] = 1. If xk > 0 and αk ∈(1, 1 + γk), then P[Xk(t) > 0,
t ≥0] = 1. (b) If bkk > 0, then P[limt→∞Xk(t) = ∞] = 1. Proof. Assertion (b) follows immediately from Theorem 3.7 (a). Let us prove assertion
(a). Since α1, . . . , αd ∈(1, 2), it follows that X has unbounded variation. Hence it suffices
to show that Theorem 3.4 is applicable. First observe that F (k)(ξ) = βkξ +
Z
Rd
+
1 −e−ξzk
ν(dz),
R(k)(ξ) = −bkkξ +
Z ∞
0
e−ξz −1 + ξz
dz
z1+αk = −bkkξ + Kξαk, F (k)(ξ) = βkξ +
Z
Rd
+
1 −e−ξzk
ν(dz),
R(k)(ξ) = −bkkξ +
Z ∞
0
e−ξz −1 + ξz
dz
z1+αk = −bkkξ + Kξαk,
where K =
R ∞
0
(e−w −1 + w)
dw
w1+αk > 0. Then (3.8) is satisfied, provided one of the following conditions holds: Next it is easily seen that R(k)(ξ) > 0,
whenever ξ >
max{0, bkk}
K
1
αk−1
. Moreover, one finds R(k)(ξ) ≤(|bkk| + K) ξαk for ξ ≥1, and hence the assertion follows
from Remark 3.6 since αk ∈(1, 1 + γk). Moreover, one finds R(k)(ξ) ≤(|bkk| + K) ξαk for ξ ≥1, and hence the assertion follows
from Remark 3.6 since αk ∈(1, 1 + γk). In Remark 3.6, if βk > 0, then we may take γk = 1 so that (3.12) is satisfied. However,
if βk = 0, then (3.12) may be still satisfied as it is shown in the following example. Example 3.9. Let γ ∈(0, 1) and set ν(dz) = 1Rd
+(z)
dz
|z|d+γ . Then
R
Rd
+(1 ∧|z|)ν(dz) < ∞ In Remark 3.6, if βk > 0, then we may take γk = 1 so that (3.12) is satisfied. However,
if βk = 0, then (3.12) may be still satisfied as it is shown in the following example. if βk = 0, then (3.12) may be still satisfied as it is shown in the following example. Example 3.9. Let γ ∈(0, 1) and set ν(dz) = 1Rd
+(z)
dz
|z|d+γ . Then
R
Rd
+(1 ∧|z|)ν(dz) < ∞
and Example 3.9. Let γ ∈(0, 1) and set ν(dz) = 1Rd
+(z)
dz
|z|d+γ . Then
R
Rd
+(1 ∧|z|)ν(dz) < ∞
and Z
Rd
+
1 −e−ξzk
dz
|z|d+γ = ξγ
Z
Rd
+
1 −e−wk
dw
|w|d+γ . So (3.12) holds for γk = So (3.12) holds for γk = γ. Hence the assumptions of Example 3.8 (a) are satisfied, if
αk ∈(1, 1 + γ). αk ∈(1, 1 + γ). It is worthwhile to mention that there exists a large class of measures which sat-
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useful comments and remarks on this work, including the suggestion to study a more
general comparison principle, as well as for pointing out several references on this topic. Acknowledgments. The authors would like to thank the anonymous referees for many
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English
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Results of open thoracoabdominal aortic replacement in patients unsuitable for or after endovascular repair with remaining disease components
|
Interactive cardiovascular and thoracic surgery
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cc-by
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ORIGINAL ARTICLE ORIGINAL ARTICLE Interactive CardioVascular and Thoracic Surgery 2022, 35(3), ivac076
https://doi.org/10.1093/icvts/ivac076
Advance Access publication 19 April 2022 Cite this article as: Kondov S, Frankenberger L, Siepe M, Keyl C, Staier K, Humburger F et al. Results of open thoracoabdominal aortic replacement in patients unsuit-
able for or after endovascular repair with remaining disease components. Interact CardioVasc Thorac Surg 2022; doi:10.1093/icvts/ivac076. VASCULAR Stoyan Kondov
a,b,*, Leon Frankenberger
b, Matthias Siepea,b, Cornelius Keylc, Klaus Staierc,
Frank Humburgera,b, Bartosz Rylskia,b, Maximilian Kreibich
a,b, Tim Bergera,b, Friedhelm Beyersdorf
a,b and
Martin Czernya,b Stoyan Kondov
a,b,*, Leon Frankenberger
b, Matthias Siepea,b, Cornelius Keylc, Klaus Staierc,
Frank Humburgera,b, Bartosz Rylskia,b, Maximilian Kreibich
a,b, Tim Bergera,b, Friedhelm Beyersdorf
a,b and
Martin Czernya,b Downloaded from https://academic.oup.com/icvts/article/35/3/ivac076/6570176 by gue a Department of Cardiovascular Surgery, Faculty of Medicine, University Heart Centre, University Hospital Freiburg, Albert-Ludw
Germany
b y
b Faculty of Medicine, Albert Ludwigs University Freiburg, Freiburg, Germany c Department of Anesthesiology & Critical Care Medicine, Medical Center - Faculty of Medicine, University of Freiburg, Freibur https://academic.oup.com/icvts/article/35/3/ivac076/6570176 by guest on 24 October 2024 * Address for correspondence: Department of Cardiovascular Surgery, University Heart Center Freiburg, Albert Ludwigs University Freiburg, Faculty of Medicine,
Hugstetterstrasse 55, D-79106 Freiburg. Tel: ++ 49 761 270 28670; fax ++ 49 761 270 25500, e-mail: stoyan.kondov@uniklinik-freiburg.de (S. Kondov). Received 3 June 2021; received in revised form 2 October 2021; accepted 15 March 2022 Abstract
OBJECTIVES: Our goal was to evaluate outcomes in all-comer patients undergoing open thoracoabdominal aortic replacement either
unsuitable for or after failed endovascular aortic repair. METHODS: Within a 4-year period, we analysed a consecutive series of 80 patients undergoing elective, urgent and emergency
thoracoabdominal aortic replacement. Preoperative data, intraoperative data and outcomes were evaluated. Specific attention was given
to technical refinements needed in patients after previous endovascular aortic repair. ( )
y
y
p
g
y
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which per-
mits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract OBJECTIVES: Our goal was to evaluate outcomes in all-comer patients undergoing open thoracoabdominal aortic replacement either
unsuitable for or after failed endovascular aortic repair. OBJECTIVES: Our goal was to evaluate outcomes in all-comer patients undergoing open thoracoabdominal aortic replacement either
unsuitable for or after failed endovascular aortic repair. METHODS: Within a 4-year period, we analysed a consecutive series of 80 patients undergoing elective, urgent and emergency
thoracoabdominal aortic replacement. Preoperative data, intraoperative data and outcomes were evaluated. Specific attention was given
to technical refinements needed in patients after previous endovascular aortic repair. V
C The Author(s) 2022. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surg
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativeco
mits unrestricted reuse distribution and reproduction in any medium provided the original work is properly cited V
C The Author(s) 2022. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. Thi i
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C The Author(s) 2022. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which per-
mits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecomm
mits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. S. Kondov et al. / Interactive CardioVascular and Thoracic Surgery 2 RESULTS: Eighty patients underwent thoracoabdominal aortic replacement: 11.3% (n = 9) had connective tissue disorders. Twenty-six
patients (32.5%) had previous endovascular aortic repair and 54 (67.5%) did not have previous endovascular repair. The mean age was
64.2 ± 12 years, and 70% (n = 56) were male. The mean EuroSCORE was 7.9 ± 2.6. Urgent or emergency operations were done in 22.5%
(n = 18). Overall mortality was 20% (n = 16); symptomatic spinal cord injury occurred in 5% (n = 4). We did not observe differences in sur-
vival according to the presence or absence of previous endovascular aortic repair (P = 0.524). Surgical technique Patients are placed in a right lateral supine position, and the op-
eration is started with open surgical exposure of the femoral ves-
sels. An 8 -mm Dacron graft is sewn to the common femoral
artery to maintain distal perfusion during cardiopulmonary by-
pass (CPB). Afterwards, a thoraco-phrenico-lumbotomy is per-
formed, and the viscera including the left kidney are placed to
the right. Afterwards, depending on the extent of the repair, the
distal aortic arch and the descending aorta are circumferentially
dissected
and
encircled
with
silastic
tapes. Then,
CPB
is The concept of distal shifting The number of patients with thoracoabdominal aortic patholo-
gies warranting treatment is increasing due to the increasing
number of patients with remaining dissection after previous type
A repair [1–3]. Whereas open surgery has been the only treat-
ment option for many years, endovascular aortic repair has
emerged as an excellent alternative [4–6]. Although in-hospital
mortality is lower, the need for aortic-related interventions is
higher, and, finally, conversion to open surgery is needed in
some cases [7]. The numbers of these types of cases are expected
to rise in the years to come due to the substantial increase in the
number of endovascular procedures being performed [8–10]. As
the volume of endovascular procedures volume rises, the age
and comorbidities of the patients increase as well. Open TAAA
(thoracoabdominal aortic aneurysm) repair remains a major sur-
gical procedure. In the modern endovascular era, it remains an
option for young patients, patients with underlying connective
tissue disorders and those who are not suitable for endovascular
solutions [11]. Furthermore, the expertise in the different centres
plays a major role in deciding which treatment strategy to
choose and finally in the clinical outcome [4, 12]. Experience has taught that extensive manipulation of the left lung
is a limiting factor during open thoracoabdominal replacement,
and the more distally the repair can be begun, the better the op-
eration is tolerated. Hence, in type I and type II TAAAs, aortic
arch replacement using the frozen elephant trunk (FET) technique
followed by thoracic endovascular aortic repair (TEVAR) or, in
the case of a sufficient proximal landing zone, TEVAR alone as an
initial step followed by metachronous open type III/type IV re-
pair, is the treatment of choice. Anaesthesiological set-up and neuroprotection Invasive blood pressure management is done at the right radial
and at the right femoral arteries. Patients receive double-lumen
intubation. A CSF drain is routinely inserted the day before sur-
gery by an anaesthesiologist. Motor-evoked potentials (MEP) and
somatosensory-evoked potentials (SSEP) are routinely monitored
during the procedure. Near infrared spectroscopy is also applied
on a routine basis. The goal of this study was to evaluate outcomes in patients un-
dergoing open thoracoabdominal aortic replacement who were
either unsuitable for or who had a failed endovascular aortic
repair. Patients Within
a
4-year
period
(January
2015–
December
2019),
we analysed a consecutive series of 80 all comers undergoing
elective, urgent and emergency thoracoabdominal aortic re-
placement. No patients were refused for anatomical reasons or
for reasons regarding previous endovascular repair. Preoperative
data, intraoperative data, outcome and survival and freedom
from aortic-related interventions were evaluated. Specific atten-
tion was given to technical refinements needed in patients after
previous endovascular aortic repair. EuroSCORE levels were
determined for all patients. Abstract Multivariate regression analysis revealed the
amount of packed red blood cell units (P = 0.009, confidence interval 1.028–1.215, odds ratio = 1.117) as a predictor of in-hospital death. Follow-up was 100% (37.9 ± 15.8 months); freedom from aortic-related reintervention was 96.3%. CONCLUSIONS: Despite an early attrition rate, survival after open thoracoabdominal aortic replacement is excellent, and freedom from
aortic-related reintervention is high. Open surgery continues to remain an essential component in the treatment armamentarium of acute
and chronic thoracoabdominal aortic pathology. Keywords: thoracoabdominal replacement • open surgery • endovascular repair ABBREVIATIONS
CPB
cardiopulmonary bypass
FET
frozen elephant trunk
MEP
motor-evoked potentials
PRBC
packed red blood cells units
RRA
right renal artery
SMA
superior mesenteric artery
SSEP
somatosensory-evoked potentials
TAAA
thoracoabdominal aortic aneurysm
TEVAR
thoracic endovascular aortic repair ABBREVIATIONS
CPB
cardiopulmonary bypass
FET
frozen elephant trunk
MEP
motor-evoked potentials
PRBC
packed red blood cells units
RRA
right renal artery
SMA
superior mesenteric artery
SSEP
somatosensory-evoked potentials
TAAA
thoracoabdominal aortic aneurysm
TEVAR
thoracic endovascular aortic repair Patient demographics Patient demographics are shown in Table 1. The mean patient
age
was
64.2 ± 12 years;
56
(70%)
were
men. The
mean
EuroSCORE was 7.9 ± 2.6 and was higher in the group with previ-
ous endovascular aortic repair (P = 0.021). The median body mass
index was 25.6 (22.4 29.4). The diagnosis of Marfan syndrome
was established in 9 patients (11.3%). Thirty-eight patients
(47.5%) had an underlying diagnosis of aortic dissection. Of these,
22 (27.5%) had an aneurysm on the basis of a residual dissection
after type A repair and 16 (20%) had an aneurysm due to a
chronic type B aortic dissection. TAAA on the basis of a chronic
type B aortic dissection occurred more often in the group with
previous endovascular aortic repair (P = 0.095). The median aortic
diameter was 70.5 (61.5; 88.3) cm in the group with previous
endovascular aortic repair and 61 (57.0; 69.3) cm in the group
with no previous endovascular aortic repair, respectively; the sig-
nificant difference was P = 0.003. Two patients (2.5%) were on di-
alysis before the operation, as were all the patients in the group
with previous endovascular aortic repair (P = 0.039). Surgical details in patients after thoracic
endovascular aortic repair During planning, if it is determined that there is a type IA or type
III endoleak, large type II endoleaks are also considered. If there
are no type IA or type III endoleaks, the strategy is to leave the
stent graft in place, to clamp it (nitinol resumes its original shape
after declamping) and to have an end-to-end anastomosis be-
tween a Dacron prosthesis and the stent graft. In large stent
grafts, the collar of an inversed Siena prosthesis is used to correct
the diameter. The side branches meant for the supraaortic vessels
as well as the perfusion branch are used for the visceral and renal
segments [13]. Nineteen (23.8%) patients were operated on under urgent or
emergency conditions, which occurred more often in the group
with previous endovascular repair (42.3% vs 14.8%, P = 0.007). According to the modified Crawford classification, the extent of
the thoracoabdominal aortic pathology was as follows: type I=16,
type II=17, type III=26, type IV=20 and type V=1 with no inter-
group difference. Definition of unsuitability for endovascular repair Unsuitability for TEVAR [11] was defined in terms of company
instructions for use regarding endovascular fenestrated aortic re-
pair and endovascular branched aortic repair as well as the indi-
vidual experience of the aortic team taking all 4 dimensional
aspects of the decision-making process into account. Previous aortic interventions/operations Sixteen (20.0%) patients had previous FET implantation. Twenty-
six patients (32.5%) underwent previous endovascular aortic
interventions in various segments. Fifteen patients underwent
TEVAR, 5 of them after a previous FET procedure; and 11 patients
underwent a previous EVAR, 5 of whom also had TEVAR later
(Table 2). Additional surgical procedures in infective
situations/organ fistulations In the case of native or prosthetic aortic infections, we follow a
concept of “complete as possible” the removal of the indwelling
prosthetic material, radical local debridement and continuity res-
toration with neoaortas made from bovine pericardium [14]. After aortic repair, the respective organ fistulation is addressed in
the same surgical setting. Surgical details Selective organ perfusion was performed in 63 patients (78.8%). The CPB time was 113.3 ± 80.1 min with no intergroup differen-
ces. Reimplantation of segmental arteries was performed in 40
patients (50%), again without intergroup differences. The need
for blood transfusions and blood products was as follows: packed
red blood cell units (PRBC) 10 (5; 14), fresh frozen plasma units
12 (7.5- 21.5) and platelet transfusion units, 4 (3–6). There was a
significant difference in the need for PRBC between the groups,
respectively
(P = 0.014). The
mean
operating
time
was
406.4 ± 121.6 min. In 31 patients (38.8%), a straight Dacron pros-
thesis was used; in 25 patients (31.2%), a Coselli prosthesis Ethics Statement The ethical committee of the University Hospital Freiburg ap-
proved the study (558/19). The inormed consent was waived due
to the retrospective nature of the study. S. Kondov et al. / Interactive CardioVascular and Thoracic Surgery 3 3 used. Categorical variables were compared using the v2 test. In
the case of a small group (n < 5), Fischer’s exact test was used. The Kaplan–Meier estimates and the log rank calculations were
performed for survival and freedom from aortic-related reinter-
ventions. Multivariate regression analysis was used to analyse the
risk factors for in-hospital deaths. Statistical analyses were per-
formed with GraphPad Prism V 8 (San Diego, CA, USA). established via the femoral vessels, the aorta is clamped at the
most proximal level needed, haemodynamic equilibrium be-
tween the upper and lower body is achieved and sequential re-
pair from proximal to distal is performed. Thoracic segmental
arteries are routinely reimplanted. Which ones to preserve is de-
cided before the operation based on the imaging results and dur-
ing the operation under the guidance of the MEPs and SSEPs. When repairing the visceral and renal segments, normothermic
blood perfusion through selective catheters via the cardioplegia
pump is performed. We do not routinely measure flow to the re-
spective end organs. Usually, the infrarenal anastomosis is done
first so that we are able to re-establish circulation continuity be-
tween the upper and the lower body. The sequence of reimplan-
tation usually is the right renal artery (RRA), the superior
mesenteric artery (SMA), the coeliac trunk (CT) and the left renal
artery. In type IV aneurysms, we do not use CPB or selective or-
gan perfusion; we perform an oblique anastomosis to the RRA,
SMA and coeliac trunk with direct reimplantation of the left renal
artery into the main prosthesis. VASCULAR Statistical analyses Continuous data are presented as the mean with the standard
deviation in cases with a normal distribution; otherwise, they are
presented as the median with quartiles (25th-75th). Normality
distribution was tested with the Kolmogorov-Smirnov test. The t-
test was applied to compare continuous variables in cases with
an equal distribution; otherwise, the Mann-Whitney test was S. Kondov et al. / Interactive CardioVascular and Thoracic Surgery 4 4 Table 1:
Patient demographics
Variables
Overall
Previous Endovascular aortic repair
No previous Endovascular aortic repair
P-value
(n = 80)
(n = 26)
(n = 54)
Age
64.2 ± 12
66.8 ± 9.1
65.5 ± 13.0
0.171
Sex (m)
56 (70%)
20 (76.9%)
36 (66.7%)
0.348
Diabetes
7 (8.8%)
2 (7.7%)
5 (9.3%)
1.000
Arterial hypertension
68 (85%)
22 (84.6%)
46 (85.2%)
1.000
Hyperlipidaemia
29 (36.3%)
7 (26.9%)
22 (40.7%)
0.229
EuroSCORE
7.9 ± 2.6
8.85 ± 2.7
7.4 ± 2.4
0.021
BMI
25.6, 22.4; 29.4
25.5, 23.1; 30.0
25.6, 22.3; 29.1
0.828
Marfan syndrome
9 (11.3%)
2 (7.7%)
7 (13%)
0.485
COPD
15 (18.8%)
7 (26.9%)
8 (14.8%)
0.194
Previous cerebrovascular events
11 (13.8%)
4 (15.4%)
7 (13 %)
0.742
Coronary artery disease
32 (40%)
14 (53.8%)
18 (33.3%)
0.079
Previous type A repair
22 (27.5%)
7 (26.9%)
15 (27.8%)
0.936
Type B aortic dissection
16 (20%)
8 (30.8%)
8 (14.8%)
0.095
Aortic diameter
63, 58.3; 75.0
70.5, 61.5; 88.3
61, 57.0; 69.3
0.003
Dialysis
2 (2.5%)
2 (7.7%)
0 (0.0%)
0.039
Urgent/emergency
19 (23.8%)
11 (42.3%)
8 (14.8%)
0.007
Elective
61 (76.2%)
15 (57.5%)
46 (85.2%)
0.007
Crawford I
16
7
9
0.372
Crawford II
17
3
14
0.242
Crawford III
26
9
17
0.803
Crawford IV
20
7
13
0.789
Crawford V
1
0
1
0.999
BMI: body mass index; COPD: chronic pulmonary disease. of multiorgan failure; 3 patients, of sequelae of haemorrhagic
shock; 2, of septic shock; 2 died on the table during the operation
for TAAA rupture; 1 died of severe intraoperative apoplexy. Table 2:
Previous aortic interventions/operations
Previous aortic
interventions/operations,
n (%)
FET
16 (20%)
Previous endovascular repair
26 (32.5%)
TEVAR
15 (18.6%)
TEVAR after FET
5 (6.3%)
EVAR
11 (13.8%)
TEVAR after EVAR
5 (6.3%)
EVAR: endovascular aortic repair; FET: frozen elephant trunk; TEVAR: tho-
racic endovascular aortic repair. Follow-up and need for aortic-related
reinterventions Follow-up was 100%. Patients were followed in our aortic outpa-
tient clinic by computed tomography angiography and clinical
examinations. The
mean
follow-up
was
37.9 ± 15.8 months. Survival was comparable in patients with previous endovascular
aortic repair compared with that in patients with no previous
endovascular aortic repair (log rank P = 0.524) (Fig. 1). Freedom
from aortic-related reintervention during follow-up was 96.3%
(Fig. 2). Three additional patients died of non-aortic related
causes during the follow-up period: 1 patient died of a malignant
tumour 16 months after surgery, and 2 died 18 months after
surgery. EVAR: endovascular aortic repair; FET: frozen elephant trunk; TEVAR: tho-
racic endovascular aortic repair. (Terumo Aortic, Scotland, UK); and in 20 patients (25%), a Siena
prosthesis (Terumo Aortic, Scotland, UK) was used. In 4 patients
(5%) with native or prosthetic aortic infection, a self-made bovine
pericardial graft used. All patients with xenopericardial grafts had
previous endovascular aortic repair. The Siena prosthesis was
more frequently used in the group with previous endovascular
aortic repair. Five patients (6.3%) needed postoperative circula-
tory support with extracorporeal life support (Table 3). (Terumo Aortic, Scotland, UK); and in 20 patients (25%), a Siena
prosthesis (Terumo Aortic, Scotland, UK) was used. In 4 patients
(5%) with native or prosthetic aortic infection, a self-made bovine
pericardial graft used. All patients with xenopericardial grafts had
previous endovascular aortic repair. The Siena prosthesis was
more frequently used in the group with previous endovascular
aortic repair. Five patients (6.3%) needed postoperative circula-
tory support with extracorporeal life support (Table 3). Perioperative outcome Overall,
in-hospital
mortality
was
20%
(n = 16). Permanent
paraplegia was observed in 4 patients (5%). Stroke was seen in
5 patients (6.3%), and a tracheostomy was performed in 19
(23.8%) patients due to postoperative respiratory insufficiency. Intermittent haemodialysis for postoperative acute kidney injury
was needed in 15 patients (18.8%) (Table 4). Eight patients died Emergency/urgent versus elective procedures When comparing emergency/urgent and elective procedures,
more deaths occurred in the emergency/urgent group, and the
need for tracheostomy was higher. However, paraplegia rates
were comparable (Supplemental Table 1). Predictors of in-hospital deaths Using multivariate logistic regression, the quantity of PRBC units
(P = 0.009, CI 1.028–1.215, OR = 1.117) used was identified as a
significant risk factor for in-hospital death (Table 5). S. Kondov et al. / Interactive CardioVascular and Thoracic Surgery 5 Table 3:
Intraoperative details comparing patients with previous endovascular aortic repair and no endovascular aortic repair
Overall
Previous endovascular aortic repair
No previous endovascular aortic repair
P-value
n = 80
n = 26
n = 54
Selective organ perfusion using partial CPB
63 (78.8%)
24 (92.3%)
39 (72.2%)
0.045
Partial CPB bypass time (min)
113.3 ± 80.1
117.1 ± 71.65
108.8 ± 84.82
0.173
Segment artery reimplantation
40 (50%)
10 (38.5%)
30 (55.6%)
0.232
PRBC
10, 5; 14
11, 8; 20
8, 4; 12
0.014
FFP
12, 7.5; 21.5
12, 9; 21
13, 6.6; 23
0.797
PT
4, 3; 6
4, 2; 5
4.5, 3.8; 6.3
0.098
Operating time
406 ± 121.6
431.8 ± 149.6
407.0 ± 122.7
0.414
Straight Dacron prosthesis
31 (38.8%)
6 (23.1%)
25 (46.3%)
0.046
Coselli prosthesis
25 (31.2%)
3 (11.5%)
22 (40.7%)
0.001
Siena prosthesis
20 (25%)
13 (50%)
7 (13%)
<0.001
Self-made bovine pericardial graft
4 (5%)
4 (15.4%)
0 (0%)
<0.001
ECLS
5 (6.3%)
2 (7.7%)
3 (5.6%)
0.658
CPB: cardiopulmonary bypass; ECLS: extracorporeal life support; FFP: fresh frozen plasma units; PRBC: packed red blood cells, units; PT: platelet transfusion, units. Table 3:
Intraoperative details comparing patients with previous endovascular aortic repair and no endovascular aortic repair Table 3:
Intraoperative details comparing patients with previous endovascular aortic repair and no endovascular aortic repair Downloaded from https://academic.oup.com/icvts/article/35/3/ivac076/6570176 by gu Table 4:
Postoperative outcome according to the Crawford type
Variables
Overall
Type I
Type II
Type III
Type IV
Type V
n = 80
n = 16
n = 17
n = 26
n = 20
n = 1
In-hospital deaths
16 (20%)
4 (25%)
4 (23.5%)
6 (23.1%)
2 (10%)
0 (0%)
Paraplegia
4 (5%)
1 (6.3%)
1 (5.9%)
1 (3.8%)
1 (5%)
0 (0%)
Stroke
5 (6.3%)
1 (6.3%)
1 (5.9%)
3 (11.5%)
0 (0%)
0 (0%)
Tracheostomy
17 (21.3%)
5 (31.3%)
2 (11.8%)
7 (26.7%)
3 (15%)
0 (0%)
Intermittent need of haemodialysis
15 (18.8%)
3 (18.8%)
3 (17.7%)
6 (23.1%)
3 (15%)
0 (0%)
Figure 1. Kaplan–Meier survival curve comparing survival in patients with previous endovascular treatment and no previous endovascular treatment. Predictors of in-hospital deaths m https://academic.oup.com/icvts/article/35/3/ivac076/6570176 by guest on 24 October 2024 Table 4:
Postoperative outcome according to the Crawford type Table 4:
Postoperative outcome according to the Crawford type Figure 1. Kaplan–Meier survival curve comparing survival in patients with previous endovascular treatment and no previous endovascular treatment. –Meier survival curve comparing survival in patients with previous endovascular treatment and no previous endovascular treatme Figure 1. Kaplan–Meier survival curve comparing survival in patients with previous endovascular treatment and no previous S. Kondov et al. / Interactive CardioVascular and Thoracic Surgery 6 Figure 2. Freedom from aortic-related reinterventions. 6
S. Kondov et al. / Interactive CardioVascular and Thoracic Surgery Figure 2. Freedom from aortic-related reinterventions. surgery even if current recommendations would substantiate it
[15–18]. One-fourth of the patients in this series were operated
on under urgent and emergency conditions whereas there was a
substantially higher number of patients with previous endovascu-
lar repair than without previous endovascular repair. This finding
further underlines the need for continuing surveillance of
patients after previous repair, in particular after endovascular re-
pair, because failure is frequent, and obviously, in many settings,
patients are not subjected to a stringent routine follow-up proto-
col [3, 18]. Table 5:
Multivariate logistic regression for in-hospital
deaths
Odds ratio
CI
P-value
Sex
1.498
0.298-7.520
0.624
Age
1.011
0.942-1.085
0.767
COPD
0.278
0.076-2.098
0.278
Type A dissection
0.228
0.055-1.193
0.083
Type B dissection
2.147
0.309-14.911
0.440
Marfan syndrome
0.159
0.019-1.372
0.095
PRBC
1.117
1.028-1.215
0.009
COPD: chronic obstructive pulmonary disease; PRBC: packed red blood
cells. Table 5:
Multivariate logistic regression for in-hospital
deaths [
]
Previous aortic interventions and operations in this series oc-
curred frequently at every level, whereas previous endovascular
repair occurred most frequently, both TEVAR and endovascular
aortic repair. The need for secondary open conversion is often
attributed to trade-offs when primarily indicating treatment. The
broad availability of endovascular therapy often supports choos-
ing this treatment modality as the primary one. However, trade-
offs regarding the adequate length of landing zones as well as re-
specting the anatomy in combination with the—by nature—
higher probability of developing endoleaks by the higher number
of modular components often lead to failure, early and late [7]. Finally, a broader application of the FET technique provides an
ideal platform for secondary distal interventions, operations or
both [3, 19]. Predictors of in-hospital deaths The three-stage concept has recently been intro-
duced as a safe, reproducible and reliable concept for treating
mega-aortic syndromes, FET, TEVAR and open distal completion. Distal shifting of the disease by TEVAR extension reduces manip-
ulation of the left lung during open thoracoabdominal replace-
ment, which is one of the major components that influences the
success or failure of this operation. Because many patients in our
setting had a previous TEVAR and because many of them had
been given large stent grafts, diameter correction between the
stent graft and the Dacron graft used for distal extension is
needed. The modification and inversed use of the Siena prosthe-
sis has turned out to be an excellent means to accomplish this
goal. Outcome according to previous endovascular
aortic repair versus no previous endovascular
aortic repair We did not find any statistically significant differences when we
compared outcomes between patients with previous endovascu-
lar aortic repair and no previous endovascular aortic repair. Table 6 shows perioperative mortality, morbidity and neurologic
injury in the 2 groups. DISCUSSION Nearly 50% of patients had an underlying diagnosis of aortic dis-
section. Patients with aneurysmal formation on the basis of a re-
sidual dissection after a previous type A repair as well as patients
with primary type B aortic dissections were equally distributed. This result mirrors the change in treatment strategies because
many patients with degenerative aneurysmal formation now un-
dergo primary endovascular fenestrated aortic repair or endovas-
cular branched aortic repair, and few are indicated for primary S. Kondov et al. / Interactive CardioVascular and Thoracic Surgery 7 Table 6:
Outcome in the patient groups with previous endovascular treatment and no previous endovascular treatment
Overall
Previous endovascular aortic repair
No previous endovascular aortic repair
P-value
n = 80
n = 26
n = 54
In-hospital deaths
16 (20%)
6 (23.1%)
10 (18.5%)
0.633
Paraplegia
4 (5%)
1 (3.9%)
3 (5.6%)
0.999
Stroke
5 (6.3%)
2 (7.7%)
3 (5.6%)
0.999
Tracheostomy
17 (21.3%)
6 (23.1%)
11 (20.4%)
0.988
Intermittent need of haemodialysis
15 (18.8%)
5 (19.2%)
10 (18.5%)
0.940
Data availability statement: All relevant data are within the manuscript and its supporting information files. Table 6:
Outcome in the patient groups with previous endovascular treatment and no previous endovascul tracheostomy because achieving respiratory weaning is more
straightforward. Because we follow a distal shifting strategy (if
feasible) by FET and secondary TEVAR extension, which leaves a
3.5 repair for the remaining segments, and because a no-touch
technique for the left lung was followed, the incidence of severe
respiratory failure needing prolonged weaning has substantially
decreased. Perioperative mortality remains substantial but has to be inter-
preted in light of the surrounding conditions. Clearly, refraining
from treating urgent and emergency cases would have enabled a
lower perioperative mortality, but we are convinced that it is the
responsibility of academic tertiary care centers to offer treatment
to all comers even if the initial probability of treatment success is
relative [21]. The paraplegia rate in this series was low and mir-
rors our concept of the routine use of CSF drainage, the preser-
vation of major spinal cord inflow and the critical segmental
arteries and the routine intraoperative monitoring of MEPs and
SSEPs. Finally, we directly monitor spinal cord perfusion pressure
and depict that on the haemodynamic monitor, which enables
early intervention in case of borderline arterial pressure condi-
tions [22]. CONCLUSION Despite an early attrition rate, survival after open thoracoabdo-
minal aortic replacement is excellent, and freedom from aortic-
related reintervention is high. Open surgery continues to remain
an essential component in the treatment armamentarium for
acute and chronic thoracoabdominal aortic pathology. DISCUSSION When comparing emergency/urgent and elective procedures,
we observed higher mortality rates among emergent cases, which
is not surprising. A positive surprise was that we did not observe
a higher paraplegia rate in those having emergency/urgent pro-
cedures. We think that this finding is due to our stringent strategy
of CSF drainage in all cases except in those in haemodynamic in-
stability due to rupture. The stroke rate was higher in elective
cases, and strokes occurred more frequently in patients with type
I or type II extent compared to others. We developed a new
strategy in these cases: We clamped the proximal aortic segment
before initiation of CPB, which obviated retrograde dislodgement
of atherosclerotic/thrombotic debris. This strategy has turned out
to be successful. Tracheostomy rates were higher in emergency/
urgent procedures. It is our strategy to go for an early Follow-up in this series is complete due to a stringent surveil-
lance in our outpatient clinic where all aortic patients with aortic
diseases are actively followed. This approach is in particular in-
strumental in identifying the few patients who need secondary
repair of any kind after thoracoabdominal replacement, which is
fortunately an extremely rare occurrence. Finally, even in the
modern endovascular era the open procedure remains a durable
solution with a low rate of aortic reinterventions. Limitations We identified the number of PRBC units as independent pre-
dictors of perioperative mortality. This approach mirrors the detri-
mental effect of high-volume turnover and the associated
reduction of immunocompetence in the early postoperative pe-
riod. Clearly, urgent or emergency scenarios are by nature associ-
ated with a high turnover, but in elective settings, every effort
should be made to optimize blood management and to reduce
the need for plasmatic and cellular transfusions to a minimum [1]. This report contains all the inherent limitations of a retrospective
analysis. However, the unique advantage of this report is that it
describes the open surgical treatment approach in an era where
alternatives are available and are used, which naturally leaves a
more complex patient cohort for open surgery. Finally, it is an
all-comers series and does not preselect either by underlying pa-
thology, frame conditions or urgency. When comparing emergency/urgent and elective procedures,
we observed higher mortality rates among emergent cases, which
is not surprising. A positive surprise was that we did not observe
a higher paraplegia rate in those having emergency/urgent pro-
cedures. We think that this finding is due to our stringent strategy
of CSF drainage in all cases except in those in haemodynamic in-
stability due to rupture. The stroke rate was higher in elective
cases, and strokes occurred more frequently in patients with type
I or type II extent compared to others. We developed a new
strategy in these cases: We clamped the proximal aortic segment
before initiation of CPB, which obviated retrograde dislodgement
of atherosclerotic/thrombotic debris. This strategy has turned out
to be successful. Tracheostomy rates were higher in emergency/
urgent procedures. It is our strategy to go for an early DISCUSSION Interestingly, we did not observe differences regarding out-
come in patients with or without previous endovascular interven-
tions. This outcome is a very positive aspect because many
settings deem patients with previous extensive endovascular re-
pair inoperable, which obviously is not the case [7]. However,
strategies have to be developed to cope with the unique chal-
lenges associated with secondary surgical conversions after failed
endovascular repair. The use of CPB varies in the literature depending on the TAAA
type and on the need for sufficient organ protection. In our pa-
tient group, organ protection using CPB was performed in 63
patients (78.8%). The left heart bypass has advantages regarding
intraoperative anticoagulation, amount of bleeding and other
conceptual components. The major challenge is volume manage-
ment, which is easier (and eventually safer) with partial CPB be-
cause volume shifts can be balanced better if pump suction and
immediate reinfusion are available. Our concept was to stick with
the partial CPB in types I, II, III and V. In type IV, we routinely
clamp and sew an oblique anastomosis to the RRA, SMA and
coeliac trunk with selective reimplantation of the left renal artery. The use of CPB varies in the literature depending on the TAAA
type and on the need for sufficient organ protection. In our pa-
tient group, organ protection using CPB was performed in 63
patients (78.8%). The left heart bypass has advantages regarding
intraoperative anticoagulation, amount of bleeding and other
conceptual components. The major challenge is volume manage-
ment, which is easier (and eventually safer) with partial CPB be-
cause volume shifts can be balanced better if pump suction and
immediate reinfusion are available. Our concept was to stick with
the partial CPB in types I, II, III and V. In type IV, we routinely
clamp and sew an oblique anastomosis to the RRA, SMA and
coeliac trunk with selective reimplantation of the left renal artery. We identified the number of PRBC units as independent pre-
dictors of perioperative mortality. This approach mirrors the detri-
mental effect of high-volume turnover and the associated
reduction of immunocompetence in the early postoperative pe-
riod. Clearly, urgent or emergency scenarios are by nature associ-
ated with a high turnover, but in elective settings, every effort
should be made to optimize blood management and to reduce
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tants to Terumo Aortic and shareholders of Ascense Medical,
Martin Czerny is consultant to Medtronic, Endospan and NEOS,
received speaking honoraria from Cryolife-Jotec and Bentley and
is shareholder of TEVAR Ltd. Conflict of interest: Martin Czerny and Bartosz Rylski are consul-
tants to Terumo Aortic and shareholders of Ascense Medical,
Martin Czerny is consultant to Medtronic, Endospan and NEOS,
received speaking honoraria from Cryolife-Jotec and Bentley and
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endovascular repair for aortic dissection: a systematic review and meta-
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Can chiropractors contribute to work disability prevention through sickness absence management for musculoskeletal disorders? - a comparative qualitative case study in the Scandinavian context
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Open Access Open Access Can chiropractors contribute to work
disability prevention through sickness
absence management for musculoskeletal
disorders? - a comparative qualitative case
study in the Scandinavian context Mette Jensen Stochkendahl1,2*, Ole Kristoffer Larsen2,3, Casper Glissmann Nim2, Iben Axén4, Julia Haraldsson5,
Ole Christian Kvammen6,7 and Corrie Myburgh2 Mette Jensen Stochkendahl1,2*, Ole Kristoffer Larsen2,3, Casper Glissmann Nim2, Iben Axén4, Julia Haraldsson5,
Ole Christian Kvammen6,7 and Corrie Myburgh2 Mette Jensen Stochkendahl1,2*, Ole Kristoffer Larsen2,3, Casper Glissmann Nim2, Iben Axén4, Julia Haraldsson5,
Ole Christian Kvammen6,7 and Corrie Myburgh2 Abstract Background: Despite extensive publication of clinical guidelines on how to manage musculoskeletal pain and back
pain in particular, these efforts have not significantly translated into decreases in work disability due to musculoskeletal
pain. Previous studies have indicated a potential for better outcomes by formalized, early referral to allied healthcare
providers familiar with occupational health issues. Instances where allied healthcare providers of comparable
professional characteristics, but with differing practice parameters, can highlight important social and organisational
strategies useful for informing policy and practice. Currently, Norwegian chiropractors have legislated sickness
certification rights, whereas their Danish and Swedish counterparts do not. Against the backdrop of legislative variation,
we described, compared and contrasted the views and experiences of Scandinavian chiropractors engaging in work
disability prevention and sickness absence management. Methods: This study was embedded in a two-phased, sequential exploratory mixed-methods design. In a comparative
qualitative case study design, we explored the experience of chiropractors regarding sickness absence management
drawn from face-to-face, semi-structured interviews. We subsequently coded and thematically restructured their
experiences and perceptions. Results: Twelve interviews were conducted. Thematically, chiropractors’ capacity to support patients in sickness
absence management revolved around four key issues: issues of legislation and politics; the rationale for being a
sickness absence management partner; whether an integrated sickness absence management pathway existed/could
be created; and finally, the barriers to service provision for sickness absence management. Conclusion: Allied health providers, in this instance chiropractors, with patient management expertise can fulfil a key
role in sickness absence management and by extension work disability prevention when these practices are
legislatively supported. In cases where these practices occur informally, however, practitioners face systemic-related
issues and professional self-image challenges that tend to hamper them in fulfilling a more integrated role as providers
of work disability prevention practices. Keywords: Chiropractic, Policy, Work disability prevention, Sickness absence, Qualitative, Interview * Correspondence: m.jensen@nikkb.dk
1Nordic Institute of Chiropractic and Clinical Biomechanics, Campusvej 55,
DK-5230 Odense M, Denmark
2Department of Sports Science and Clinical Biomechanics, University of
Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark
Full list of author information is available at the end of the article © The Author(s). Stochkendahl et al. Chiropractic & Manual Therapies (2018) 26:15
https://doi.org/10.1186/s12998-018-0184-0 Stochkendahl et al. Chiropractic & Manual Therapies (2018) 26:15
https://doi.org/10.1186/s12998-018-0184-0 Background guidelines on how to manage musculoskeletal pain in
general and back pain in particular, these efforts have
not significantly translated into decreases in work dis-
ability due to musculoskeletal pain as evident by the
continuously high costs to society. With the substantial
cost implications of work disabilities for national econ-
omies, there is a need for improvement in the way
healthcare systems and their actors incorporate work
disability prevention (WDP) in their service for individ-
uals with musculoskeletal conditions. Moreover, there is
a need for improving the communication and collabor-
ation between the healthcare actors, employees and
workplaces. Previous studies have indicated a potential
for better work disability outcomes by formalized, early
referral patterns to AHPs familiar with occupational
health issues [20–22]. Therefore, one potential strategy
could be to integrate WDP in the model of care pro-
vided by AHPs [20–22] for patients with musculoskeletal
disorders. Musculoskeletal pain is a major cause of work disability
with enormous socioeconomic consequences. Back pain-
related disorders alone are costly and responsible for up
to one quarter of days off work in European countries
like Sweden [1] and Denmark [2], and in Norway four
out of ten sickness certifications are based on a muscu-
loskeletal diagnosis [3]. For patients with musculoskeletal pain or other work-
related problems, general practitioners (GPs) are the
traditional
gatekeepers
to
workers’
compensation
through sickness certification in the majority of Euro-
pean countries, but studies from the UK and Scandi-
navia have indicated that GPs question the relevance of
work-related issues to their primary healthcare provider
role [4–8]. Restraints in terms of time and resources and
of lack of knowledge around judging capacity to work
have been identified as major barriers for GPs to engage
with social workers and workplaces [9, 10]. Furthermore,
some GPs would prefer not to be part of the sickness
certification system, suggesting the alternative of an au-
thoritative individual to whom they could refer patients
[7, 11]. This leaves a missed potential for relevant work-
place assessments, and for engaging in dialogue with the
patient and the employer regarding work accommoda-
tions. Further, to provide evidence-based guidance to
encourage early self-management and a continuation, or
early resumption of work activities [12], such a dialogue
is necessary. Background The GPs’ solitary role in sickness certifica-
tion may also result in lack of collaboration between
clinicians and other stakeholders, which has been
identified as detrimental for a positive return to work
outcome [13]. Chiropractors’ sickness absence management practices
across Scandinavia Occupational groups operating within similar social con-
texts, but with varied legislated practice parameters, pro-
vide an opportunity to observe the impact of systematic
variation [23]. More specifically, highlighting the social
sequelae of different sick leave management practices is
useful for informing policy and practice [24]. Chiropractic is a growing musculoskeletal health pro-
fession in Norway, Denmark, and Sweden. It is con-
cerned with the diagnosis, treatment and prevention of
mechanical disorders of the musculoskeletal system. Members of the respective national chiropractic associa-
tions hold a 4- or 5-years Master’s degree in musculo-
skeletal health, which, when followed by a 1-year
internship, qualifies for the respective national board of
health certifications as independent healthcare providers. In all three Scandinavian countries, chiropractors func-
tion as first points of contact for patients with musculo-
skeletal disorders, but under different regulations and
levels of integration in the welfare systems. In Sweden,
chiropractors are largely private musculoskeletal practi-
tioners outside the national health service with limited
integration into the national healthcare system, while in
Denmark and Norway, the chiropractors work inside the
respective national health services as AHPs. The use of allied healthcare providers (AHP), like
physiotherapists, chiropractors and manual therapists,
within the field of musculoskeletal pain is gaining popu-
larity amongst patients, especially in the working popu-
lation [14]. AHP are also more often sought out as the
first points of contact and principal providers of health-
care for individuals with musculoskeletal conditions [15,
16]. This poses a challenge for the continuity and coord-
ination of care when sick leave certification is required
as many of these patients may not see another practi-
tioner about their back pain [17], while others may also
consult their GP. In the context of work, the integration
of healthcare professionals may be even more challen-
ging as outcomes are not merely dependent on high
quality healthcare, but also the collaboration of multiple
stakeholders inside and outside the healthcare sector
and the workplace [14]. In Norway, chiropractors and manual therapists (i.e. physiotherapists with a degree at the masters level) re-
ceived authorization to issue sickness certifications for
0–8 weeks in 2006, and since 2008, for 0–12 weeks [25]. However, in the two other Scandinavian countries,
Denmark and Sweden, no such regulation currently ex-
ists (see Table 1). Abstract 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Page 2 of 12 Page 2 of 12 Stochkendahl et al. Chiropractic & Manual Therapies (2018) 26:15 Interviews An interview guide with prompts was developed where
the team drew on their experience of work disability
programs as researchers and as clinicians, and on recent
research on the topic. Face-to-face, semi-structured inter-
views were conducted where the chiropractors were asked
to draw on examples of cases from their own experience
[28]. A rolling interview schedule was used. That is,
questions were modified over the course of the interviews
to ensure that responses to key cross-case topics were elic-
ited (see Table 2). In the interviews, participants were
asked to talk about their personal experience and level of
involvement in SAM of patients with musculoskeletal Chiropractors’ sickness absence management practices
across Scandinavia In Denmark, in 2009, the traditional
sickness certification was replaced by a fitness for work As the population ages and the current health reforms
focus on shifting secondary care services into the com-
munity, demands on GPs and primary healthcare con-
tinue to rise [18, 19]. Despite the publication of clinical Stochkendahl et al. Chiropractic & Manual Therapies (2018) 26:15 Page 3 of 12 Table 1 Key facts relating to the chiropractors’ role in sickness absence management across Norway, Denmark, and Sweden
Denmark
Norway
Sweden
Healthcare system
Public funding (tax-funded)
Yes
Yes
Yes
Sickness absence legislation
Certification required
No
Yes, if more than 3 days
Yes, if more than 7 days
Sickness benefits paid by employera
30 days
16 days
2–14 days
Sickness benefits paid by social servicesa
> 30 days- 23 weeks
> 16 days −1 year
> 14 days – 1 year
Chiropractors
Sick leave certification rights
Not applicable (“fit-note”)
Yes (0–12 weeks)
No
Referral rights to imaging
(e.g., x-ray or MRI) and medical
specialists
Yes, partially
(musculoskeletal related)
Yes, partially
(musculoskeletal related)
No
Practice forms
Allied healthcare within the national
healthcare system
Allied healthcare within the national
healthcare system
Private musculoskeletal
healthcare providers
Regulated under the national
health services
Yes
Yes
Largely outsideb
Privately owned clinics
Yes
Yes
Yes
Patient fee for service
Partial
Partial
Full (private) and
partial (public)b
aPresented are overall rules. Specifics may apply\
bApproximately 20% of clinics have agreements under the national health services aPresented are overall rules. Specifics may apply\ Presented are overall rules. Specifics may apply\
bApproximately 20% of clinics have agreements under the national health services the experience of Swedish, Norwegian and Danish chiro-
practors regarding SAM. certificate (“fit note”), which describes how the patient’s
condition influences their work situation and work role
functioning. The employee, employer and GP all con-
tribute information to the certificate, and the purpose of
the fit note is to facilitate return to work. Thus, the GP
no longer must sanction sickness absence in order for
the employee to receive benefits. A detailed description
of the three countries’ legislations and regulations is pre-
sented in Table 1. Sampling and recruitment During the period of June 2015 to March 2016, we pur-
posively sampled chiropractors across the three countries
with experiences regarding SAM who were willing to
share these [28]. Specifically, we recruited chiropractors
with a recent experience in managing patients with work
disability and were seeking a variety of practice types
(solo/group/multidisciplinary),
location
(country),
and
“other interests” (additional occupation/board member-
ship). Chiropractors who were identified by a project gate-
keeper from the research group’s network were invited by
email. Further, chiropractors were invited via a snowball
technique through the chiropractors’ networks [29]. In the context of changing healthcare policies and organ-
isational structures, there is an increasing need for evalua-
tions of the impact of role extensions, and the potential
barriers and facilitators for implementation of such a change. The objectives of this study were to: 1) Describe the experiences of chiropractors engaging
in sickness absence management (SAM). 2) Compare and contrast chiropractors’ integration of
SAM in their model of care in a context with
legislated sickness certification rights (Norway) and
in two contexts without sickness certification rights
(Sweden and Denmark). Methods This study formed part of a two-phased sequential,
exploratory mixed-methods design (the results of the
quantitative phase will be reported separately) [26]. Using a postpositivistic lens, a comparative qualitative
case study [27] was conceptualized in order to analyse
and understand detailed and in-depth descriptions about Stochkendahl et al. Chiropractic & Manual Therapies (2018) 26:15 Page 4 of 12 Table 2 Topics discussed and examples of trigger questions during the interviews
Topics discussed
Examples of questions
Current role in SAM
• How are you currently involved in managing sickness absence of your patients? • What is your experience regarding sickness absence of your patients? • In what kind of patients do you typically consider sickness absence? • How often do you certify/advise about sickness absence? • How do you assess the patients regarding SAM? • How do feel equipped to engage in SAM? Collaboration with stakeholders
• How do you collaborate with the general practitioner/work place/social services regarding SAM? Barriers and facilitators for SAM
• Which challenges have you met in managing sickness absence? • What do you see as facilitators for managing sickness absence? Future role
• Given the possibility, which initiatives or collaborations would support you in your work regarding SAM? • How do you envision the future collaboration between the general practitioners and you – who has which role? • How do you think you can contribute to managing sickness absence? - What role would you like to play? SAM Sickness absence management Given the possibility, which initiatives or collaborations would support you in your work regarding SAM? How do you envision the future collaboration between the general practitioners and you – who has which role? How do you think you can contribute to managing sickness absence? - What role would you like to play? SAM Sickness absence management experienced qualitative researcher (CM) who provided
peer consultation about the qualitative data analysis
process [28]. A qualitative data analysis package (NVivo,
Version 10, QSR International) was used to organize,
code and interpret text data. A fourth investigator (MJS),
scrutinised sample transcripts, reviewed the
coding
scheme and analytical decisions, and developed a the-
matic map. Through an iterative process using memo
sharing and consensus meetings, involving all investiga-
tors, data was recoded, code families created, and finally,
themes were reviewed for coherence. pain. The interviews were designed to map out the chiro-
practors’ general experience in clinical practice. Methods The par-
ticipants were then asked to share their perception of
their current role and competencies, and to talk about the
support and training they would need to better assist an
individual patient in these matters. At the commencement of each interview, demographic
data (i.e. gender, age, practice type, and other work func-
tions) were collected. Interviews were conducted in the
native language of the participants by one or two out of
three interviewers from the research team at locations
convenient for the participants. Each interview was
audio-recorded and transcribed verbatim by the research
team into computer-readable text files. During the inter-
view phase of the project, the researchers documented
their reflections about the interviews in a journal. These
included notes about informal conversations prior to or
after the interviews, as well as other information not
captured in an audio transcript. In instances where new topics emerged, follow-up
questions were e-mailed to the previous participants
who were asked to state their experience or percep-
tion regarding the newly emerged topics. This data
was then incorporated into the data analysis to ensure
data saturation (i.e., the point where it was felt that
no
additional
information
would
be
produced
by
increasing the sample size) [28]. Ethical considerations
d
h The interviews were analysed in the language of the par-
ticipants. The three Scandinavian languages have similar
roots and are understood across the three countries,
which enabled coding and interpretation of the tran-
scripts in the original language. Further, the team con-
sisted of three bilingual (fluent in Danish/Norwegian;
Danish/Swedish; and Danish/English) team members
and one trilingual (fluent in Danish/Norwegian/Swedish)
team member. Quotes were translated into English by
the team members in combination in a way that trans-
ferred content equivalence in translation while maintain-
ing semantic equivalence [30]. The research team’s
linguistic skills, and cultural and content knowledge
assured the accuracy in translation [31]. In Sweden, the regional ethics committee evaluated the
project and found that the study did not need ethical
permission (advisory statement 2016/3:1). In Denmark,
the Regional ethics of Southern Denmark gave approval
for the study and declared that the study does not fall
within the scope of the Medical Research Involving Hu-
man Subject Act (§14). Approval for data handling and
storage covering both Denmark and Norway under the
European Economic Area collaboration was granted
from the Danish Data Protection Agency. Prior to the
interviews, written and oral information about the study
were provided to the participants. Written informed
consent was obtained from all participants. All partici-
pants were advised that conversations were to be audio
or video recorded, and assured of confidentiality and
anonymity in reporting of the results. Two members of the research team (OKL and CGN)
independently coded the transcripts using content ana-
lysis before discussing the codes and categories with an Page 5 of 12 Stochkendahl et al. Chiropractic & Manual Therapies (2018) 26:15 Stochkendahl et al. Chiropractic & Manual Therapies (2018) 26:15 Results do not write a sick leave certification, but they can
get a document, which says I [the patient] have
actually seen a chiropractor with this problem. And
it [the problem] has to be rectified or I need help
with this. (S4-6) Twelve chiropractors participated in the study with in-
terviews lasting between 12 and 65 min with a mean of
44 min. The descriptive participant characteristics of our
sample are presented in Table 3, below. Four themes emerged from our analysis: Legislation
and politics shape sick leave practice, a rationale for the
chiropractor as sickness absence manager, an integrated
sick leave management pathway and the emergence of
the chiropractor as a sick leave manager. Danish chiropractors, the group that occupies the le-
gislative “middle ground”, expressed a degree of un-
certainty about where the responsibility lay for SAM. In particular, the group’s perception was that current
Danish legislation left most of the responsibility to
the patient, but that patients were not always aware
of the system. They also frequently received requests
for assistance from not only patients, but also their
place of work with respect to job modification, but
they felt unsure whether this type of activity lay
within their scope of practice. This issue is illustrated
by the following passage from DK4’s interview:… a lot
of citizens are perhaps downright uncertain about,
how is it…who can certify sick leave and who will
do it. It’s a bit of a grey area, where people are
perhaps a little unsure about how the system
works, and maybe it’s also an area, where we as
chiropractors are a little afraid to open up in that
respect. (DK4-1) and … if you want a card to play, which makes
chiropractors someone to trust, someone who is
known, and who delivers measurable results… (S3-5) … a disadvantage or a limitation [of the present
system], is for the patient, they have to first seek
our help and then they have to go further to seek
help at the GP who can give them a sick leave
certification, if they assess that as necessary. So, it
is both the cost, that they meet two health
professionals, when it could be enough with one. (S2-7) Mirroring the legal status in the two countries, the Nor-
wegians saw the sick leave certification rights as a final ap-
proval, the Swedish struggled to gain recognition and
mentioned sick leave certification rights as a means to be-
come integrated into the national healthcare system. Legislation and politics shape sick leave practice No
one is educated in that system, but we refer to the GP
then. (S1-4) who are used for those kinds of assessments…. No
one is educated in that system, but we refer to the GP
then. (S1-4) This filter results in a situation where chiropractors
tend not to see patients with a low socioeconomic sta-
tus, who in turn, are perceived by the chiropractors to
be more complicated to manage. Across all three contexts, practitioners cited frequent
patient contact as a facilitating element in SAM. They
explained how this allowed them to get to know the pa-
tients through continuous dialogue and to establish
trust. As a Norwegian participant explains, routine con-
tact was also thought to provide the opportunity to opti-
mally monitor progression and adapt plans accordingly: As a consequence, the incentive to engage in compli-
cated SAM was considered low. This was particularly
observed in the Danish context: But it’s the financial part, because you do not get a dime,
and it’s actually quite time consuming…and having to
write an employer, it takes a long time. You could easily
see one, two, three patients instead. (DK2-4) You can continue one week, maybe two weeks, and
then have the dialogue with the patient all the time,
during treatments too. So, like that you have more
options than when you say “I’ll put you off work for
three weeks. See you.” And that’s when they return
[the patients]. Instead, you have a continuous dialogue
about progress. (NO2-4) For the Norwegian and Swedish chiropractors, sick
leave certification rights were perceived as a “seal of ap-
proval” of the profession. The rights were perceived as a
way of becoming fully integrated into the healthcare sys-
tem, and a way of changing the scope of practice from
(alternative) therapy to being recognized for diagnostic
and management skills too. Being able to both handle the patients’ musculoskeletal
complaint and certify sickness absence was also per-
ceived by chiropractors from all three countries as a
means to prevent chronicity. Across contexts our partic-
ipants argued that the risk of chronicity was a weakness
of the current sick leave management system. By adding
a chiropractor, the risk would reduce by shortening the
chain of management. As S2 explains: … then they [the patients] appreciate it [the
chiropractors’ sickness certification rights], they also
see as a quality stamp that we can do it. (NO2-3) Legislation and politics shape sick leave practice Norwegian and Danish chiropractors appeared mindful
of maintaining the traditional gatekeeper role of GPs, as
they consistently recognized the importance of inform-
ing the GPs regarding sick leave issues. There were,
however, a variety of opinions about which patients or
situations were best coordinated by the GP and which
should be supervised by the chiropractor. When we talk about long-term sick leave, the GP is
perhaps a good basis, also because they, they kind of
have the whole package [of knowledge]. (DK4-16) When it’s related to patients within our area of
expertise, then it is us who are in control, it is us who
know about the course of treatment and follows the
patient closely, and it is us who have the main
competencies. (DK2-10) Informal roles are furthermore observable in the Da-
nish context as the patient functions as a messenger and
the chiropractor takes on the role of arbitrator, balancing
agreeing with the patient about sick leave and the need
to “push” the patient back to work to prevent unneces-
sary absence. This related mainly to situations where co-morbidity,
such as mental disorder had been identified as well as
long duration sickness absences. In all three contexts, fee-for-service significantly influ-
ences SAM: By contrast, efforts to contribute to SAM in the Swed-
ish context are characterized by uncertainty. We are private, it is an expense for the patient, and
there are many who do not consult us because of that. … they rather go to the public healthcare, and then
they get the sick certification that way. (S2-4) I write why the patient is here and what has been
done and what would be good for the patient in the
future. So, in that way, you can say it is a grey area. I Table 3 Participant characteristics (n = 12)
Norway (n = 4)
Denmark (n = 4)
Sweden (n = 4)
Gender, male/female
3/1
3/1
2/2
Age, years, mean (range)
47 (42–55)
43 (39–50)
38 (36–42)
Type of practice
Solo/group/multidisciplinary
1/0/3
1/0/3
0/2/2
Other functions
Active in research projects
1
2
0
Practice consultant
2
0
National association board member
2
0
1
Method of recruitment
Gatekeeper/snowballing
2/2
4/0
3/1 Table 3 Participant characteristics (n = 12) Page 6 of 12 Page 6 of 12 Stochkendahl et al. Chiropractic & Manual Therapies (2018) 26:15 who are used for those kinds of assessments…. An integrated SAM pathway But if it is a person with a more active profession,
which does not include heavy lifting and where it
is optional to sit, stand, walk etc., then I
recommend them to go to work, because if you
stay home, you will become inactive and then you
have problems for a longer period compared to if
you stay active. (S2-2) Norwegian
and
Danish
chiropractors
described
a
spectrum of sick leave-related conditions, ranging from
uncomplicated musculoskeletal conditions to more com-
plex cases with various degrees of psychosocial and
work-related factors, whereas, the Swedish chiropractors
predominantly referred to patients with few apparent
psychosocial problems. Both Norwegian and Danish chiropractors described
how early and timely communication between stake-
holders was key to successful return-to-work. Well, it is different, right? An office worker, it might
be an arm, shoulder or the neck. If the work regards
heavy loads, it might be the lower back, for example. (NO3-19) Then I write to the GP and state “this is my opin-
ion” so the GP is informed, so at least there is a
common ground, because if we start to say something
different, then all of the sudden it becomes difficult. (DK2–18). And then, of course, there are the ones where stress
management and all thoughts of things, where it is
psychological and the work environment, or a lot of
other stuff... (DK4-5) The standard method of communication with the GPs
was via the official electronic platforms, but many felt
that the communication was unidirectional, and re-
quested more information going from the GP to the
chiropractors. Well, the patients visiting me who are in need of sick
leave are very, very few…most of the patients we see ei-
ther have had chronic pain for a long time, but are still
working,.., or come in with more acute conditions and
get symptom free quite fast. (S2–1). They never write anything. (DK2-34) A rationale for the chiropractor as sickness absence
manager Despite some reluctance to engage in SAM due to lack
of financial incentive, Danish chiropractors expressed a
sense of obligation toward society, and expressed a
moral dilemma between service for the greater good or
their own pocket. According to DK1: Generally speaking, Danish and Norwegian chiropractors
perceived themselves as musculoskeletal specialists due to
their university degree and as such, as competent as SAM
partners for patients with musculoskeletal problems. They
felt comfortable in assessing musculoskeletal function and
saw the assessments as integral in routine practice. It’s really an area that is important also socio-
economically, and that’s why I think, at one point,
we need to step up to the plate as a profession and
give it the prestige that it really has, and say
“listen, this means something with respect to us
saving tax money.” So that Denmark becomes a
cheaper country to live in, rather than saying “we
focus on our own game, our own everyday life”,
but takes on, perhaps something more like a
societal supportive function. (DK1-37) I think we have a good basis for doing the evaluation,
because we actually know the patients well. And we do
it [assessments] already at the first visit. (DK3–18). However, despite the same level of training, the Swed-
ish chiropractors expressed more hesitation regarding
their competencies. According to a Swedish respondent: I think we have a good basis for doing the evaluation,
because we actually know the patients well. And we do
it [assessments] already at the first visit. (DK3–18). However, despite the same level of training, the Swed-
ish chiropractors expressed more hesitation regarding
their competencies. According to a Swedish respondent: If you look at long term sick leave and the level of
workability and so on, chiropractors are not a group Page 7 of 12 Page 7 of 12 Stochkendahl et al. Chiropractic & Manual Therapies (2018) 26:15 Stochkendahl et al. Chiropractic & Manual Therapies (2018) 26:15 They never write anything. (DK2-34) This is contrasted by the Swedish chiropractors, which
for the most part did not have access to electronic com-
munication platforms, and did not communicate directly
with GPs. Further, the Danish and Norwegian chiropractors
more often described multifaceted patient-centred and
work-related action plans, And then there are situations where you, ehm, again,
you have to look at the whole situation. Are there any
other factors in play, their employer, their workability,
other than just the musculoskeletal? What is the big
picture, and so on? (DK3-17) …as the GP, or as everybody is listed in various places
is impossible to have communication with everyone. (S2-10) …as the GP, or as everybody is listed in various places
is impossible to have communication with everyone.
(S2-10) The Norwegians also described a unidirectional flow
of information to the social services, whereas the Danish
had very little contact or communication with the social
services. whereas, the Swedish chiropractors described a focus
on manual therapy as their primary tool. The chiropractors had a general approach to the pa-
tients, where emphasis was laid on functional disabilities
rather than diagnoses or pain location. But, perhaps, I would like more collaboration with
NAV [social services]. That there was a closer
dialogue basically. (NO4-12) But, perhaps, I would like more collaboration with
NAV [social services]. That there was a closer
dialogue basically. (NO4-12) So really, I look at the function of people, even
though they have a lot of pain, but I always ask them
“Is it worse when you go to work?”…but it starts to
hurt after 2 PM [mimics the patient], well, then
perhaps you should ask about a shorter working day,
right? Or ask if is possible for you to lay down
somewhere to rest, like every other hour. Do you have
an adjustable table, can you stand and sit, like. So, it’s
dependent on what option people have for
adjustments. (DK2-20) A noticeable difference between the countries was that
the Norwegians positively described how SAM was inte-
gral to practice. They used SAM as an integral tool and
natural part of the care package that they offered their
patients. They described SAM as an additional tool in
the clinical tool box that they used if they felt it was
relevant, and, therefore, as an add-on to clinical practice. They perceived this extra tool as essential to clinical
practice, but also an instrument that lead to higher levels
of involvement in their patients’ care, which in turn was
perceived as challenging and personally and profession-
ally rewarding. Not only did the chiropractors discuss return-to-work,
but often mentioned the importance of the patient stay-
ing at work despite some degree of pain or disability. Page 8 of 12 Page 8 of 12 Stochkendahl et al. Chiropractic & Manual Therapies (2018) 26:15 Professional practice
Systematised channels of communication (Sweden)
For the Swedes, the lack of systematic channels of com-
munication was an important barrier for communication
and involvement. It’s my consistent, positive experience with sickness
certification,…. and if I was to move to another
country without these rights, I would feel a little
helpless and naked, I think. S4–11: Other healthcare professions. S4–11: Other healthcare professions. …as the GP, or as everybody is listed in various places
is impossible to have communication with everyone.
(S2-10) It would take some
getting used to because it is such an important
tool, because it is so closely linked to the result of
what you do and the process, and how you plan a
treatment course. It’s not like we’re just therapists
anymore, but there is also the advice part, right? (NO3-17) It is a waste of social resources in some way. That the
patient all the time has to contact another authority
to….Mostly we tell the patients they should contact
the GP and then they make the call themselves. We
don’t have direct communication with the GP. (S4-5) This was in contrast to most of the Danish and
Swedish chiropractors, who were more hesitant about
engaging in SAM. The Danes generally described
SAM as a tedious process that they were somewhat
reluctant to engage in. Further, the Swedish chiropractors, perceived sickness
certification rights as a direct platform for increasing the
general communication with GPs and other healthcare
professionals: S4–11: … [SL rights] would be a good commercial for
us. Yes, that would initiate an automatic dialogue. Not if you ask the chiropractors, because the
paperwork takes up too much. And that’s where you
don’t want to spend your time. It’s, it’s the
bureaucratic hassle. (DK3-8) Interviewer: A dialogue with? Administrative burden and collaboration (Denmark) Functioning in the role of a sick leave manager is, as
the previous themes indicate, influenced by the indi-
vidual practitioner, the profession’s state of practice
and the social systems that facilitate day-to-day imple-
mentation. Our final theme then, chronicles the expe-
riences of chiropractors across the three case settings
where these variables are present or absent. Administrative burden and collaboration (Denmark)
Danish chiropractors had reservations regarding en-
gaging in SAM because of the administrative burden as-
sociated with SAM. The task was considered time
consuming and bureaucratic, and lacking the necessary
administrative support systems. Especially regarding the
collaboration with the social services, which was consid-
ered non-existent, unsatisfactory or even adversarial. The chiropractors also reported how they perceived the
case managers to have a specific agenda relating to cut-
ting costs to a minimum. (Denmark) Danish chiropractors expressed some degree of am-
bivalence regarding embracing the SAM role. It was
on one hand perceived as a natural progression of the
profession, a responsibility to be taken for the greater
good, as an obligation or to honour six years of free
university schooling, but on the other hand as bother-
some and with no financial incentive. Well, in respect to the Jobcenter [social services], it’s
my experience that if you get hold of a specific case
worker there on a specific case, then you can make it
work, but otherwise it’s often, I think, that the
Jobcenter has its own agenda, at least in the area
where I work, where I get most of my patients from,
they have an agenda, which is fastest return to work. (DK1-13) And that [the chiropractors’ role] will slowly
change, and that’s why I think there’ll be a big
difference between clinics along the way. How
many are willing to venture into this, and how
many can’t be bothered, and there will be some
who can’t be bothered…. We also know there are
some [chiropractors] who won’t see chronic
patients. And you can’t say that when you have six
years of university schooling. Well, you just can’t. (DK2-26) The Danish chiropractors alluded to the notion that to
avoid paying for the chiropractors’ services, the social
services case managers did not stick to procedure
protocols. Then some jolly caseworker emails me and asks about
things in relation to the [confidential] social security
number and this and that in an unencrypted email. Page 9 of 12 Stochkendahl et al. Chiropractic & Manual Therapies (2018) 26:15 Stochkendahl et al. Chiropractic & Manual Therapies (2018) 26:15 Then sometimes I write them to please don’t include
full social security numbers in an open email. And
with respect to the actual case, it would be better if
we communicated via Status [secure, encrypted
communication platform] or similar. (DK1-40) I think it is missing, in some way, enlightenment
about who we are, and where we come from and
respect for the education, we actually have. That kind
of enlightenment… (S4-8) I still have patients who doesn’t know that I prescribe
sick leave. and But actually, some patients are not really set on
paying for just a piece of advice, they think it’s strange
to pay to go for longer walks. (DK2-24) You have to think about how you can make it
work, what it is you accept. How do you make it
work? That’s the hardest part. And then make sure
you have 100% support before you start. Make sure
everybody is onboard. Also those who don’t give a
damn. (DK3-30) Patient fee and expectations (Denmark) The final barriers, mentioned by most of the Danish chi-
ropractors, were the patient fee and patients’ expecta-
tions of manual treatment. It’s still not our second nature to be part of the team
now. But we’re still a little like a lonesome cowboy
who’s been given a responsibility …but I know of
colleagues who thinks of certification rights as a
hassle. They would rather treat, examine and treat
and continue their workday. But that’s why I think it’s
more and more important that you embrace the role
as a primary care provider. (NO2-40) …I know that, at least some [patients] think whether
they can afford the consultations or find an
alternative… (DK4-21) (Denmark) (NO1-35) Professional self-image and adaptation in thinking (all)
A salient issue in all three countries is the transition in
self-image and adaptation in thinking and behaviour
from chiropractors being manual therapists or even al-
ternative care providers to becoming fully integrated
members in the primary healthcare sector, which in-
cludes more responsibility in terms of communication,
collaboration and patient management. It was quite
clearly addressed by the chiropractors that they did not
see the profession as united in this issue (difference of
opinions), and they did not think that all colleagues de-
livered the same standard of care. It was clear how the
Norwegians, to a larger degree, talked about embracing
the role, whereas the Danes were more ambivalent, and
the Swedes more hesitant. In Denmark, it was also mentioned that the political
climate ran counter to changing the system, and that
there wasn’t a willingness to provide the necessary fund-
ing for collaboration between stakeholders and for hold-
ing dialogue meetings. And in reality that’s where….if you want it to work,
then you would need those round table discussions,
right?....I don’t know how often they are held, but I
don’t think it’s often, and again because it’s so damn
expensive. (DK2-8) Discussion The practitioners in this study described different levels
of integration in their respective healthcare settings and
how the legislation impacts the clinical encounter and
the level of their involvement in SAM. In Norway, SAM
was described as highly integrated in the clinical en-
counter and as “part of the tool box”, whereas in
Denmark the participants described SAM as a pending
issue and questioned “Is it worth the trouble?” In
Sweden, where chiropractors usually are not part of the
national health services, SAM is not integrated in rou-
tine practice, and the profession is still struggling to gain
broad public recognition. In both Norway and Sweden,
the participants described sick leave certification rights
as a seal of approval of the profession. The social systems that facilitate day-to-day
implementation Scope of practice awareness (all)
Public knowledge of chiropractors and their scope of
practice
were
frequently
mentioned
as
barriers
in
Norway and Sweden. and That is how it is. Then, well, I don’t think the
limitations are disinterest, I just think we have not
been ready for it. (S3-8) Scope of practice awareness (all) The sickness certification rights in Norway were negoti-
ated as part of a larger agreement regarding reimbursement Page 10 of 12 Page 10 of 12 Stochkendahl et al. Chiropractic & Manual Therapies (2018) 26:15 schemes and referral rights to imaging and medical special-
ties between the Norwegian Chiropractic Association and
the Norwegian government. The sickness certification
rights were included in the negotiations as an “expendable”
item (personal communication). Much to the surprise of
many Norwegian chiropractors, the certification rights were
never discussed in detail, but granted without further ado
(personal communication). This situation serves as an ex-
ample of how professional territories are acquired by polit-
ical manoeuvres rather than by virtue of the content of
clinical work [32]. It also shows how the negotiations of this
particular role extension were shaped by national policies
in relation to public services (i.e. the reimbursement and
referral rights). Our interviews reflect that, despite the certi-
fication rights in Norway, it is still not clear to all partici-
pants whose responsibility it is to assist the patient in a
given situation. This is especially evident when talking
about SAM of complex cases and cases with psychosocial
components. Uncertainty around roles and confusion about
accountability between GPs and AHPs are not uncommon
in primary care settings [33], and themes from our synthe-
sis appear to echo the general findings from integration ex-
perience amongst other healthcare professionals [19, 34]. considering “the full picture” and the routine practice of
frequent contacts with the patients. In previous studies in-
volving the nursing profession, references to “patient-
centred” and “holistic care” were made, clearly as a form
of professional rhetoric designed to support their bid for
legitimacy in claiming role exclusivity, or at least primacy
[19], and similar have been reported in a study of GPs and
physiotherapists [36]. We identified a range of barriers for the chiropractors
to engage in SAM. Especially salient were organizational
or systemic factors, such as the patients’ fee-for-service,
which was voiced as a barrier for seeing patients with
lower socioeconomic status. Affordability has been iden-
tified as a significant gap in health system coverage [37],
and it is a problematic barrier in the context of a social
welfare system were the pursuit of equality in health in-
cluding access to healthcare is an overriding goal and
principle [38]. Scope of practice awareness (all) Communication was described as an essential part of
SAM, but the chiropractors described a predominantly
unidirectional flow to other stakeholders and perceived
this to be an important barrier for SAM. For the Swed-
ish chiropractors in particular, lack of communication
because of the absence of formal communication plat-
forms was especially pertinent. In Denmark, we found
that the lack of financial incentives and the administra-
tive burden were important barriers for engaging in
SAM. In a systematic review, Kilgour et al. [39] sug-
gested that reduction of organizational pressure and im-
proving communications between stakeholders could
ensure that healthcare providers are more amenable to
operating in compensation systems. The likely benefit
would be a corresponding positive influence on patients’
recovery and return to work [39]. p
g
p
The legitimacy that sickness certification rights add to
the profession was perceived differently in the three coun-
tries. Over the past 25 years, the Danish chiropractic com-
munity has raised its professional profile to that of an
acknowledged contributor to local musculoskeletal health-
care. Thus, it is perhaps the first example of the chiroprac-
tic profession being accepted into mainstream healthcare as
an equal partner [35]. This was reflected in our findings,
where issues of professional legitimacy were raised, pre-
dominantly by the Swedish and Norwegian participants. While the Swedish chiropractors struggle to establish legit-
imacy to integrate fully with established healthcare (primary
legitimacy), the Norwegians battle for factors to boost legit-
imacy to the level of benchmark healthcare professions
(secondary legitimacy), e.g., GPs [35]. Methodological considerations and future directions Methodological considerations and future directions
This study used a qualitative description with in-depth,
semi-structured interviews. This provided a broad per-
spective as well as a deep understanding of the partici-
pants’
experiences
and
perceptions
regarding
this
previously uncharted topic. The study was conducted as
part of a mixed-method study, and will inform a quantita-
tive phase. Therefore, we see the present results as a first
exploration of SAM among Scandinavian chiropractors. Further interviews may have provided other perspectives. A specific concern is the lack of triangulation with other
stakeholders such as GPs, patients or case-workers. In the
quantitative phase, we will extend the data collection to
representative samples from all three countries. The rationale of chiropractors as SAM partners put for-
ward by the participants are highly linked to issues of le-
gitimacy. Respondents based their claims on perceptions
of their own and other professions’ specialist expertise to
justify their part in the certification process and the role
extension concept [36]. The chiropractors highlighted
their skill in musculoskeletal conditions and alluded to
GP’s knowledge gap and lack of time to legitimize role ex-
tension claims. In studies of role extension, the use of a
common discourse to discredit the competitor profession,
either on the basis of their approach to clinical care or
their skills or competence is common [19, 34, 36]. As an-
other rationale for chiropractors as SAM partners, the
Norwegian and Danish chiropractors argued the need for
adoption of a holistic approach towards SAM. They legiti-
mized their role through highlighting their approach of Healthcare systems, employment settings and work le-
gislation vary considerably internationally, and the find-
ings from this study may not be applicable to settings
outside the Scandinavian countries. However, we have
found commonalities with existing literature in the area Page 11 of 12 Stochkendahl et al. Chiropractic & Manual Therapies (2018) 26:15 Page 11 of 12 of professional legitimacy and role extension. The chiro-
practic’s professional development is considered a test
case with the potential to affect other AHPs with aspira-
tions to move towards mainstream healthcare [35]. Therefore, the tension between complementary and
alternative medicine providers and ordinary healthcare
systems,
and
the
Danish
chiropractors’ consequent
moves toward mainstream inclusion have been observed
with interest by contemporary social scientists. [35]. Competing interests IA is an associate editor and MJS is a society representatives of Chiropractic
and Manual Therapies, but had no influence over the review assignment or
process. The authors declare that they have no further competing interests. Ethics approval and consent to participate pp
p
p
In Sweden, the regional ethics committee evaluated the project and found that
the study did not need ethical permission (advisory statement 2016/3:1). In
Denmark, the Regional ethics of Southern Denmark gave approval for the study
and declared that the study does not fall within the scope of the Medical
Research Involving Human Subject Act (§14). Approval for data handling and
storage covering both Denmark and Norway under the EEA-collaboration was
granted from the Danish Data Protection agency. Prior to the interviews, written
and oral information about the study were provided to the participants. Written
informed consent was obtained from all participants. All participants were
advised that conversations were to be audio or video recorded, and assured of
confidentiality and anonymity in reporting of the results. advised that conversations were to be audio or video recorded, and assured of
confidentiality and anonymity in reporting of the results. Availability of data and materials All data generated and analysed during this study are included in this
published article. 5. von Knorring M, Sundberg L, Lofgren A, Alexanderson K. Problems in
sickness certification of patients: a qualitative study on views of 26
physicians in Sweden. Scand J Prim Health Care. 2008;26:22–8. Conclusion AHPs, in this instance chiropractors, with patient man-
agement expertise can fulfil a key role in SAM and by
extension work disability prevention when these prac-
tices are legislatively supported. In cases where these
practices occur informally, however, practitioners face
systemic-related issues and professional self-image chal-
lenges that tend to hamper them in fulfilling a more
integrated role as providers of WDP practices. Received: 29 November 2017 Accepted: 4 April 2018 Received: 29 November 2017 Accepted: 4 April 2018 Authors’ contributions 6. Nilsing E, Soderberg E, Bertero C, Oberg B. Primary healthcare professionals’
experiences of the sick leave process: a focus group study in Sweden. J Occup Rehabil. 2013;23:450–61. MJS, OKL, CN, IA, OCK, CM contributed to the concept and design of the
study, which was led by MJS. MJS, OKL, CN and CM developed the interview
guides. OKL and CN conducted the Danish interviews. OKL conducted the
Norwegian interviews. OKL and OCK provided content about the Norwegian
settings. JH conducted the Swedish interviews and provided content about
the Swedish setting together with IA. OKL, CN, MJS and CM analysed and
interpreted the data. MJS drafted the manuscript and all authors critically
revised the article for important intellectual content and gave final approval
of the version to be published. 7. Wynne-Jones G, Mallen CD, Main CJ, Dunn KM. What do GPs feel about
sickness certification? A systematic search and narrative review. Scand J
Prim Health Care. 2010;28:67–75. 7. Wynne-Jones G, Mallen CD, Main CJ, Dunn KM. What do GPs feel about
sickness certification? A systematic search and narrative review. Scand J
Prim Health Care. 2010;28:67–75. 8. Breen A, Austin H, Campion-Smith C, Carr E, Mann E. “you feel so hopeless”:
a qualitative study of GP management of acute back pain. Eur J Pain. 2007;
11:21–9. 8. Breen A, Austin H, Campion-Smith C, Carr E, Mann E. “you feel so hopeless”:
a qualitative study of GP management of acute back pain. Eur J Pain. 2007;
11:21–9. 9. Johansen K, Andersen JS, Mikkelsen S, Lynge E. Decision making and
co-operation between stakeholders within the process of sick leave. A
case study in a Danish municipality. J Interprof Care. 2011;25:59–65. 9. Johansen K, Andersen JS, Mikkelsen S, Lynge E. Decision making and
co-operation between stakeholders within the process of sick leave. A
case study in a Danish municipality. J Interprof Care. 2011;25:59–65. Author details
1 1Nordic Institute of Chiropractic and Clinical Biomechanics, Campusvej 55,
DK-5230 Odense M, Denmark. 2Department of Sports Science and Clinical
Biomechanics, University of Southern Denmark, Campusvej 55, DK-5230
Odense M, Denmark. 3Horten, Norway. 4Unit of Intervention and
Implementation Research for Worker Health, Institute of Environmental
Medicine, Karolinska Institutet, Stockholm, Sweden. 5Falkenberg, Sweden. 6Sandefjord, Norway. 7Institute of Health and Society, University of Oslo,
Kirkeveien 166, Frederik Holsts hus, 0450 Oslo, Norway. Funding
h
d This study was funded by in kind contributions from the authors’ institutions. The funding bodies had no role in the design of the study; collection,
analysis, and interpretation of data; in writing of the manuscript; or in the
decision to submit the article for publication. 4. Cohen D, Marfell N, Webb K, Robling M, Aylward M. Managing long-
term worklessness in primary care: a focus group study. Occup Med
(Lond). 2010;60:121–6. 4. Cohen D, Marfell N, Webb K, Robling M, Aylward M. Managing long-
term worklessness in primary care: a focus group study. Occup Med
(Lond). 2010;60:121–6. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. Abbreviations
AHP Alli d h
l 2. Mortensen OS, Andersen JH, Ektor-Andersen J, Eriksen HR, Fallentin N,
Frost P, et al. White paper on sickness absence and return-to-work due
to musculoskeletal disorders – causes and 0ptions. National Research Centre
for the working Environment. 2008. http://www.arbejdsmiljoforskning.dk/~/
media/boeger-og-rapporter/hvidbog-sygefravaer.pdf. Accessed 17 Nov 2017. Abbreviations
AHP: Allied healthcare provider; CAM: Complementary and alternative
medicine; GP: General practitioner; SAM: Sickness absence management;
WDP: Work disability prevention 3. The Norwegian Labour and Welfare Administration (NAV). Statistics on
certified sickness absence 2017 [in Norwegian]. https://www.nav.no/no/NAV
+og+samfunn/Statistikk/Sykefravar+-+statistikk/Tabeller/legemeldte-
sykefrav%C3%A6rsdagsverk-2-kv-2008-2017-diagnose-og-kj%C3%B8nn. Acceessed 17 Nov 2017. 3. The Norwegian Labour and Welfare Administration (NAV). Statistics on
certified sickness absence 2017 [in Norwegian]. https://www.nav.no/no/NAV
+og+samfunn/Statistikk/Sykefravar+-+statistikk/Tabeller/legemeldte-
sykefrav%C3%A6rsdagsverk-2-kv-2008-2017-diagnose-og-kj%C3%B8nn. Acceessed 17 Nov 2017. References
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Morphometric and allometric relations of cage-reared Iguaçu surubim
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Notas Científicas
Morphometric and allometric relations
of cage-reared Iguaçu surubim Notas Científicas
Morphometric and allometric relations
of cage-reared Iguaçu surubim Aldi Feiden(1), Altevir Signor(1), Wilson Rogério Boscolo(1), Adilson Reidel(2),
Anderson Coldebella(2), Arcangelo Augusto Signor(2) and Sidnei Klein(3) (1)Universidade Estadual do Oeste do Paraná, Grupo de Estudos de Manejo na Aquicultura, Rua da Faculdade, no 645, Jardim Santa
Maria, CEP 85903‑000 Toledo, PR, Brazil. E‑mail: aldifeiden@gmail.com, altevir.signor@gmail.com, wilsonboscolo@hotmail.com (2)Instituto
Federal do Paraná, Avenida Araucária, no 780, Vila A, CEP 85860‑000 Foz do Iguaçu, PR, Brazil. E‑mail: adilson.reidel@ifpr.edu.br,
anderson.coldebella@ifpr.edu.br, angelo_signor@hotmail.com
(3)Instituto Água Viva, Estrada da Usina, Km 05, Tecnoparque,
CEP 85970‑900 Toledo, PR, Brazil. E‑mail: skpesca@hotmail.com Abstract – The objective of this work was to evaluate the morphometric and allometric relations of Iguaçu
surubim (Steindachneridion melanodermatum) cultivated in net cages. One hundred and twenty specimens
were cultivated at a density of 50 fish per square meter in three 6 m³ net cages. Fish were fed three times a
day with commercial feed. Thirty fish were evaluated at 60, 120, 180, and 360 days of cultivation as to the
variables: total body, head, clean trunk, viscera, skin, and fin weight; total, standard, and head length; and head
and body height. The Iguaçu surubim shows later development of the clean trunk and early development of the
other body parts. Pesq. agropec. bras., Brasília, v.48, n.8, p.1154-1158, ago. 2013
DOI: 10.1590/S0100-204X2013000800052 Morphometric and allometric relations of cage-reared Iguaçu surubim length and the different body parts were performed
according to Ricker (1973). The knowledge on the morphometric and allometric
parameters of a species allows to elaborate new
strategies on the industrialization and utilization of
related residues and to improve the understanding of
its biological characteristics. Data were subjected to homogeneity and normality
tests, and to analysis of regression. When significant
differences
were
observed,
Tukey’s
multiple
comparison test, at 1% probability, was applied. Data
were analyzed using the SAS software (SAS Institute,
Cary, NC, USA). The objective of this work was to evaluate the
morphometric and allometric relations of Iguaçu
surubim
(Steindachneridion
melanodermatum)
cultivated in net cages. Regression analysis showed lower incorporation rate
of fat and viscera and greater clean trunk weight in fish
with 360 days of rearing than in those with 180 days
(Table 1). At 60, 120, and 180 days of cultivation, there
was a lower incorporation rate of fat and viscera and a
larger clean trunk in fish subjected to a longer rearing
period. The obtained results indicate that the growth of
the Iguaçu surubim is slow and its productivity is low. The experiment was conducted at the Centro de
Difusão de Desenvolvimento Tecnológico do Rio
Iguaçu, which is a branch of the Grupo de Estudos de
Manejo na Aquicultura (Gemaq), in the municipality
of Boa Vista da Aparecida, PR, Brazil, from January to
December, 2001. A total of 900 Iguaçu surubim fingerlings, with
approximately 60 days of age, average initial weight of
6.77±3.25 g, and average initial length of 8.34±1.63 cm,
were distributed in three cages with volume capacity of
6 m³ at a density of 50 fish per square meter. The fish
were fed ad libitum with commercial feed composed of
different diets, offered at different time slots during the
experiment. The diets contained: 48% crude protein
(CP) and 4,000 kcal of gross energy (GE) per kg of
animal feed during the first 60 days of cultivation;
40% CP between 60 and 180 days of cultivation; and
36% CP between 180 and 360 days of cultivation. This
feed management was used due to the variation in the
nutritional requirements of fish during development. Morphometric parameters showed that the Iguaçu
surubim has excellent characteristics for processing
as a consumable product. Significant reductions were
observed in head, viscera, and visceral fat weight,
along with increases in clean trunk weight. Pesq. agropec. bras., Brasília, v.48, n.8, p.1154-1158, ago. 2013
DOI: 10.1590/S0100-204X2013000800052 Relações morfométricas e alométricas do surubim‑do‑iguaçu
cultivado em tanques‑rede Resumo – O objetivo deste trabalho foi avaliar as relações morfométricas e alométricas do surubim‑do‑iguaçu
(Steindachneridion melanodermatum) cultivado em tanques‑rede. Cento e vinte espécimes foram cultivados
em densidade de 50 peixes por metro quadrado, em três tanques‑rede de 6 m3. Os peixes foram alimentados três
vezes ao dia com ração comercial. Foram avaliados 30 peixes aos 60, 120, 180 e 360 dias de cultivo quanto às
variáveis: peso total do corpo, da cabeça, do tronco limpo, das vísceras, da pele e das nadadeiras; comprimento
total, padrão e da cabeça; e altura da cabeça e do corpo. O surubim‑do‑iguaçu apresenta desenvolvimento
tardio de tronco limpo e precoce das demais partes corporais. Termos para indexação: Steindachneridion melanodermatun, peso corporal, desenvolvimento, processamento,
classe de peso, rendimento. life stages and can be used to assess body development
traits. In addition, the morphometric variables of fish
are useful to evaluate yield characteristics of different
body parts (Meyer, 2009; Karachle & Stergiou, 2012)
and can be used for the selection of species with
potential for cultivation and processing for human
consumption purposes (Cadrin, 2000; Kalayci et al.,
2007). These parameters vary widely between different
animals and are directly related to the size, shape, and
dimensions of the body and to their respective relations
throughout the developmental process (Santos et al.,
2007). The
Iguaçu
surubim
(Steindachneridion
melanodermatum) was considered at risk of extinction
for many years, which lead to repopulation programs
in the region and to studies on the development of
farming systems for this species (Feiden et al., 2006b). It has high growth potential, excellent meat quality,
high market value, and is an interesting alternative
to leverage the development of regional fish farming
(Feiden et al., 2006a). However, its morphometric and
allometric relations are still unknown. According to Santos et al. (2001), allometric studies
explain quantitative differences in the various animals Morphometric and allometric relations of cage-reared Iguaçu surubim 1155 Morphometric and allometric relations of cage-reared Iguaçu surubim Since the negative
heterogonic relationship of the different body parts
presented by this species was directly correlated to the
increase in weight in the clean trunk, which showed a
positive heterogonic relationship during development, the Iguaçu surubim stands out as a potential species for
cultivation, processing, and production of good quality
fish meat. The Iguaçu surubim shows later development of the
clean trunk and early development of the head, viscera,
and visceral fat. In general, it also presents excellent
characteristics for cultivation and processing. Table 1. Bodily parameters of Iguaçu surubim (Steindachneridion melanodermatum) cultivated in net cages(1). Morphometric and allometric relations of cage-reared Iguaçu surubim The Iguaçu
surubim presented early development of the head,
visceral fat, viscera, and skin; however, a late growth
of the trunk. Farmed fish with an early development
of the trunk must be slaughtered earlier than fish with
a later development, which must remain in cultivation
for a longer period of time (Santos et al., 2007). This
indicates that, to achieve high meat yields, the Iguaçu
surubim should be slaughtered after a long cultivation
period. The averages of water physical and chemical
properties, such as pH, electrical conductivity, dissolved
oxygen, and transparency, were 7.78, 31.0 mS cm‑1,
8.7 mg L‑1, and 2.70 m, respectively. These parameters
are suitable for tropical fish farming (Sipaúba‑Tavares
et al., 2003).i The proportional increase in weight and the
percentage of the clean trunk in relation to fish
development are desirable features in farmed fish
(Silva et al., 2009) and are correlated to increased meat
yield (Power et al., 2007). It was observed that, in this
species, the development of the different body parts
was proportional to the growth in weight; however, the
development in length of these same body parts was
smaller than the development in weight. Thirty fish were collected and transported in
rectangular containers, with 500 L capacity and
constant aeration, to the laboratory at Gemaq, at
60, 120, 180, and 360 days of cultivation, for the
evaluation of the morphometric and allometric
relations. Fish were numbed in ice, and measurements
of total weight, total length, head length, head height,
head weight, body height, visceral weight, visceral
fat weight, and clean trunk weight were recorded. These measurements were compared with fish total
weight and total length to determine growth in the
different body parts and their relations with fish body
yield. Allometric evaluations between weight and The instantaneous growth rate ranged from 1.42
to 0.10 in fish cultivated from 60 to 360 days,
respectively. However, the regression equation of the
allometric weight‑length relation indicated annual
growth rate of 0.95. Growth through the weight‑length
relation revealed that this species exhibits negative
allometric development with a coefficient of 2.59. Similarly, the growth in weight of the different body
parts of Iguaçu surubim, in the different phases of Pesq. agropec. bras., Brasília, v.48, n.8, p.1154-1158, ago. 2013
DOI: 10.1590/S0100-204X2013000800052 A. Feiden et al. 1156 A. Feiden et al. the cultivation period, showed negative heterogonic
growth (Figure 1), except for clean trunk weight,
with heterogonic development. )Means±SD followed by equal letters, in the rows, do not differ by Tukey’s test, at 1% probability. Pesq. agropec. bras., Brasília, v.48, n.8, p.1154-1158, ago. 2013
DOI: 10.1590/S0100-204X2013000800052 tters, in the rows, do not differ by Tukey’s test, at 1% probability. Morphometric and allometric relations of cage-reared Iguaçu surubim Evaluated parameters
Evaluation time points
CV
p
60 days
120 days
180 days
360 days
(%)
Average total weight (g)
101.66d
238.97c
366.27b
441.53a
15.19
<0.0001
(±15.24)
(±34.08)
(±49.92)
(±59.13)
Average total length (cm)
18.89d
25.41c
29.29b
33.87a
5.76
<0.0001
(±0.98)
(±1.98)
(±1.30)
(±1.19)
Average standard length (cm)
16.55d
22.17c
25.96b
29.48a
5.84
<0.0001
(±0.78)
(±1.55)
(±1.72)
(±0.92)
Head height (cm)
2.00d
2.58c
2.94b
3.34a
10.26
<0.0001
(±0.30)
(±0.28)
(±0.22)
(±0.21)
Head length (cm)
4.99d
6.17c
6.77b
7.48a
6.48
<0.0001
(±0.31)
(±0.45)
(±0.46)
(±0.35)
Body height (cm)
3.59c
4.59b
5.57a
5.04ab
12.72
<0.0001
(±0.49)
(±0.54)
(±0.62)
(±0.74)
Head weight (g)
23.90d
43.47c
66.96b
85.57a
14.97
<0.0001
(±3.16)
(±6.37)
(±8.86)
(±11.75)
Viscera, skin, and fin weight (g)
25.87c
44.02b
68.64a
72.84a
18.30
<0.0001
(±4.03)
(±6.86)
(±13.84)
(±11.02)
Eviscerated body weight (g)
53.80d
134.68c
232.97b
274.27a
19.36
<0.0001
(±8.50)
(±27.29)
(±43.49)
(±37.89)
Visceral fat weight (g)
10.22c
21.71b
27.04a
23.55ab
23.33
<0.0001
(±2.12)
(±4.81)
(±4.43)
(±7.01)
Rate of length relative to weight (%)
18.88a
10.75b
8.08c
7.76c
11.49
<0.0001
(±2.13)
(±1.02)
(±0.76)
(±0.76)
Rate of head height relative to weight (%)
2.00a
1.10b
0.81c
0.77c
17.32
<0.0001
(±0.34)
(±0.15)
(±0.11)
(±0.09)
Rate of head height relative to length (%)
10.60
10.20
10.06
9.87
11.09
<0.1135
(±1.48)
(±1.09)
(±0.82)
(±0.67)
Rate of body height relative to weight (%)
3.56a
1.93b
1.53c
1.15d
10.72
<0.0001
(±0.35)
(±0.18)
(±0.12)
(±0.16)
Rate of body height relative to length (%)
18.98a
18.97a
18.06a
14.88b
10.49
<0.0033
(±2.03)
(±1.78)
(±1.57)
(±2.15)
Rate of head length relative to weight (%)
5.00a
2.61b
2.07c
1.55d
14.09
<0.0001
(±0.69)
(±0.27)
(±0.23)
(±0.14)
Rate of visceral weight relative to weight (%)
25.67a
18.45b
18.73b
16.58b
12.62
<0.0001
(±3.45)
(±1.70)
(±2.56)
(±2.06)
Rate of head weight relative to total weight (%)
23.68a
18.20b
18.43b
19.45b
9.61
<0.0001
(±2.29)
(±1.28)
(±2.42)
(±1.65)
Rate of the clean trunk relative to total weight (%)
52.91c
56.00bc
64.11a
62.14ab
11.94
<0.0007
(±2.38)
(±6.69)
(±12.36)
(±2.74)
Rate of visceral fat relative to total weight (%)
10.11a
9.04a
7.47b
5.32c
18.01
<0.0001
(±1.87)
(±1.31)
(±1.39)
(±1.32)
Daily growth (g)
1.69b
1.99a
2.03a
1.23c
14.46
<0.0001
(±0.25)
(±0.57)
(±0.83)
(±0.31)
(1)Means±SD followed by equal letters, in the rows, do not differ by Tukey’s test, at 1% probability. Iguaçu surubim (Steindachneridion melanodermatum) cultivated in net cages(1). Morphometric and allometric relations of cage-reared Iguaçu surubim
1157 Morphometric and allometric relations of cage-reared Iguaçu surubim 1157 Figure 1. Morphometric and allometric relations of cage-reared Iguaçu surubim 0
1
2
3
4
0
100
200
300
400
500
600
700
Weight (g)
Head height (cm)
0
2
4
6
8
10
0
100
200
300
400
500
600
700
Weight (g)
Head lenght (cm)
y = 0.255x + 0.4315 R = 0.8274
2
y = 0.3268x - 0.8284 R = 0.7781
2
0
1
2
3
4
5
6
7
8
0
100
200
300
400
500
600
700
Weight (g)
0
20
40
60
80
100
120
0
100
200
300
400
500
600
700
Weight (g)
Body height (cm)
Visceral weight (g)
y = 0.7226x - 0.1151 R = 0.918
2
y = 0.2996x - 0.1206 R = 0.7258
2
0
20
40
60
80
100
120
0
100
200
300
400
500
600
700
Weight (g)
0
100
200
300
400
0
100
200
300
400
500
600
700
Weight (g)
y = 0.8247x - 0.6687 R = 0.9526
2
y = 1.1151x - 1.1799 R = 0.9688
2
Head weight (g)
Eviscerated body weight (g)
15
115
215
315
415
515
615
15
20
25
30
35
40
Length (cm)
0
10
20
30
40
50
0
100
200
300
400
500
600
700
Weight (g)
Weight (g)
Visceral fat weight (g)
y = 2.5934x - 2.9558 R = 0.956
2
y = 0.678x - 0.7751 R = 0.7638
2 0
1
2
3
4
0
100
200
300
400
500
600
700
Weight (g)
Head height (cm)
y = 0.3268x - 0.8284 R = 0.7781
2 0
2
4
6
8
10
0
100
200
300
400
500
600
700
Weight (g)
Head lenght (cm)
y = 0.255x + 0.4315 R = 0.8274
2 0
2
4
6
8
10
0
100
200
300
400
500
600
700
Weight (g)
Head lenght (cm)
y = 0.255x + 0.4315 R = 0.8274
2
0
1
2
3
4
5
6
7
8
0
100
200
300
400
500
600
700
W i ht ( )
Body height (cm)
y = 0.2996x - 0.1206 R = 0.7258
2 Head lenght (cm) Head lenght (cm) Weight (g)
0
20
40
60
80
100
120
0
100
200
300
400
500
600
700
Weight (g)
Visceral weight (g)
y = 0.7226x - 0.1151 R = 0.918
2 Weight (g) Weight (g) Weight (g)
0
20
40
60
80
100
120
0
100
200
300
400
500
600
700
Weight (g)
y = 0.8247x - 0.6687 R = 0.9526
2
Head weight (g) Weight (g)
0
100
200
300
400
0
100
200
300
400
500
600
700
Weight (g)
y = 1.1151x - 1.1799 R = 0.9688
2
Eviscerated body weight (g) Eviscerated body weight (g) Weight (g) Weight (g)
0
10
20
30
40
50
0
100
200
300
400
500
600
700
Weight (g)
Visceral fat weight (g)
y = 0.678x - 0.7751 R = 0.7638
2 Weight (g)
15
115
215
315
415
515
615
15
20
25
30
35
40
Length (cm)
Weight (g)
y = 2.5934x - 2.9558 R = 0.956
2 Figure 1. Morphometric and allometric relations of cage-reared Iguaçu surubim Allometric relations of Iguaçu surubim (Steindachneridion melanodermatum) cultivated in net cage systems during
one year. Pesq. agropec. bras., Brasília, v.48, n.8, p.1154-1158, ago. 2013
DOI: 10.1590/S0100-204X2013000800052 Acknowledgements To Fundação Araucária from the government of the
state of Paraná, Brazil, for financial support. POWER, J.H.; BURGER, M.J.; STOKES, A.M. Mass, volume, and
length relationships in plaice (Pleuronectes platessa L.) juveniles. Journal of Sea Research, v.57, p.230‑235, 2007. DOI: 10.1016/j. seares.2006.09.001. POWER, J.H.; BURGER, M.J.; STOKES, A.M. Mass, volume, and
length relationships in plaice (Pleuronectes platessa L.) juveniles. Pesq. agropec. bras., Brasília, v.48, n.8, p.1154-1158, ago. 2013
DOI: 10.1590/S0100-204X2013000800052 Morphometric and allometric relations of cage-reared Iguaçu surubim Allometric relations of Iguaçu surubim (Steindachneridion melanodermatum) cultivated in net cage systems during
one year. Pesq. agropec. bras., Brasília, v.48, n.8, p.1154-1158, ago. 2013
DOI: 10.1590/S0100-204X2013000800052 ,
,
,
, p
, g
DOI: 10.1590/S0100-204X2013000800052 1158 A. Feiden et al. Acknowledgements MEYER, A. Morphometrics and allometry in the trophically
polymorphic cichlid fish, Cichlasoma citrinellum: alternative
adaptations and ontogenetic changes in shape. Zoology, v.221,
p.237‑260, 2009. Received on January 29, 2012 and accepted on July 4, 2013 References Journal of Sea Research, v.57, p.230‑235, 2007. DOI: 10.1016/j. seares.2006.09.001. CADRIN, S.X. Advances in morphometric identification of fishery
stocks. Reviews in Fish Biology and Fisheries, v.10, p.91‑112,
2000. DOI: 10.1023/A:1008939104413. RICKER, W.E. Linear regressions in fishery research. Journal of
the Fisheries Research Board of Canada, v.30, p.409‑434, 1973. DOI: 10.1139/f73‑072. FEIDEN, A.; HAYASHI, C.; BOSCOLO, W.R. Desenvolvimento
de
larvas
de
surubim‑do‑iguaçu
(Steindachneridion
melanodermatum) submetidas a diferentes dietas. Revista
Brasileira de Zootecnia, v.35, p.2203‑2210, 2006a. DOI: 10.1590/
S1516‑35982006000800002. SANTOS, C.L.; PEREZ, J.R.O.; GERASEEV, L.C.; PRADO,
O.V.; MUNIZ, J.A. Estudo do crescimento alométrico dos cortes
de carcaça de cordeiros da raça Santa Inês e Bergamácia. Ciência
e Agrotecnologia, v.25, p.149‑158, 2001. SANTOS, V.B. dos; FREITAS, R.T.F. de; LOGATO, P.V.R.;
FREATO, T.A.; ORFÃO, L.H.; MILLIOTI, L.C. Rendimento do
processamento de linhagens de tilápias (Oreochromis niloticus)
em função do peso corporal. Ciência e Agrotecnologia, v.31,
p.554‑562, 2007. DOI: 10.1590/S1413‑70542007000200041. FEIDEN, A.; HAYASHI, C.; BOSCOLO, W.R.; REIDEL,
A. Desenvolvimento de larvas de Steindachneridion sp. em
diferentes condições de refúgio e luminosidade. Pesquisa
Agropecuária Brasileira, v.41, p.133‑137, 2006b. DOI: 10.1590/
S0100‑204X2006000100018. SILVA, F.V. e; SARMENTO, N.L. de A.F. e; VIEIRA, J.S.;
TESSITORE, A.J. de A.; OLIVEIRA, L.L. dos S.; SARAIVA,
E.P. Características morfométricas, rendimentos de carcaça, filé,
vísceras e resíduos em tilápias‑do‑nilo em diferentes faixas de
peso. Revista Brasileira de Zootecnia, v.38, p.1407‑1412, 2009. DOI: 10.1590/S1516‑35982009000800003. KALAYCI, F.; SAMSUN, N.; BILGIN, S.; SMSUN, O. Length‑weight relationship of 10 fish species caught by bottom
trawl and midwater trawl from the Middle Black Sea, Turkey. Turkish Journal of Fisheries and Aquatic Sciences, v.7, p.33‑36,
2007. KALAYCI, F.; SAMSUN, N.; BILGIN, S.; SMSUN, O. Length‑weight relationship of 10 fish species caught by bottom
trawl and midwater trawl from the Middle Black Sea, Turkey. y
Turkish Journal of Fisheries and Aquatic Sciences, v.7, p.33‑36,
2007. SIPAÚBA‑TAVARES, L.H.; GOMES, J.P.F.; BRAGA, F.M. de S. Effect of liming management on the water quality in
Colossoma macropomum (“Tambaqui”) ponds. Acta Limnologica
Brasiliensia, v.15, p.95‑103, 2003. KARACHLE, P.K; STERGIOU, K.I. Morphometrics and allometry
in fish. In: WAHL, C.M. Morphometrics. New York: Cornell
University, 2012. p.65‑68. (Agricultural and biological science). DOI: 10.5772/34529. Received on January 29, 2012 and accepted on July 4, 2013
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English
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Cytoplasmic Retention of a Nucleocytoplasmic Protein TBC1D3 by Microtubule Network Is Required for Enhanced EGFR Signaling
|
PloS one
| 2,014
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cc-by
| 10,612
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Abstract This is an open-access article distributed under the terms of the Creative Commons Attribution License,
use, distribution, and reproduction in any medium, provided the original author and source are credited. supported by grants (No. 81272261, No. 81071803, and No. 30971144) from the National Natural Science Foundation of China (http://
unders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Funding: This work was supported by grants (No. 81272261, No. 81071803, and No. 30971144) from the National Natural Science F
www.nsfc.gov.cn/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the Competing Interests: The authors have declared that no competing interests exist. * E-mail: shencl37@163.com . These authors contributed equally to this work. . These authors contributed equally to this work. . These authors contributed equally to this work. ubiquitination [4,5]. There appears to be two distinct mechanisms
underlying the ubiquitination of EGFR by Cbl. The first one is
mediated by the C-terminal phosphorylated tyrosine residue Tyr
1045 of EGFR, which directly binds to the N-terminal tyrosine
kinase binding domain of Cbl [6,7]. The alternative mechanism
involves the tyrosine residues Tyr 1068 and Tyr 1086 of the
activated receptor, which indirectly recruits Cbl through its
interaction with the SH3 domain of Grb2 [8]. With Cbl serving
as an adaptor to bridge Ubc4/5 E2 ubiquitin-conjugating enzyme
interaction with EGFR, ubiquitin is transferred directly from the
E2 to distinct lysine residues within the kinase domain of EGFR,
including six major ubiquitin conjugation sites (Lys692, Lys713,
Lys, 730, Lys843, Lys905, and Lys946) [9,10]. Although sufficient
but not essential for EGFR internalization [11–13], EGFR
ubiquitination is indeed required for its sorting onto intraluminal
vesicles of multivesicular endosomes/bodies and subsequent
lysosomes for efficient degradation [12,14]. Cytoplasmic Retention of a Nucleocytoplasmic Protein
TBC1D3 by Microtubule Network Is Required for
Enhanced EGFR Signaling Ze He1,2., Tian Tian1., Dan Guo1, Huijuan Wu1, Yang Chen1, Yongchen Zhang1, Qing Wan1, Huzi Zhao1,
Congyang Wang1, Hongjing Shen3, Lei Zhao1, Xiaodong Bu1, Meiling Wan1, Chuanlu Shen1* 1 Department of Pathology and Pathophysiology, Medical School, Southeast University, Nanjing, Jiangsu, People’s Republic of China, 2 Key Laboratory of Developmental
Genes and Human Diseases, Ministry of Education, Institute of Life Sciences, Southeast University, Nanjing, Jiangsu, People’s Republic of China, 3 Department of
Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, New York, United States of America Abstract The hominoid oncogene TBC1D3 enhances epidermal growth factor receptor (EGFR) signaling and induces cell
transformation. However, little is known regarding its spatio-temporal regulation and mechanism of tumorigenesis. In
the current study, we identified the microtubule subunit b-tubulin as a potential interaction partner for TBC1D3 using
affinity purification combined with mass spectrometry analysis. The interaction between TBC1D3 and b-tubulin was
confirmed by co-immunoprecipitation. Using the same method, we also revealed that TBC1D3 co-precipitated with
endogenous a-tubulin, another subunit of the microtubule. In agreement with these results, microtubule cosedimentation
assays showed that TBC1D3 associated with the microtubule network. The b-tubulin-interacting site of TBC1D3 was
mapped to amino acids 286,353 near the C-terminus of the TBC domain. Deletion mutation within these amino acids was
shown to abolish the interaction of TBC1D3 with b-tubulin. Interestingly, the deletion mutation caused a complete loss of
TBC1D3 from the cytoplasmic filamentous and punctate structures, and TBC1D3 instead appeared in the nucleus. Consistent with this, wild-type TBC1D3 exhibited the same nucleocytoplasmic distribution in cells treated with the
microtubule depolymerizing agent nocodazole, suggesting that the microtubule network associates with and retains
TBC1D3 in the cytoplasm. We further found that deficiency in b-tubulin-interacting resulted in TBC1D3’s inability to inhibit
c-Cbl recruitment and EGFR ubiquitination, ultimately leading to dysregulation of EGFR degradation and signaling. Taken
together, these studies indicate a novel model by which the microtubule network regulates EGFR stability and signaling
through tubulin dimer/oligomer interaction with the nucleocytoplasmic protein TBC1D3. Citation: He Z, Tian T, Guo D, Wu H, Chen Y, et al. (2014) Cytoplasmic Retention of a Nucleocytoplasmic Protein TBC1D3 by Microtubule Network Is Required for
Enhanced EGFR Signaling. PLoS ONE 9(4): e94134. doi:10.1371/journal.pone.0094134
Edi
Y
H
S
U i
i
f L
b
k G uo D, Wu H, Chen Y, et al. (2014) Cytoplasmic Retention of a Nucleocytoplasmic Protein TBC1D3 by Microtubule Network Is Required for
. PLoS ONE 9(4): e94134. doi:10.1371/journal.pone.0094134 Received December 25, 2012; Accepted March 13, 2014; Published April 8, 2014 Copyright: 2014 He et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: 2014 He et al. Citation: He Z, Tian T, Guo D, Wu H, Chen Y, et al. (2014) Cytoplasmic Retention of a Nucleocytoplasmic Protein TBC1D3 by Microtubule Network Is Required for
Enhanced EGFR Signaling. PLoS ONE 9(4): e94134. doi:10.1371/journal.pone.0094134 Microtubule Network Regulates TBC1D3-Enhanced EGFR Signaling Diverse negative or positive regulators of EGFR ubiquitination
have been identified, including Sprouty2, Cdc42, intersectin-1,
protein-tyrosine kinase 6 (PTK6) and TBC1D3. Among them,
phosphorylated Sprouty2 and activated Cdc42, in complex with
the Cbl-interacting protein of 85 kDa and Cool-1, respectively,
negatively regulates the ubiquitination of EGFR through seques-
tration of Cbl away from the activated receptor [15–17]. Conversely, the multi-domain scaffolding protein intersectin-1
stimulates its ubiquitination through competitively inhibiting
Sprouty2 from binding to Cbl [18]. In contrast to association
with Cbl, PTK6, a non-receptor protein-tyrosine kinase competes
with Cbl for binding to the phosphorylated Y1045 on EGFR,
leading to disruption of EGFR-Cbl interaction and inhibition of
EGFR ubiquitination [19]. Signaling Technology, antibody against c-Cbl (#21549) from
Signalway Antibody Co., Ltd., antibodies against a-tublin (T9026)
and acetylated a-tublin (T7451) from Sigma, antibodies against
GAPDH (AP0063) and HDAC6 (BS1165) from Bioworld Tech-
nology, Inc. and anti-ubiquitin (Ub) antibody (FK2) from Enzo
Life Sciences. The secondary antibodies used were Rhodamine (TRITC)-
conjugated goat anti-mouse immunoglobulin G (IgG; heavy and
light chain; Jackson Immunoresearch Laboratories) and DyLight
594 AffiniPure goat anti-rabbit immunoglobulin G (IgG; heavy
and light chain; EarthOx, LLC). Cell Culture and Transfections Human SMMC7721 hepatocellular carcinoma cells were
obtained from the Committee on Type Culture Collection of
Chinese Academy of Sciences (Shanghai, China) and grown in
Dulbecco’s
Modified
Eagle’s
Medium
(DMEM,
Invitrogen)
containing 2 mM of glutamine and supplemented with 10%
heat-inactivated fetal bovine serum, 100 U/ml of penicillin and
100 mg/ml of streptomycin at 37uC in 5% CO2. SMMC7721 cells
were transfected with LipoD293 Reagent (SignaGen) according to
manufacturer’s instructions. Approximately 24 h after transfec-
tion, cells were processed as described for each experiment. Human SMMC7721 hepatocellular carcinoma cells were
obtained from the Committee on Type Culture Collection of
Chinese Academy of Sciences (Shanghai, China) and grown in
Dulbecco’s
Modified
Eagle’s
Medium
(DMEM,
Invitrogen)
containing 2 mM of glutamine and supplemented with 10%
heat-inactivated fetal bovine serum, 100 U/ml of penicillin and
100 mg/ml of streptomycin at 37uC in 5% CO2. SMMC7721 cells
were transfected with LipoD293 Reagent (SignaGen) according to
manufacturer’s instructions. Approximately 24 h after transfec-
tion, cells were processed as described for each experiment. In the current study, we report that tubulin/microtubule
interacts with TBC1D3. We further identify TBC1D3 as a
nucleocytoplasmic protein and its association with tubulin/
microtubule regulates its nucleocytoplasmic distribution. Finally,
our work reveals a novel model by which microtubule network
regulates EGFR stability and signaling through tubulin dimer/
oligomer interaction with TBC1D3. GGA3 forward primer, 59-GAGAATTCATGGCGGAGGCG-
GAAGG-39; GGA3
reverse
primer,
59-CTCTCGAGTCA-
TAGGTTCCCCCACTGTTC-39. After their sequences were confirmed by direct automated DNA
sequencing, the entire open reading frame (ORF) of TBC1D3 was
inserted into BamH I/EcoR I site of HA-pcDNA3.0 and pEBG
vectors to generate HA- and GST-tagged fusion proteins (HA-
TBC1D3 and GST-TBC1D3), respectively. The entire ORF of
GGA3 was ligated into EcoR I/Xho I site of pGEX-6P-1 vector to
generate a GST-tagged fusion protein (GST-GGA3). pEGFP-
TBC1D3 encodes a EGFP-tagged TBC1D3 protein (EGFP-
TBC1D3) and was generated by PCR. HA-TBC1D3(D252-285)/
pcDNA3,
HA-TBC1D3(D286-319)/pcDNA3,
HA-
TBC1D3(D320-353)/pcDNA3,
and
pEGFP-TBC1D3(D320-
353), harboring internal deletions of the indicated residues, were
generated
by
overlap
extension
PCR. HA-TBC1D3(251)/
pcDNA3,
HA-TBC1D3(353)/pcDNA3,
HA-TBC1D3(476)/
pcDNA3,
pEGFP-TBC1D3(251),
pEGFP-TBC1D3(353),
pEGFP-TBC1D3(476) and pGEX-GGA3(298) encode the indi-
cated N-terminal residues of the corresponding genes and were
generated by PCR. Further details are available upon request. After their sequences were confirmed by direct automated DNA
sequencing, the entire open reading frame (ORF) of TBC1D3 was
inserted into BamH I/EcoR I site of HA-pcDNA3.0 and pEBG
vectors to generate HA- and GST-tagged fusion proteins (HA-
TBC1D3 and GST-TBC1D3), respectively. The entire ORF of
GGA3 was ligated into EcoR I/Xho I site of pGEX-6P-1 vector to
generate a GST-tagged fusion protein (GST-GGA3). pEGFP-
TBC1D3 encodes a EGFP-tagged TBC1D3 protein (EGFP-
TBC1D3) and was generated by PCR. HA-TBC1D3(D252-285)/
pcDNA3,
HA-TBC1D3(D286-319)/pcDNA3,
HA-
TBC1D3(D320-353)/pcDNA3,
and
pEGFP-TBC1D3(D320-
353), harboring internal deletions of the indicated residues, were
generated
by
overlap
extension
PCR. HA-TBC1D3(251)/
pcDNA3,
HA-TBC1D3(353)/pcDNA3,
HA-TBC1D3(476)/
pcDNA3,
pEGFP-TBC1D3(251),
pEGFP-TBC1D3(353),
pEGFP-TBC1D3(476) and pGEX-GGA3(298) encode the indi-
cated N-terminal residues of the corresponding genes and were
generated by PCR. Further details are available upon request. TBC1D3 reverse primer, 59-GCGAATTCTAGAAGCCTG-
GAGGGAACTG-39; GGA3 forward primer, 59-GAGAATTCATGGCGGAGGCG-
GAAGG-39; Introduction The epidermal growth factor receptor (EGFR) is the first
identified member of the ErbB receptor tyrosine kinase family. The receptor activates a wide variety of signaling pathways, with
the Ras-ERK pathway as perhaps the best characterized of these
pathways. EGFR signaling controls numerous critical cellular
processes, such as cell survival, proliferation, differentiation and
locomotion [1,2]. After activation, the receptor must be inactivat-
ed to prevent prolonged stimulation of cells via feedback control
mechanisms, including activation of phosphatases, post-transla-
tional modifications and endocytosis of the receptor. Excessive
activation of EGFR has been associated with the development and
progression of numerous tumors [3]. As one of the most common post-translational modifications,
ubiquitination of EGFR plays a critical role in endocytic
trafficking and lysosomal degradation of the activated receptor. Cbl is a RING domain E3 ubiquitin ligase responsible for EGFR 1 April 2014 | Volume 9 | Issue 4 | e94134 PLOS ONE | www.plosone.org TBC1D3 forward primer, 59-TAGGATCCATGGACGTGG-
TAGAGGTCGC-39; TBC1D3 forward primer, 59-TAGGATCCATGGACGTGG-
TAGAGGTCGC-39; TBC1D3 reverse primer, 59-GCGAATTCTAGAAGCCTG-
GAGGGAACTG-39; TBC1D3 reverse primer, 59-GCGAATTCTAGAAGCCTG-
GAGGGAACTG-39; Plasmids Human TBC1D3 and GGA3 cDNAs were amplified by reverse
transcription-PCR from total RNA using the following primers: Human TBC1D3 and GGA3 cDNAs were amplified by reverse
transcription-PCR from total RNA using the following primers: TBC1D3 (also called prostate cancer gene 17, PRC17) is a
hominoid-specific gene that was originally identified as a novel
amplified oncogene, based on its ability to confer tumorigenicity to
NIH 3T3 cells [20–22]. The oncogene is amplified in 15% of
primary prostate tumors and in approximately 50% of metastatic
prostate tumors [22]. Consistent with many other hominoid-
specific genes, TBC1D3 underwent several segmental duplications
during primate evolution, resulting in the existence of eight
paralogues organized in two clusters within the 17q12 genomic
region [23]. TBC1D3 possesses an N-terminal TBC (Tre-2, Bub2,
Cdc16) domain, which generally encodes GTPase-activating
proteins (GAPs) for Rab family GTPases [24]. However, no
GAP activity could be detected because the TBC domain of
TBC1D3 lacks the conserved arginine and glutamine residues
essential for catalytic activity [25]. Instead, TBC1D3 promotes
EGF and insulin signaling by inhibiting or substantially delaying
ubiquitination and subsequent degradation of EGFR and insulin
receptor substrate-1 (IRS-1), respectively, thereby triggering cell
proliferation [26,27]. The delay in IRS-1 ubiquitination has been
linked to TBC1D3-induced activation of protein phosphatase 2A
(PP2A). Following the activation, PP2A dephosphorylates and
inactivates p70 S6 kinase (S6K), leading to suppression of
phosphorylation at key sites required for IRS-1 binding to Cul7
E3 ubiquitin ligase [27]. Similarly, TBC1D3 reduces EGFR
ubiquitination through suppression of Cbl recruitment and
binding to the receptor [26]. Most recently, TBC1D3 was shown
to be ubiquitinated and degraded by the Cul7 E3 ligase in
response to serum stimulation [28]. However, little is known
regarding the mechanism by which TBC1D3 inhibits the
recruitment of Cbl. Furthermore, spatio-temporal regulation of
TBC1D3 remains undefined. Antibodies Erk1/2 activation and EGFR degradation were measured in
cells serum-starved for 24 h. Cells were incubated in the presence
of 100 ng/ml EGF (PeproTech) at 37uC for different time points,
washed with ice-cold phosphate-buffered saline (PBS), and lysed in
RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.1% SDS,
0.5% sodium deoxycholate, 1% TritonX-100, 1 mM phenyl-
methylsulfonyl fluoride, 5 mg/ml leupeptin, 2 mg/ml aprotinin,
5 mg/ml pepstatin, 1 mM sodium orthovanadate, 10 mM sodium
fluoride). The lysates were clarified by centrifugation, and protein For immunofluorescence of b-tubulin, anti-b-tubulin antibody
(E021040, Earthox) was used; for immunoprecipitation, anti-HA
(sc-805, Santa Cruz) and anti-b-tubulin (#21335, Signalway
Antibody) antibodies were used. Anti-EGFR antibody (#21074,
Signalway Antibody) was used for both immunofluorescence and
immunoprecipitation. For immunoblotting, antibodies against
EGFR (#4267), HA (#2367), p44/42 MAPK (#9107), and
phospho-p44/42 MAPK (#9106) were purchased from Cell April 2014 | Volume 9 | Issue 4 | e94134 PLOS ONE | www.plosone.org 2 Microtubule Network Regulates TBC1D3-Enhanced EGFR Signaling Microtubule Network Regulates TBC1D3-Enhanced EGFR Signaling aliquots of tubulin (100 mg of protein dissolved in General Tubulin
Buffer containing 1 mM GTP) and incubated at 35uC for 20 min. Newly formed microtubules were stabilized by the addition of
200 ml of General Tubulin Buffer containing 20 mM Taxol. Clarified lysates were then added to the Taxol supplemented
General Tubulin Buffer alone (MTs(-)) or together with polymer-
ized microtubules (MTs(+)). The microtubule binding protein
fraction (MAPF, containing MAP2 and tau proteins) and BSA
were used as positive and negative controls, respectively. After
30 min of incubation at room temperature, samples were added
on top of the Cushion Buffer containing Taxol and centrifuged at
100,0006g for 40 min at room temperature. The pellet (P) and
supernatant (S) fractions were collected and separated by SDS-
PAGE. The blots were then stained with Ponceau S followed by
Western blotting with the anti-HA antibody or with Coomassie
brilliant blue (MAP2 and BSA controls). concentrations were determined using the Bradford assay. Peptide Mass Fingerprinting Analysis Peptide Mass Fingerprinting Analysis
SMMC7721 cells were transfected with GST-TBC1D3 or
control GST. The following day, cell extracts were pulled down
(PD) with glutathione-Sepharose 4B beads. After SDS PAGE, the
gels were stained with Coomassie brilliant blue. The differentially
bound protein bands were then excised from the SDS PAGE gel,
in-gel digested by trypsin, and analyzed by Shanghai GeneCore
BioTechnologies Co., Ltd. using the Proteomics Solution 1 System
(Applied Biosystems Co., Ltd., USA). To identify the proteins,
peptide masses from MALDI-TOF MS were matched with the
theoretical molecular weight of peptides for proteins in the NCBI
database using MASCOT search tools (www.matrixscience.com). SMMC7721 cells were seeded at 26103 cells per well in 96-well
plates
and
transfected
with
HA-TBC1D3,
HA-
TBC1D3(320,353) and control HA vector, respectively. After
the indicated times, complete medium was replaced with 90 ml of
fresh serum-free DMEM medium and 10 ml of Cell Counting Kit-
8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan) was added
to each well. After incubation in the dark at 37uC for 1 hour, the
absorbance was measured with excitation at 450 nm (OD450)
using the ELISAmate spectrophotometer (MULTISKAN MK3,
Thermo Labsystems). Microtubule Cosedimentation Assays Results were expressed as mean values 6 standard deviation
(mean 6 SD). One-way analysis of variance (ANOVA) was
performed to calculate statistical significance. After transfected with HA-TBC1D3, SMMC7721 cells were
scraped in 0.5 ml of General Tubulin Buffer (80 mM PIPES,
pH 7.0, 2 mM MgCl2, 1 mM EGTA) supplemented with 1 mM
GTP, 1 mM phenylmethylsulfonyl fluoride, 5 mg/ml leupeptin,
2 mg/ml aprotinin, 5 mg/ml pepstatin, 1 mM sodium orthovana-
date, 10 mM sodium fluoride, collected and then sonicated 5 times
at 4uC for 15 seconds with a 1 minute cooling period between each
burst. The lysates were centrifuged at 100,0006g for 40 min at
4uC to remove any microtubules. Clarified lysates were then used
for microtubule binding protein spin-down assays using a
commercially available kit (Cytoskeleton Inc. Denver, CO, cat.#
BK029). Briefly, 2 ml of Cushion Buffer (80 mM PIPES, pH 7.0,
1 mM MgCl2, 1 mM EGTA, 60% glycerol) was added to 20 ml GST Pull-down and Co-immunoprecipitation GST Pull down and Co immunoprecipitation
Cells were lysed in ice-cold lysis buffer (20 mM Tris-Cl, pH 7.5,
0.5% Triton X-100, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA,
10 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM
phenylmethylsulfonyl fluoride, 5 mg/ml pepstatin, 5 mg/ml leu-
peptin, 2 mg/ml aprotinin). For analysis of EGFR ubiquitination,
additional 10 mM N-ethylmaleimide was added into lysis buffer. After incubation on ice for 15 min, the lysates were centrifuged at
13,000 rpm for 10 min at 4uC. For GST pull-down, an aliquot of
the clarified supernatant was removed for direct immunoblotting,
and the remainder was incubated with GST, GST-TBC1D3, or
GST-GGA3(298) fusion proteins attached to the glutathione-
Sepharose beads (Amersham) for 4 h at 4uC with constant mixing. After centrifugation, the beads were washed three times in the lysis
buffer. Samples were boiled, fractionated by SDS PAGE, stained
with 1% Coomassie brilliant blue or Ponceau S followed by
immunoblotting with anti-HA antibody, and then detected by
enhanced chemiluminescence (Pierce). For co-immunoprecipita-
tion, the clarified lysates were incubated with the indicated
antibodies for 1 h and then with Protein G PLUS-Agarose beads
(Santa Cruz) for 4 h or overnight at 4uC. Beads were washed three
times in the lysis buffer. Samples were separated by SDS PAGE
and then immunoblotted with the indicated antibodies. Antibodies Proteins
were separated by SDS PAGE and electroblotted onto a 0.45-mm
pore PVDF membrane, which was blocked in 2% bovine serum
albumin in TBST buffer (25 mM Tris [pH 7.4], 150 mM NaCl,
0.05% Tween 20), probed with anti-EGFR, anti-p44/42 MAPK,
anti-phospho-p44/42 MAPK, anti-HA and anti-a-tubulin anti-
bodies, washed with TBST and incubated with horseradish
peroxidase-conjugated goat anti-rabbit IgG (Santa Cruz) or goat
anti-mouse IgG (Bioworld) for detection by SuperSignal West Pico
Chemiluminescent Substrate (Pierce). Immunoblotting data were
quantified by Image-Pro Plus 6.0 software (Media Cybernetics,
Inc.). Confocal Immunofluorescence Microscopy Cells grown on coverslips were fixed in 4% paraformaldehyde
in PBS for 15 min at room temperature and then washed three
times in PBS carefully. Cells were permeabilized in PBS
containing 0.25% Triton X-100 (PBST) for 10 min followed by
blocking for 30 min in PBS containing 1% BSA at room
temperature. Coverslips were incubated for 2 h with anti-b-
tubulin and/or anti-EGFR antibodies at room temperature and
then washed three times in PBST. Samples were incubated with
the secondary antibodies Rhodamine (TRITC)-conjugated goat
anti-mouse immunoglobulin G (Jackson) and/or DyLight 594
AffiniPure goat anti-rabbit immunoglobulin G (Earthox) for 1 h at
room temperature and then washed three times in PBST. After
incubation with 5 mg/ml Hoechst 33258 (sigma) for 5 min at room
temperature, coverslips were mounted in ProLong Gold antifade
reagent (Invitrogen). Confocal microscopy images were acquired
using an Olympus FluoView FV1000 confocal laser scanning
microscope (Olympus, Tokyo, Japan) equipped with an oil-
immersion Plan apochromat 1.4-NA 6100 objective lens. TBC1D3 interacts with b-tubulin Cell extracts were pulled dow
glutathione-Sepharose 4B beads (PD), washed, resolved by 10% SDS-PAGE and then stained with Coomassie brilliant blue. The bait protei
(GST-TBC1D3 or control GST) and b-tubulin, one of the TBC1D3-interacting proteins identified by mass spectrometry, are indicated by arrow
rectangle, respectively. Molecular weight markers (Marker) are shown on left in kDa. WCL, whole cell lysate. (B and C) SMMC7721 cells were se
three 60-mm dishes. The following day, cells were transfected with HA-tagged TBC1D3 (HA-TBC1D3) (dishes 1 and 3) or control HA vector (
(dish 2). Lysates were immunoprecipitated (IP) with control immunoglobulin G (IgG) (dish 1 in B and C), anti-HA (dishes 2 and 3 in B) or anti-b-
(dishes 2 and 3 in C) antibodies. After SDS PAGE and transfer, the blots were stained with Ponceau S (bottom panels) prior to immunoblott
with anti-HA and anti-b-tubulin antibodies (top panels). (D) SMMC7721 cells were transfected with EGFP-tagged TBC1D3 (top left, green). Ce
subjected to indirect immunofluorescence with anti-b-tubulin antibody (top central, red), and then analyzed by confocal micro
Immunofluorescent images were merged (top right), with yellow revealing overlap (Merge). The indicated areas of the outlined boxes in t
panels are shown in higher magnification in the bottom panels. Arrows indicate co-localizations between TBC1D3 and b-tubulin on filam
structures. Scale bar, 10 mm. doi:10.1371/journal.pone.0094134.g001 Figure 1. TBC1D3 interacts with b-tubulin. (A) A representative image of the resolved protein eluate following the GST pull-down assay. SMMC7721 cells were transfected with GST-tagged TBC1D3 (GST-TBC1D3) or control GST vector (GST). Cell extracts were pulled down with
glutathione-Sepharose 4B beads (PD), washed, resolved by 10% SDS-PAGE and then stained with Coomassie brilliant blue. The bait protein band
(GST-TBC1D3 or control GST) and b-tubulin, one of the TBC1D3-interacting proteins identified by mass spectrometry, are indicated by arrows and a
rectangle, respectively. Molecular weight markers (Marker) are shown on left in kDa. WCL, whole cell lysate. (B and C) SMMC7721 cells were seeded in
three 60-mm dishes. The following day, cells were transfected with HA-tagged TBC1D3 (HA-TBC1D3) (dishes 1 and 3) or control HA vector (Vector)
(dish 2). Lysates were immunoprecipitated (IP) with control immunoglobulin G (IgG) (dish 1 in B and C), anti-HA (dishes 2 and 3 in B) or anti-b-tubulin
(dishes 2 and 3 in C) antibodies. TBC1D3 interacts with b-tubulin To gain insights into TBC1D3 function and regulation, we
sought to identify its interaction partners using a GST pull-down
assay combined with mass spectrometry analysis. SMMC7721
cells were transfected with GST-tagged fusion of human TBC1D3
(GST-TBC1D3) and control GST alone (GST), which were used
as bait proteins to screen prey proteins composed of total cellular
proteins. When the pattern of protein bands was compared, some April 2014 | Volume 9 | Issue 4 | e94134 PLOS ONE | www.plosone.org 3 Microtubule Network Regulates TBC1D3-Enhanced EGFR Signaling bands were unique to the GST-TBC1D3 but not GST interacting
assays on Coomassie brilliant blue-stained gel (Figure 1A). The
differentially bound protein bands were analyzed by using a
MALDI-TOF mass spectrometer and several interacting proteins
were identified (Data not shown). Among them, one protein (about
55 kDa, indicated by a rectangle in Figure 1A) matched best with
a variety of mammalian b-tubulin isoforms (Table 1), which are
multifunctional cytoskeletal proteins. We decided to investigate b-
tubulin in this study because of its potential connection with
EGFR signaling as described in the discussion section. vector-expressing cells. Also, the reciprocal immunoprecipitation
was performed. TBC1D3 co-immunoprecipitated with endoge-
nous b-tubulin
but not control IgG in SMMC7721 cells
(Figure 1C). Collectively, these data reveal that b-tubulin is a
potential interaction partner for TBC1D3. As an alternative approach to determine if TBC1D3 interacted
with b-tubulin, their subcellular localization was analyzed by
confocal microscopy. Since anti-TBC1D3 antibody is not com-
mercially
available,
EGFP-fusion
version
of
TBC1D3
was
produced and then transiently transfected into SMMC7721 cells. An indirect immunofluorescence with anti-b-tubulin antibody was
performed. As seen in Figure 1D (left panel), TBC1D3 exhibited
localization at the plasma membrane, on filamentous structures,
and punctate structures that were often aligned along the
filaments. On the other hand, b-tubulin was stained in a radial
or reticular array in the cytoplasm (middle panel in Figure 1D). As an independent means of confirming this association, we
performed the co-immunoprecipitation with anti-HA antibody. As
seen in Figure 1B, endogenous b-tubulin was not co-immunopre-
cipitated with control immunoglobulin G (IgG) in SMMC7721
cells transiently transfected with HA-TBC1D3, but did exist in
anti-HA immunoprecipitates from HA-TBC1D3 but not control Figure 1. TBC1D3 interacts with b-tubulin. (A) A representative image of the resolved protein eluate following the GST pull-down
SMMC7721 cells were transfected with GST-tagged TBC1D3 (GST-TBC1D3) or control GST vector (GST). TBC1D3 interacts with b-tubulin After SDS PAGE and transfer, the blots were stained with Ponceau S (bottom panels) prior to immunoblotting (IB)
with anti-HA and anti-b-tubulin antibodies (top panels). (D) SMMC7721 cells were transfected with EGFP-tagged TBC1D3 (top left, green). Cells were
subjected to indirect immunofluorescence with anti-b-tubulin antibody (top central, red), and then analyzed by confocal microscopy. Immunofluorescent images were merged (top right), with yellow revealing overlap (Merge). The indicated areas of the outlined boxes in the top
panels are shown in higher magnification in the bottom panels. Arrows indicate co-localizations between TBC1D3 and b-tubulin on filamentous
structures. Scale bar, 10 mm. d i 10 1371/j
l
0094134 001 April 2014 | Volume 9 | Issue 4 | e94134 PLOS ONE | www.plosone.org 4 Microtubule Network Regulates TBC1D3-Enhanced EGFR Signaling Table 1. Identification of the TBC1D3-interacting protein with molecular weight of about 55 kDa. Accession
Mass
Score
Description
Queries matched
1. gi|57209813
47736
250
tubulin, beta polypeptide [Homo sapiens]
23
2. gi|7106439
49639
247
tubulin, beta 5 [Mus musculus]
23
3. gi|18088719
49640
247
Tubulin, beta [Homo sapiens]
23
4. gi|2119276
48848
238
beta-tubulin - human (fragment)
22
5. gi|338695
49727
237
beta-tubulin
22
6. gi|55731354
41715
235
hypothetical protein [Pongo pygmaeus]
21
7. gi|5174735
49799
218
tubulin, beta, 2 [Homo sapiens]
20
8. gi|23958133
49808
218
Tubulin, beta 2C [Homo sapiens]
20
9. gi|20809886
49776
218
Tubulin, beta 2C [Homo sapiens]
20
10. gi|27368062
49721
218
class IVb beta tubulin [Homo sapiens]
20
doi:10.1371/journal.pone.0094134.t001 ble 1. Identification of the TBC1D3-interacting protein with molecular weight of about 55 kDa. There are partial co-localization between TBC1D3 and b-
tubulin/microtubule on the filamentous structures (right panel in
Figure 1D). This co-localization pattern suggested at least two
possibilities concerning the relationship between TBC1D3 and b-
tubulin/microtubule. One possibility is that TBC1D3 indirectly
interacts with b–tubulin/microtubule via other proteins such as
MAPs. This indirect interaction pattern would lead to co-
localization or a lack of co-localization between TBC1D3 and
b–tubulin/microtubule in the presence or absence of MAPs,
respectively. Another possibility is that localization of TBC1D3 at
the plasma membrane is, at least partly, independent of its
interaction with b–tubulin/microtubule. Notably, Figure 1D
showed that substantial amount of TBC1D3 lacks co-localization
with b–tubulin/microtubule at the plasma membrane. anti-HA and anti-b-tubulin antibodies. As a negative control, HA-
TBC1D3 did not bind to endogenous b-tubulin in the presence of
IgG (Figure 3B). TBC1D3 associates with a-tubulin and microtubules a- and b-tubulin form heterodimers and assemble dynamic
microtubules, which are involved in maintenance of cell structure
and transport of intracellular vesicles and organelles as well as
cellular signal transduction. Since TBC1D3 associated with b-
tubulin, we examined whether it associates with a-tubulin. To test
this, we performed the co-immunoprecipitation with anti-a-
tubulin antibody. As shown in Figure 2A, HA-TBC1D3 was not
present in anti-a-tubulin immunoprecipitates from control vector-
expressing cells. In contrast, TBC1D3 did co-immunoprecipitate
with endogenous a-tubulin but not control IgG in SMMC7721
cells transiently transfected with HA-TBC1D3 (Figure 2A),
indicating that TBC1D3 associates with a-tubulin. TBC1D3 interacts with b-tubulin TBC1D3(476) and TBC1D3(353) mutants bound
strongly to b-tubulin in the presence of anti-b-tubulin antibody as
efficiently as full-length of TBC1D3 (Figure 3B). In contrast, a
construct containing the first 251 amino acids (TBC1D3(251))
failed to associate with b-tubulin (Figure 3B). These data suggest
that the b-tubulin-interacting site resides at or near amino acids
252,353, which comprises the C-terminus of TBC/Rab GAP
homology domain (Figure 3A). gy
(
g
)
To pinpoint the site of interaction, internal deletion mutants
were generated (Figure 3A). As shown in Figure 3C, an internal
deletion of amino acids 252,285 had no effect on interaction
between TBC1D3 and endogenous b-tubulin while deletion of
amino acids 286,319 or 320,353 abolished interaction of
TBC1D3. To ensure that both of the internal mutants were well
folded and functional, we confirmed their ability to associate with
a GST-tagged C-terminal truncation mutant encoding the first
298 amino acids of GGA3, which contains the known TBC1D3-
binding site, as efficiently as wild-type TBC1D3 (Figure 3D). In
contrast, as a negative control, wild-type TBC1D3 did not bind to
GST protein (Figure 3D). Taken together, these results indicate
that optimal interaction between TBC1D3 and b-tubulin requires
amino acids 286,353 near the C-terminus of the TBC1D3
domain. The subcellular distribution of TBC1D3 is regulated by
microtubules We next performed microtubule cosedimentation assays using
polymerized microtubules. As shown in Figure 2B (right panel),
TBC1D3 bound to and was co-sedimented with the microtubules,
while TBC1D3 alone did not pellet. In control sedimentation
assay (Figure 2B, left panel), MAP2 bound to microtubules, but
BSA did not. Neither of the two proteins pelleted in the absence of
microtubules. In aggregate, these results suggest that TBC1D3
associates with tubulin dimer/oligomer and microtubules. Microtubules provide a spatially extensive docking platform for
many signal molecules and spatially regulate signal transduction
by at least three distinct mechanisms: MT sequestering and
release, MT delivery and MT scaffolding of the molecules [29,30]. Since TBC1D3 associates with tubulin/microtubules, it was
unclear whether interaction with tubulin/microtubule might be
required for TBC1D3’s subcellular distribution. To address this
issue, we generated EGFP-fusion version of TBC1D3 and mutants
encoding the indicated N-terminal residues of TBC1D3 or
harboring internal deletions of the indicated residues, then
transiently transfected SMMC7721 cells with these constructs,
and analyzed their localization using confocal microscopy. Similar
to wild-type TBC1D3, the deletion mutants TBC1D3(476) and
TBC1D3(353) exhibited localization at the plasma membrane, on
cytoplasmic filamentous and punctate structures (Figure 4A). In
contrast, TBC1D3(251) and TBC1D3(D320-353), which have no Mapping of the b-tubulin-interacting site in TBC1D3 Cell lysates were subjected to
ultracentrifugation to remove polymerized microtubules. Clarified lysates were then added to the Taxol supplemented General Tubulin Buffer alone
(MTs(-)) or together with polymerized microtubules (MTs(+)). After incubation at room temperature, samples were placed on top of the Cushion
buffer containing Taxol and centrifuged at 100,0006g. The pellet (P) and supernatant (S) fractions were collected and separated by SDS PAGE. The
blots were then stained with Ponceau S (middle panel) followed by immunoblotting (IB) with anti-HA antibody (right panel). Positive (MAP2) and
negative controls (BSA and no protein (-)) were also performed and detected by Coomassie brilliant blue staining (left panel). doi:10.1371/journal.pone.0094134.g002 Figure 2. TBC1D3 associates with a-tubulin and microtubules. (A) SMMC7721 cells were seeded in three 60-mm dishes. The next day cells
were transfected with HA-TBC1D3 (dishes 1 and 3) or control HA vector (dish 2). Lysates were immunoprecipitated (IP) with control IgG (dish 1), or
anti-a-tubulin (dishes 2 and 3) antibodies. After SDS PAGE and transfer, the blots were stained with Ponceau S (right panel) prior to immunoblotting
(IB) with anti-HA and anti-a-tubulin antibodies (left panel). Molecular weight markers are shown on left in kDa. WCL, whole cell lysate. (B) SMMC7721
cells were transfected with HA-TBC1D3 and lysed in General Tubulin Buffer (see details in Materials and Methods). Cell lysates were subjected to
ultracentrifugation to remove polymerized microtubules. Clarified lysates were then added to the Taxol supplemented General Tubulin Buffer alone
(MTs(-)) or together with polymerized microtubules (MTs(+)). After incubation at room temperature, samples were placed on top of the Cushion
buffer containing Taxol and centrifuged at 100,0006g. The pellet (P) and supernatant (S) fractions were collected and separated by SDS PAGE. The
blots were then stained with Ponceau S (middle panel) followed by immunoblotting (IB) with anti-HA antibody (right panel). Positive (MAP2) and
negative controls (BSA and no protein (-)) were also performed and detected by Coomassie brilliant blue staining (left panel). doi:10.1371/journal.pone.0094134.g002 membrane and in the cytoplasmic endosomes [9]. TBC1D3
inhibits the recruitment of c-Cbl and ubiquitination of EGFR [26]. However, the localization where TBC1D3 inhibits the c-Cbl-
mediated ubiquitination was not examined. It was also unknown
whether microtubules are involved in TBC1D3-mediated sup-
pression of the c-Cbl recruitment and EGFR ubiquitination. Mapping of the b-tubulin-interacting site in TBC1D3 To
address these issues, we examined the c-Cbl recruitment and
EGFR ubiquitination in SMMC7721 cells expressing control
vector, wild-type TBC1D3, and b-tubulin interacting-deficient
TBC1D3 mutants which disappeared from cytoplasmic filamen-
tous and punctate structures but not plasma membrane. As seen in
Figure 5, TBC1D3 expression substantially reduced the c-Cbl
recruitment and EGFR ubiquitination compared to control vector. In contrast, the TBC1D3 mutants lost the inhibitory effect
(Figure 5). As a negative control, endogenous EGFR and c-Cbl
were not co-immunoprecipitated with control IgG in SMMC7721
cells transiently transfected with control vector (Figure 5, top right
panel). Together, these data indicate that association with
microtubules is required for TBC1D3 to suppress the c-Cbl
recruitment and EGFR ubiquitination. Since the cytoplasmic
TBC1D3-positive
punctate
and
filamentous
structures
may
represent endosomal and tubular endosomal compartments,
respectively, these results also suggest that TBC1D3 inhibits the
c-Cbl-mediated EGFR ubiquitination in the cytoplasmic endo-
somes, not at the plasma membrane. b-tubulin-interacting site, appeared mainly in the nucleus instead
of cytoplasmic filamentous and punctate structures although their
plasma membrane localization persisted (Figure 4A). Since EGFP-
fusion proteins are often used in many studies, it was unclear
whether nuclear localization of the TBC1D3 mutants was not
based from the nature of EGFP fusion. To explore this issue, we
tested HA-tagged TBC1D3 and mutants for their distribution in
SMMC7721 cells. As seen in Figure 4B, HA fusion variants
exhibited the same distribution as their respective EGFP-fusion
constructs. These data suggest that association with tubulin/
microtubule is required for cytoplasmic filamentous and punctate
localization
but
not
for
plasma
membrane
localization
of
TBC1D3. Since TBC1D3 associates with microtubules, we next examined
whether disruption of microtubules affected wild-type TBC1D3
distribution. In the presence of the microtubule depolymerizing
drug nocodazole, cytoplasmic TBC1D3-positive filaments and
punctates were completely abolished and wild-type TBC1D3
instead appeared in the nucleus (Figure 4C). In contrast, plasma
membrane localization of TBC1D3 persisted (Figure 4C). Unlike
nocodazole, EGF stimulation had no effect on wild-type TBC1D3
distribution (Data not shown). Together, these results indicate that
the localization of TBC1D3 is spatially regulated by microtubules. Without the microtubule-mediated sequestration, TBC1D3 trans-
locates from the cytoplasm into the cell nucleus. Mapping of the b-tubulin-interacting site in TBC1D3 Mapping of the b-tubulin-interacting site in TBC1D3
To determine the b-tubulin-interacting site of TBC1D3, a series
of nested deletion mutants were generated, which encodes the
indicated N-terminal residues of TBC1D3 (Figure 3A), and
transfected into SMMC7721 cells. Co-immunoprecipitations with
anti-b-tubulin antibody or control IgG were performed, and
association of TBC1D3 mutants was then immunoblotted with PLOS ONE | www.plosone.org April 2014 | Volume 9 | Issue 4 | e94134 5 Microtubule Network Regulates TBC1D3-Enhanced EGFR Signaling Figure 2. TBC1D3 associates with a-tubulin and microtubules. (A) SMMC7721 cells were seeded in three 60-mm dishes. The next day cells
were transfected with HA-TBC1D3 (dishes 1 and 3) or control HA vector (dish 2). Lysates were immunoprecipitated (IP) with control IgG (dish 1), or
anti-a-tubulin (dishes 2 and 3) antibodies. After SDS PAGE and transfer, the blots were stained with Ponceau S (right panel) prior to immunoblotting
(IB) with anti-HA and anti-a-tubulin antibodies (left panel). Molecular weight markers are shown on left in kDa. WCL, whole cell lysate. (B) SMMC7721
cells were transfected with HA-TBC1D3 and lysed in General Tubulin Buffer (see details in Materials and Methods). Cell lysates were subjected to
ultracentrifugation to remove polymerized microtubules. Clarified lysates were then added to the Taxol supplemented General Tubulin Buffer alone
(MTs(-)) or together with polymerized microtubules (MTs(+)). After incubation at room temperature, samples were placed on top of the Cushion
buffer containing Taxol and centrifuged at 100,0006g. The pellet (P) and supernatant (S) fractions were collected and separated by SDS PAGE. The
blots were then stained with Ponceau S (middle panel) followed by immunoblotting (IB) with anti-HA antibody (right panel). Positive (MAP2) and
negative controls (BSA and no protein (-)) were also performed and detected by Coomassie brilliant blue staining (left panel). doi:10.1371/journal.pone.0094134.g002 Figure 2. TBC1D3 associates with a-tubulin and microtubules. (A) SMMC7721 cells were seeded in three 60-mm dishes. The next day cells
were transfected with HA-TBC1D3 (dishes 1 and 3) or control HA vector (dish 2). Lysates were immunoprecipitated (IP) with control IgG (dish 1), or
anti-a-tubulin (dishes 2 and 3) antibodies. After SDS PAGE and transfer, the blots were stained with Ponceau S (right panel) prior to immunoblotting
(IB) with anti-HA and anti-a-tubulin antibodies (left panel). Molecular weight markers are shown on left in kDa. WCL, whole cell lysate. (B) SMMC7721
cells were transfected with HA-TBC1D3 and lysed in General Tubulin Buffer (see details in Materials and Methods). TBC1D3 Ubiquitination of EGFR plays a critical role in protection
against excessive activation of the activated receptor. EGFR is
ubiquitinated by c-Cbl, an E3 ligase, both at the plasma Having determined that association with microtubules is
essential for TBC1D3 to suppress EGFR ubiquitination, we next April 2014 | Volume 9 | Issue 4 | e94134 PLOS ONE | www.plosone.org 6 Microtubule Network Regulates TBC1D3-Enhanced EGFR Signaling examined role of microtubules in TBC1D3-medicated enhance-
than 50% of EGFR proteins persisted after 2 hours (Figure 6A and
Figure 3. Mapping of b-tubulin-interacting site in TBC1D3. (A) Schematic representation of full-length TBC1D3 and various deletion mutants. The length or internal deletion (D) of each isoform in amino acids is indicated. TBC/Rab GAP homology domain is shown in gray. (B and C) SMMC7721
cells were transfected with HA-tagged TBC1D3 and mutants encoding the indicated N-terminal residues of TBC1D3 (B) or harboring internal deletions
of the indicated residues (C). Lysates were immunoprecipitated (IP) with anti-b-tubulin antibody or control IgG. After SDS PAGE, the blots were
stained with Ponceau S (bottom panels) followed by immunoblotting (IB) with anti-HA and anti-b-tubulin antibodies (top panels). Molecular weight
markers are shown on left in kDa. WCL, whole cell lysate. (D) SMMC7721 cells were seeded in four 60-mm dishes. The following day, cells wre
transfected with HA-tagged TBC1D3 (dishes 1 and 2) or mutants harboring internal deletions of the indicated residues (dishes 3 and 4). Cell extracts
were pulled down (PD) with bacterially purified GST-GGA3(298) (dishes 2, 3 and 4) or GST (dish 1) immobilized on glutathione-Sepharose 4B beads. After SDS PAGE, the blots were stained with Ponceau S (right panel) followed by immunoblotting (IB) with anti-HA antibody (left panels). doi:10.1371/journal.pone.0094134.g003 Figure 3. Mapping of b-tubulin-interacting site in TBC1D3. (A) Schematic representation of full-length TBC1D3 and various deletion mutants. The length or internal deletion (D) of each isoform in amino acids is indicated. TBC/Rab GAP homology domain is shown in gray. (B and C) SMMC7721
cells were transfected with HA-tagged TBC1D3 and mutants encoding the indicated N-terminal residues of TBC1D3 (B) or harboring internal deletions
of the indicated residues (C). Lysates were immunoprecipitated (IP) with anti-b-tubulin antibody or control IgG. After SDS PAGE, the blots were
stained with Ponceau S (bottom panels) followed by immunoblotting (IB) with anti-HA and anti-b-tubulin antibodies (top panels). Molecular weight
markers are shown on left in kDa. WCL, whole cell lysate. TBC1D3 (D) SMMC7721 cells were seeded in four 60-mm dishes. The following day, cells wre
transfected with HA-tagged TBC1D3 (dishes 1 and 2) or mutants harboring internal deletions of the indicated residues (dishes 3 and 4). Cell extracts
were pulled down (PD) with bacterially purified GST-GGA3(298) (dishes 2, 3 and 4) or GST (dish 1) immobilized on glutathione-Sepharose 4B beads. After SDS PAGE, the blots were stained with Ponceau S (right panel) followed by immunoblotting (IB) with anti-HA antibody (left panels). doi:10.1371/journal.pone.0094134.g003 than 50% of EGFR proteins persisted after 2 hours (Figure 6A and
6B). However, deficiency in b-tubulin interacting resulted in
TBC1D3’s inability to delay EGFR degradation; about 40% and
over 75% of the EGFR signal were lost after 30 min and 2 hours,
respectively (Figure 6A and 6B). These results indicate that
association with microtubule is required for regulation of EGFR
stability by TBC1D3. examined role of microtubules in TBC1D3-medicated enhance-
ment of EGFR stability and signaling. As seen in Figure 6A and
6B,
cells
transfected
with
control
vector
showed
a
rapid
degradation of EGFR; after 30 minutes of EGF stimulation, over
40% of the EGFR signal was lost, and only about 20% of EGFR
proteins were left after 2 hours. In contrast, EGFR degradation
was significantly delayed in cells expressing TBC1D3; merely 20%
of the receptor population was degraded after 30 min, and more April 2014 | Volume 9 | Issue 4 | e94134 7 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org Microtubule Network Regulates TBC1D3-Enhanced EGFR Signaling Microtubule Network Regulates TBC1D3-Enhanced EGFR Signaling increased in all of the transfected cells. The EGF-induced Erk1/2
activation was enhanced by expression of TBC1D3, but not
TBC1D3 mutants deficient in b-tubulin interacting (Figure 6A
and 6C). Subsequently, the phosphorylation levels of Erk1/2
decreased and Erk1/2 signaling in both control vector and the
TBC1D3 mutant-transfected cells subsided after 5 hours, whereas
TBC1D3-expressing cells sustained their Erk1/2 activation during
this period (Figure 6A and 6C). Consistent with these results,
TBC1D3 expression stimulated proliferation of SMMC7721 cells,
but TBC1D3 mutant deficient in b-tubulin interacting had no
such effect (Figure 6D). Taken together, these results indicate that
association with microtubule is required for TBC1D3 to regulate
EGFR stability and signaling. Figure 4. The subcellular distribution of TBC1D3 is regulated
by the microtubule network. Discussion Our studies identify tubulin/microtubule network as a novel
regulator of TBC1D3. Interaction of b-tubulin is mediated by
amino acids 286,353 within TBC1D3. Deletion mutations in
these amino acids significantly attenuate interaction with b-
tubulin. Furthermore, through combined analysis of b-tubulin
interacting-deficient TBC1D3 mutants and the pharmacological
agent nocodazole that interferes with the polymerization of
microtubules, we identify TBC1D3 as a novel nucleocytoplasmic
protein. Inability to bind b-tubulin and disruption of the
microtubule network result in disappearance of TBC1D3 from
cytoplasmic filamentous and punctate structures and TBC1D3
instead transports into the nucleus. Finally, our results indicate
that the cytoplasmic retention of TBC1D3 by the microtubule
network is required for enhanced EGFR stability and signaling. Figure 4. The subcellular distribution of TBC1D3 is regulated
by the microtubule network. SMMC7721 cells were transfected with
EGFP (A and C, green) or HA (B) -tagged TBC1D3 and mutants encoding
the indicated N-terminal residues of TBC1D3 or harboring internal
deletions of the indicated residues. EGFP-tagged TBC1D3-transfected
SMMC7721 cells were treated with nocodazole (40 mg/ml) for 2 h (C). Cells were subjected to indirect immunofluorescence with anti-b-
tubulin antibody (blue) alone (A and C), or together with anti-HA
antibody (B, green), stained with Hoechst 33258 (red), and then
analyzed by confocal microscopy. Immunofluorescent images were
merged with yellow revealing overlap (Merge). Scale bar, 10 mm. doi:10.1371/journal.pone.0094134.g004 q
y
g
g
The
precise
mechanism
by
which
b-tubulin/microtubule
network modulates the activation of EGFR signaling by TBC1D3
remains to be determined. The fact that TBC1D3 mutants
deficient in b-tubulin interacting had no ability to delay EGFR
degradation and to enhance EGFR signaling indicates that
interaction between TBC1D3 and b-tubulin is required. Two
possible mechanisms are by regulating inhibitory impact of
TBC1D3 on microtubule acetylation or EGFR ubiquitination. Microtubule acetylation is under the control of balanced tubulin
acetyltransferases/deacetylases. Acetylatransferases (such as ELP3
and ARD1) and deacetylases (including HDAC6 and SIRT2)
associate with microtubules and function to acetylate and
deacetylate a-tubulin, respectively [31]. Before EGF stimulation,
EGFR associates with HDAC6, which inhibits microtubule
acetylation. Upon EGF binding, EGFR phosphorylates and
deactivates HDAC6, thereby promoting microtubule acetylation
[32], which increases the affinity and processivity of microtubule
motors [33,34], and results in accelerated segregation of EGFR
from early endosomes and premature delivery of EGFR to the late
endosomal and lysosomal compartments for degradation [35]. Also, c-Cbl and Cbl-b interact with b-tubulin and promote
microtubule acetylation by displacing HDAC6 from b-tubulin
[36]. TBC1D3 SMMC7721 cells were transfected with
EGFP (A and C, green) or HA (B) -tagged TBC1D3 and mutants encoding
the indicated N-terminal residues of TBC1D3 or harboring internal
deletions of the indicated residues. EGFP-tagged TBC1D3-transfected
SMMC7721 cells were treated with nocodazole (40 mg/ml) for 2 h (C). Cells were subjected to indirect immunofluorescence with anti-b-
tubulin antibody (blue) alone (A and C), or together with anti-HA
antibody (B, green), stained with Hoechst 33258 (red), and then
analyzed by confocal microscopy. Immunofluorescent images were
merged with yellow revealing overlap (Merge). Scale bar, 10 mm. doi:10.1371/journal.pone.0094134.g004 Discussion Anti-ubiquitin and anti-HA antibodies were
used to monitor ubiquitinated EGFR, HA-TBC1D3 and its mutant. Molecular weight markers are shown on left in kDa. WCL, whole cell lysate. (B) The
bar graph is derived from densitometric analysis of Western blots as typified in top right panel in A. Ubiqitinated EGFR and c-Cbl are normalized to
EGFR total amounts. The data are presented as means 6 SD of three independent experiments (**, p,0.01). doi:10.1371/journal.pone.0094134.g005 Figure 5. Microtubules are involved in TBC1D3-mediated regulation of EGFR ubiquitination. (A) SMMC7721 cells were seeded in four 60-
mm dishes. The following day, cells were transfected with control HA vector (dishes 1 and 2), HA-TBC1D3 (dish 3) and HA-TBC1D3(D320–353) (dish 4),
respectively. The next day cells were serum-starved and then treated with 100 ng/ml of EGF for 15 minutes. Lysates were immunoprecipitated (IP)
with anti-EGFR antibody (dishes 2, 3 and 4) or control IgG (dish 1). After SDS PAGE, the blots were stained with Ponceau S (bottom panels) followied
by immunoblotting (IB) with anti-c-Cbl, anti-EGFR, anti-HA and anti-ubiquitin antibodies (top panels). Anti-ubiquitin and anti-HA antibodies were
used to monitor ubiquitinated EGFR, HA-TBC1D3 and its mutant. Molecular weight markers are shown on left in kDa. WCL, whole cell lysate. (B) The
bar graph is derived from densitometric analysis of Western blots as typified in top right panel in A. Ubiqitinated EGFR and c-Cbl are normalized to
EGFR total amounts. The data are presented as means 6 SD of three independent experiments (**, p,0.01). doi:10.1371/journal.pone.0094134.g005 of the motor protein dynein to promote the nuclear translocation
of proteins such as hypoxia inducible factor-1, p53, parathyroid
hormone-related protein, retinoblastoma protein, and the rabies
virus P-protein [41–48]. Dynein transports various cellular cargoes
on microtubules towards the minus-end of the microtubule, which
is usually oriented from the cell periphery to the nucleus [49]. The
microtubule network and dynein, therefore, are thought to
facilitate nuclear import [44,46,48]. In contrast, the microtubule
network have also been implicated in binding and subsequently
sequestering at least three nucleocytoplasmic proteins, c-myc,
MIZ-1 and SRY-related HMG box-9 (SOX9), in the cytoplasm,
thereby inhibiting their nuclear import and resulting in suppres-
sion of their transcriptional activity [41,50–53]. Discussion However, we observed that TBC1D3 had no impact on b-
tubulin binding to both HDAC6 and c-Cbl (data not shown), and
microtubule acetylation (Figure S1), demonstrating the former
mechanism is unlikely. Nevertheless, it remains possible that
TBC1D3 inhibits directly the interaction of microtubule with its
motors
including
dynein
and
kinesin
1
in
a
microtubule
acetylation-independent manner. Functional interference with
motor proteins or cellular trafficking systems will contribute to
clarification of the function. Figure 4. The subcellular distribution of TBC1D3 is regulated
by the microtubule network. SMMC7721 cells were transfected with
EGFP (A and C, green) or HA (B) -tagged TBC1D3 and mutants encoding
the indicated N-terminal residues of TBC1D3 or harboring internal
deletions of the indicated residues. EGFP-tagged TBC1D3-transfected
SMMC7721 cells were treated with nocodazole (40 mg/ml) for 2 h (C). Cells were subjected to indirect immunofluorescence with anti-b-
tubulin antibody (blue) alone (A and C), or together with anti-HA
antibody (B, green), stained with Hoechst 33258 (red), and then
analyzed by confocal microscopy. Immunofluorescent images were
merged with yellow revealing overlap (Merge). Scale bar, 10 mm. doi:10.1371/journal.pone.0094134.g004 Phosphorylation of Erk1/2 on T202 and Y204 is a well-
established downstream marker for activation of EGFR signaling. Since microtubule involved in TBC1D3-enhanced EGFR stability,
we next examined phosphorylation level of Erk1/2 in SMMC7721
cells transfected with control vector, HA-TBC1D3 and b-tubulin
interacting-deficient TBC1D3 mutants. Total cellular contents of
Erk1/2 were used to normalize the results in each sample
(Figure 6A). As seen in Figure 6A and 6C, following EGF
stimulation for 30 minutes phosphorylation levels of Erk1/2 It appears more likely that b-tubulin interaction promotes
TBC1D3-enhanced EGFR signaling by regulating TBC1D3’s
ability to inhibit EGFR ubiquitination. Intriguingly, TBC1D3 April 2014 | Volume 9 | Issue 4 | e94134 PLOS ONE | www.plosone.org 8 Microtubule Network Regulates TBC1D3-Enhanced EGFR Signaling Figure 5. Microtubules are involved in TBC1D3-mediated regulation of EGFR ubiquitination. (A) SMMC7721 cells were seeded in four 60-
mm dishes. The following day, cells were transfected with control HA vector (dishes 1 and 2), HA-TBC1D3 (dish 3) and HA-TBC1D3(D320–353) (dish 4),
respectively. The next day cells were serum-starved and then treated with 100 ng/ml of EGF for 15 minutes. Lysates were immunoprecipitated (IP)
with anti-EGFR antibody (dishes 2, 3 and 4) or control IgG (dish 1). After SDS PAGE, the blots were stained with Ponceau S (bottom panels) followied
by immunoblotting (IB) with anti-c-Cbl, anti-EGFR, anti-HA and anti-ubiquitin antibodies (top panels). Discussion At variance with
these proteins, which function in the nucleus as transcriptional
factors, we observed that deficiency in b-tubulin interacting or
disruption of the microtubule network caused not only disappear-
ance of TBC1D3 from the cytoplasm but also inability to enhance
EGFR
signaling,
demonstrating
that
TBC1D3
is
a
novel
nucleocytoplasmic protein that functions in the cytoplasm retained
by the microtubule network. However, we cannot exclude the
possibility that TBC1D3 also functions in the nucleus, which may
provide novel insights into its function in that case. mutants deficient in b-tubulin interacting not only lost the ability
to inhibit c-Cbl recruitment and EGFR ubiquitination, but also
disappeared from the cytoplasm although plasma membrane
localization of TBC1D3 persisted. These results touch upon the
poorly understood issue of where TBC1D3 inhibits EGFR
ubiquitination [26]. It has been speculated that c-Cbl ubiquitinates
EGFR both at the plasma membrane and in the cytoplasmic
endosomes [9]. Although sufficient for EGFR internalization,
EGFR ubiquitination at the plasma membrane is not essential for
the process [11–13]. EGFR ubiquitination in the endosomes,
however, is indeed required for its sorting onto intraluminal
vesicles of multivesicular endosomes/bodies and subsequent
lysosomes for efficient degradation [12,14]. Notably, the TBC1D3
mutants had no ability to enhance EGFR stability and signaling,
suggesting that TBC1D3 may function in the cytoplasmic
endosomes, not at the plasma membrane, to inhibit c-Cbl
recruitment and EGFR ubiquitination. Consistent with this
notion, EGFR internalization was not inhibited, but enhanced,
by TBC1D3 [26], although these results might be explained by
increased expression and recycling of EGFR in TBC1D3-
expressing cells. In addition, there are at least two deubiquitinating
enzymes including AMSH [37,38] and Cezanne-1 [39], which
remove ubiquitin from EGFR in endosomes. It is possible that
TBC1D3 functions in the cytoplasmic endosomes to stimulate the
action of the enzymes and prevent ubiqutin-mediated degradation
of EGFR. Supporting Information Figure S1
TBC1D3 does not affect the acetylation of a-
tubulin. SMMC7721 cells were transfected with HA-TBC1D3
and control HA vector, respectively. After 24 h, cells were serum-
starved and then stimulated with 100 ng/ml of EGF for the indicted
times. Lysates were immunoblotted with anti-c-Cbl, anti-HDAC6,
anti-HA, anti-acetyl-a-tubulin and anti-GAPDH antibodies. (TIF) In this study, we identify TBC1D3 as a novel nucleocytoplasmic
protein that is retained by the microtubule network in the
cytoplasm. It has been speculated that there exist two opposing
modes of microtubule-regulated nucleocytoplasmic transport,
including microtubule-facilitated and -inhibited nuclear import
[40]. In the first mode, the microtubule network functions as tracks April 2014 | Volume 9 | Issue 4 | e94134 PLOS ONE | www.plosone.org 9 Microtubule Network Regulates TBC1D3-Enhanced EGFR Signaling Figure 6. Microtubule network modulates the enhancement of EGFR stability and activation of EGFR signaling by TBC1D3. (A)
SMMC7721 cells were transfected with HA-TBC1D3, HA-TBC1D3(D320–353) and control HA vector, respectively. After 24 h, cells were serum-starved,
pretreated with cycloheximide (40 mg/ml) for 2 h, and then stimulated with 100 ng/ml of EGF for the indicted times. After cell lysates were resolved
by SDS-PAGE, the blot was stained with Ponceau S (bottom panel) followed by Western blotting (WB) with anti-EGFR, anti-Erk1/2, anti-phospho-Erk1/
2 (p-Erk1/2), anti-HA and anti-GAPDH antibodies (top panel). Molecular weight markers are shown on left in kDa. The line (B) and bar (C) graphs are
derived from densitometric analysis of Western blots as typified in top panel in A. The percentage of the amount of EGFR present at the indicated
time of EGF stimulation over the initial amount in each group was quantified in B. p-Erk1/2 is normalized to Erk1/2 total amounts in C. The data are
presented as means 6 SD of three independent experiments (*, p,0.05; **,p,0.01). (D) SMMC7721 cells were transfected with HA-TBC1D3, HA-
TBC1D3(D320–353) and control HA vector, respectively. After incubation in 10% FBS-DMEM for the indicated times, their survival was determined by
the CCK8 assay. Cell growth rate is expressed as absorbance at 450 nm. Data are presented as Means 6 SD of sextuplicates from one experiment
representative of three performed (*, p,0.05; **, p,0.01). doi:10.1371/journal.pone.0094134.g006 Figure 6. Microtubule network modulates the enhancement of EGFR stability and activation of EGFR signaling by TBC1D3. (A)
SMMC7721 cells were transfected with HA-TBC1D3, HA-TBC1D3(D320–353) and control HA vector, respectively. Supporting Information After 24 h, cells were serum-starved,
pretreated with cycloheximide (40 mg/ml) for 2 h, and then stimulated with 100 ng/ml of EGF for the indicted times. After cell lysates were resolved
by SDS-PAGE, the blot was stained with Ponceau S (bottom panel) followed by Western blotting (WB) with anti-EGFR, anti-Erk1/2, anti-phospho-Erk1/
2 (p-Erk1/2), anti-HA and anti-GAPDH antibodies (top panel). Molecular weight markers are shown on left in kDa. The line (B) and bar (C) graphs are
derived from densitometric analysis of Western blots as typified in top panel in A. The percentage of the amount of EGFR present at the indicated
time of EGF stimulation over the initial amount in each group was quantified in B. p-Erk1/2 is normalized to Erk1/2 total amounts in C. The data are
presented as means 6 SD of three independent experiments (*, p,0.05; **,p,0.01). (D) SMMC7721 cells were transfected with HA-TBC1D3, HA-
TBC1D3(D320–353) and control HA vector, respectively. After incubation in 10% FBS-DMEM for the indicated times, their survival was determined by
the CCK8 assay. Cell growth rate is expressed as absorbance at 450 nm. Data are presented as Means 6 SD of sextuplicates from one experiment
representative of three performed (*, p,0.05; **, p,0.01). doi:10 1371/journal pone 0094134 g006 Author Contributions Conceived and designed the experiments: CS ZH TT. Performed the
experiments: ZH TT DG HW. Analyzed the data: ZH TT CS XB. Contributed reagents/materials/analysis tools: YC YZ QW HZ CW HS
LZ MW. Wrote the paper: CS ZH. Conceived and designed the experiments: CS ZH TT. Performed the
experiments: ZH TT DG HW. Analyzed the data: ZH TT CS XB. Contributed reagents/materials/analysis tools: YC YZ QW HZ CW HS
LZ MW. Wrote the paper: CS ZH. We thank Dr. Jie Zheng for critical reading of the manuscript. g
g
g
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24. References 1. Lemmon MA, Schlessinger J (2010) Cell signaling by receptor tyrosine kinases. Cell 141: 1117–1134. 1. Lemmon MA, Schlessinger J (2010) Cell signaling by receptor tyrosine kinases. Cell 141: 1117–1134. 6. Levkowitz G, Waterman H, Ettenberg SA, Katz M, Tsygankov AY, et al. (1999)
Ubiquitin ligase activity and tyrosine phosphorylation underlie suppression of
growth factor signaling by c-Cbl/Sli-1. Mol Cell 4: 1029–1040. 2. Sibilia M, Kroismayr R, Lichtenberger BM, Natarajan A, Hecking M, et al. (2007) The epidermal growth factor receptor: from development to tumorigen-
esis. Differentiation 75: 770–787. 2. Sibilia M, Kroismayr R, Lichtenberger BM, Natarajan A, Hecking M, et al. (2007) The epidermal growth factor receptor: from development to tumorigen-
esis. Differentiation 75: 770–787. 7. Waterman H, Levkowitz G, Alroy I, Yarden Y (1999) The RING finger of c-Cbl
mediates desensitization of the epidermal growth factor receptor. J Biol Chem
274: 22151–22154. 3. Blume-Jensen P, Hunter T (2001) Oncogenic kinase signalling. Nature 411:
355–365. 3. Blume-Jensen P, Hunter T (2001) Oncogenic kinase signalling. Nature 411:
355–365. 4. Levkowitz G, Waterman H, Zamir E, Kam Z, Oved S, et al. (1998) c-Cbl/Sli-1
regulates endocytic sorting and ubiquitination of the epidermal growth factor
receptor. Genes Dev 12: 3663–3674. 5. Schmidt MH, Dikic I (2005) The Cbl interactome and its functions. Nat Rev
Mol Cell Biol 6: 907–918. April 2014 | Volume 9 | Issue 4 | e94134 PLOS ONE | www.plosone.org 10 Microtubule Network Regulates TBC1D3-Enhanced EGFR Signaling Yamasaki C, Tashiro S, Nishito Y, Sueda T, Igarashi K (2005) Dynamic
cytoplasmic anchoring of the transcription factor Bach1 by intracellular
hyaluronic acid binding protein IHABP. J Biochem 137: 287–296. 32. Deribe YL, Wild P, Chandrashaker A, Curak J, Schmidt MH, et al. (2009)
Regulation of epidermal growth factor receptor trafficking by lysine deacetylase
HDAC6. Sci Signal 2: ra84. hyaluronic acid binding protein IHABP. J Biochem 137: 287– 53. Ziegler EC, Ghosh S (2005) Regulating inducible transcription through
controlled localization. Sci STKE 2005: re6. April 2014 | Volume 9 | Issue 4 | e94134 PLOS ONE | www.plosone.org 11
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Von Genen zu Chromosomen: Pflanzenzüchtung mit CRISPR-CAS
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613 613 Chromosome Engineering
Von Genen zu Chromosomen:
Pfl anzenzüchtung mit CRISPR-CAS spektrum des CRISPR-Cas-Systems haben
sich in den letzten Jahren immer wieder neue
Ansätze zur Genomveränderung bei Pfl anzen
ergeben [5]. spektrum des CRISPR-Cas-Systems haben
sich in den letzten Jahren immer wieder neue
Ansätze zur Genomveränderung bei Pfl anzen
ergeben [5]. REBECCA WETZEL, PATRICK SCHINDELE, HOLGER PUCHTA
BOTANISCHES INSTITUT, KARLSRUHER INSTITUT FÜR TECHNOLOGIE Using the CRISPR-Cas system, it has been possible to introduce dif-
ferent kinds of mutations in single or multiple genes for trait improve-
ment in crops. Last year, for the fi rst time, the CRISPR-Cas-mediated
induction of different kinds of targeted heritable chromosomal rear-
rangements has been achieved in plants. This novel application has
the potential to revolutionize plant breeding as genetic exchange and
linkage drag are now becoming controllable in a targeted manner. Induktion chromosomaler
Veränderungen mittels CRISPR-Cas Natürliche chromosomale Veränderungen
spielen eine wichtige Rolle in der Anpassung
und Artenbildung von Pfl anzen. So tragen sie
beispielsweise zur Ausbildung genetischer
Barrieren bei, da in der Meiose der geneti-
sche Austausch zwischen elterlichen Chro-
mosomen in solchen Bereichen unterbunden
ist [4]. Dieser fehlende genetische Austausch
stellt aber gerade in Bezug auf die herkömm-
liche Pfl anzenzüchtung ein großes Problem
dar, da sich diese Bereiche über große Teile
der Chromosomen erstrecken und somit
wichtige Merkmale für die Züchtung nicht
genützt werden können [6]. Folglich ist es
von großem Interesse, solche Bereiche wie-
der für die meiotische Rekombination und DOI: 10.1007/s12268-021-1644-y
© Die Autorinnen und Autoren 2021 ó Schon von Anbeginn der Zeit hat die
Menschheit Pflanzen mit gewünschten
Eigenschaften selektiert und gezüchtet. Hier-
bei wurde meist auf reichen Ertrag und guten
Geschmack geachtet. Andere wichtige gene-
tische Eigenschaften, wie Stresstoleranz und
Anfälligkeit gegen Krankheiten rückten
dabei aber in den Hintergrund oder gingen
ganz verloren [1]. Leider sind die Grenzen
der herkömmlichen Züchtung erreicht, sol-
che Merkmale wieder in unsere Kulturpfl an-
zen einzubringen. So ist die Verbesserung
von Nutzpfl anzen durch herkömmliche Züch-
tungsmethoden eingeschränkt. Dies liegt
einerseits an der geringen genetischen Viel-
falt der modernen Kultursorten, anderseits
an der gemeinsamen Vererbung von
gewünschten mit unerwünschten Merkma-
len durch die Genkopplungen bei Kreuzun-
gen. Bei Kulturpfl anzen können bereits seit
einigen Jahren einzelne Eigenschaften mit
der molekularen Schere CRISPR-Cas (clus-
tered regularly interspaced short palindromic
repeats-CRISPR-associated protein) verbessert
werden [2]. Die durch die Cas-Nuklease indu-
zierten Doppelstrangbrüche (DSB) werden
im Anschluss über homologe Rekombination
(HR) oder die fehlerhafte nicht homologe
Endverknüpfung (NHEJ) repariert, wobei die
DSB in somatischen Zellen vorwiegend über
NHEJ repariert werden [3]. Bei einzelnen
DSB kommt es dabei vor allem zu kleineren
Insertionen oder Deletionen an der Bruch- stelle, sodass hierdurch offene Leserahmen
zerstört werden können. Werden jedoch zeit-
gleich zwei DSB induziert, kann es zu chro-
mosomalen Veränderungen kommen, wie
Deletionen, Inversionen oder Translokatio-
nen (Abb. 1, [4]). Durch die einfache Hand-
habung und das vielfältige Anwendungs- ˚ Abb. 1: Gene Editing versus Chromosome Engineering. Beim Gene Editing werden einzelne
Doppenstrangbrüche (DSB) innerhalb von Genen induziert, infolgedessen es bei ihrer Reparatur
zu kleinen Veränderungen (Insertionen bzw. Deletionen) am Bruch kommt, welche zum Funktions-
verlust der genetischen Information führen können. Beim Chromosome Engineering werden meh-
rere DSB gleichzeitig entweder auf demselben oder auf unterschiedlichen Chromosomen indu-
ziert, infolgedessen es zu chromosomalen Umstrukturierungen kommt. NHEJ: nicht homologe
Endverknüpfung. BIOspektrum | 06.21 | 27. Jahrgang Induktion chromosomaler
Veränderungen mittels CRISPR-Cas konnten hier erfolgreich eine 75,5 Mbp große
Inversion in einer Maissorte revertieren, wel-
che ungefähr ein Drittel des gesamten Chro-
mosoms 2 umfasst [9]. Diese Region ist damit
auch wieder für den genetischen Austausch
bei Kreuzungen zugänglich. so wurde ein bulk screening-Verfahren ange-
wandt, um dann so die relativ seltenen Chro-
mosomenveränderungen optimal in einer
größeren Zahl von Nachkommen zu detektie-
ren. Überraschenderweise wurde bei dieser
Studie auch festgestellt, dass bei den Inver-
sionen die Bruchenden meist ohne Verlust
von Basen perfekt miteinander ligiert wur-
den. Die wohl bekannteste natürliche Inver-
sion in A. thaliana ist die parazentrische
Inversion des hk4S, welche sich auf Chromo-
som 4 befi ndet. Dabei handelt es sich um
einen heterochromatischen Bereich mit der
Größe von 1,17 Mbp, der vor ca. 4.000 Jahren
entstanden ist und u. a. im Ökotyp Columbia
vorkommt. Eine Reihe an anderen Ökotypen,
wie beispielsweise Landsberg, trägt das
Chromosom noch unverändert im Original-
zustand. Deshalb ist auch ein genetischer
Austausch zwischen diesen beiden Ökotypen
in diesem Bereich während der Meiose nicht
mehr möglich. Mit der neu etablierten Tech-
nologie konnte diese natürliche Inversion in
A. thaliana jetzt wieder revertiert werden. So
konnten dann zum ersten Mal überhaupt
genetische Austausche zwischen den beiden
Ökotypen in diesem Chromosomenbereich
erhalten werden [8]. Zusätzlich zu diesen
Erfolgen ist es bereits gelungen, die erste
chromosomale Veränderung in einer Kultur-
pfl anze zu induzieren. C. Schwartz et al. somit den genetischen Austausch zugänglich
zu machen (Abb. 2). Darüber hinaus ist der
genetische Austausch nur dann für die Züch-
tung nutzbar, wenn die Gene für wünschens-
werte und nachteilige Merkmale nicht nah
beieinander auf ein und demselben Chromo-
som liegen. Ist dies der Fall, sind die Gene
genetisch gekoppelt und beide Merkmale
werden immer gemeinsam an Nachkommen
vererbt. Dank CRISPR-Cas ist es nun mög-
lich, gezielte Veränderungen auf der Chro-
mosomenebene zu induzieren, um diese
räumliche Nähe aufzuheben. Neben Inversionen spielen bei der Genom-
evolution auch Translokationen eine wichti-
ge Rolle, die ebenso zur Einschränkung des
genetischen Austauschs in Pfl anzen und zur
Spezifi kation beitragen [4, 10]. Zudem sind
sie ein wichtiges Werkzeug, um die geneti-
sche Kopplung von Genen zu regulieren,
wodurch sie besonders interessant für die
Pflanzenzüchtung sind. So ist es auf der
einen Seite möglich, unerwünschte geneti-
sche Kopplungen aufzuheben, aber auf der
anderen Seite eben auch Gene zu koppeln,
die gemeinsam vererbt werden sollen
(Abb. 3). Induktion chromosomaler
Veränderungen mittels CRISPR-Cas ˚ Abb. 1: Gene Editing versus Chromosome Engineering. Beim Gene Editing werden einzelne
Doppenstrangbrüche (DSB) innerhalb von Genen induziert, infolgedessen es bei ihrer Reparatur
zu kleinen Veränderungen (Insertionen bzw. Deletionen) am Bruch kommt, welche zum Funktions-
verlust der genetischen Information führen können. Beim Chromosome Engineering werden meh-
rere DSB gleichzeitig entweder auf demselben oder auf unterschiedlichen Chromosomen indu-
ziert, infolgedessen es zu chromosomalen Umstrukturierungen kommt. NHEJ: nicht homologe
Endverknüpfung. ˚ Abb. 1: Gene Editing versus Chromosome Engineering. Beim Gene Editing werden einzelne
Doppenstrangbrüche (DSB) innerhalb von Genen induziert, infolgedessen es bei ihrer Reparatur
zu kleinen Veränderungen (Insertionen bzw. Deletionen) am Bruch kommt, welche zum Funktions-
verlust der genetischen Information führen können. Beim Chromosome Engineering werden meh-
rere DSB gleichzeitig entweder auf demselben oder auf unterschiedlichen Chromosomen indu-
ziert, infolgedessen es zu chromosomalen Umstrukturierungen kommt. NHEJ: nicht homologe
Endverknüpfung. BIOspektrum | 06.21 | 27. Jahrgang WISSENSCHAFT · SPECIAL: GENOME EDITING 614 ˚ Abb. 2: Bedeutung von Inversionen für die Pfl anzenzucht. Invertierte
Bereiche sind für den genetischen Austausch über Crossover (CO) zwi-
schen elterlichen Chromosomen nicht zugänglich. Über die gezielte
Induktion von Inversionen können nun bisher genetisch nicht zu tren-
nende Merkmale einzeln für die Züchtung zugänglich gemacht werden,
sodass sie unab hängig voneinander vererbt werden. Ebenso können
chromosomale Bereiche invertiert und so attraktive Merkmale mit-
einander genetisch gekoppelt werden. ˚ Abb. 3: Bedeutung von Translokationen für die Pfl anzenzucht. Merk-
male, die auf einem Chromosom nah beieinanderliegen, werden mit
sehr hoher Wahrscheinlichkeit gemeinsam vererbt. Über die Induktion
von reziproken Translokationen können diese Merkmale gezielt gene-
tisch voneinander getrennt werden. Ebenso ist auch eine Neukopplung
von attraktiven Merkmalen möglich. ˚ Abb. 2: Bedeutung von Inversionen für die Pfl anzenzucht. Invertierte
Bereiche sind für den genetischen Austausch über Crossover (CO) zwi-
schen elterlichen Chromosomen nicht zugänglich. Über die gezielte
Induktion von Inversionen können nun bisher genetisch nicht zu tren-
nende Merkmale einzeln für die Züchtung zugänglich gemacht werden,
sodass sie unab hängig voneinander vererbt werden. Ebenso können
chromosomale Bereiche invertiert und so attraktive Merkmale mit-
einander genetisch gekoppelt werden. ˚ Abb. 3: Bedeutung von Translokationen für die Pfl anzenzucht. Merk-
male, die auf einem Chromosom nah beieinanderliegen, werden mit
sehr hoher Wahrscheinlichkeit gemeinsam vererbt. Über die Induktion
von reziproken Translokationen können diese Merkmale gezielt gene-
tisch voneinander getrennt werden. Ebenso ist auch eine Neukopplung
von attraktiven Merkmalen möglich. BIOspektrum | 06.21 | 27. Jahrgang Korrespondenzadresse:
Prof. Dr. Holger Puchta
Botanisches Institut
Karlsruher Institut für Technologie (KIT)
Fritz-Haber-Weg 4
D-76128 Karlsruhe
holger.puchta@kit.edu Induktion chromosomaler
Veränderungen mittels CRISPR-Cas Auch bei Translokationen konnte
dank CRISPR-Cas kürzlich ein Meilenstein
erreicht werden. N. Beying et al. konnten
erstmals in Pfl anzen vererbbare, reziproke
Translokationen zwischen heterologen Chro-
mosomen im Mbp-Bereich induzieren [10]. Hierbei wurde ein ähnlicher Ansatz wie bei
der Reversion der Inversion verwendet. Die
Autoren konnten so reziproke Chromoso-
menarmaustausche zwischen Chromosom 1
und 2 sowie 1 und 5 erzielen, die auch beide
vererbt wurden. Kürzlich konnte erstmals in der Modell-
pfl anze Arabidopsis thaliana gezeigt werden,
dass sich mithilfe des CRISPR-Cas9-Systems
chromosomale Veränderungen tatsächlich
im Mbp-Bereich induzieren und Pfl anzen, die
diese chromosomalen Veränderungen stabil
tragen, erzeugen lassen. Kleine Inversionen
im kbp-Bereich konnten zuvor bereits erfolg-
reich in Pfl anzen induziert werden [7]. Diese
erste Untersuchung zeigte, dass chromoso-
male Veränderungen im Gegensatz zu einfa-
chen Mutationen nur sehr selten vererbt
werden. Um die Effi zienz zu steigern, wurde
deshalb die sehr potente Cas9-Nuklease aus
Staphylococcus aureus (SaCas9) unter der
Expression eines eizellspezifi schen Promo-
tors verwendet, um so für eine effi ziente Ver-
erbung der Veränderungen zu sorgen. Eben- BIOspektrum | 06.21 | 27. Jahrgang Fazit [7] Schmidt C, Pacher M, Puchta H (2019) Effi cient
induction of heritable inversions in plant genomes using
the CRISPR/Cas system. Plant J 98: 577–589
[8] Schmidt C, Fransz P, Rönspies M et al. (2020)
Changing local recombination patterns in Arabidopsis by
CRISPR/Cas mediated chromosome engineering. Nat
Commun 11: 4418 [7] Schmidt C, Pacher M, Puchta H (2019) Effi cient
induction of heritable inversions in plant genomes using
the CRISPR/Cas system. Plant J 98: 577–589
[8] Schmidt C, Fransz P, Rönspies M et al. (2020)
Changing local recombination patterns in Arabidopsis by
CRISPR/Cas mediated chromosome engineering. Nat
Commun 11: 4418
[9] Schwartz C, Lenderts B, Feigenbutz L et al. (2020)
CRISPR-Cas9-mediated 75.5-Mb inversion in maize. Nat
Plants 6: 1427–1431
[10] Beying N, Schmidt C, Pacher M et al. (2020)
CRISPR-Cas9-mediated induction of heritable chromo-
somal translocations in Arabidopsis. Nat Plants 6: 638–
645
[11] Lee K, Wang K (2020) Level up to chromosome
restructuring. Nat Plants 6: 600–601 Mit den hier beschriebenen Studien wur-
de eine neue Ebene der Anwendung von
CRISPR-Cas für die Pfl anzenzüchtung,
aber auch die Grundlagenforschung
erreicht. Die hier vorgestellten Fortschrit-
te werfen natürlich noch weitreichendere
Fragen auf, was in Zukunft machbar, aber
eben für die Pfl anzenzüchtung sinnvoll
ist. Korrespondenzadresse:
Prof. Dr. Holger Puchta
Botanisches Institut
Karlsruher Institut für Technologie (KIT)
Fritz-Haber-Weg 4
D-76128 Karlsruhe
holger.puchta@kit.edu [6] Rönspies M, Dorn A, Schindele P et al. (2021)
CRISPR-Cas-mediated chromosome engineering for crop
improvement and synthetic biology. Nat Plants 7:566–
573 Induktion chromosomaler
Veränderungen mittels CRISPR-Cas So scheint es nun im Bereich des Mög-
lichen, dass wir mit dieser Technologie
nicht nur die Anzahl der Chromosomen in
Pfl anzenspezies ändern, sondern auch
durch
globale
Chromosomen-
restrukturierungen neue Spezies erhalten
können [11]. ó [10] Beying N, Schmidt C, Pacher M et al. (2020)
CRISPR-Cas9-mediated induction of heritable chromo- somal translocations in Arabidopsis. Nat Plants 6: 638–
645 [11] Lee K, Wang K (2020) Level up to chromosome
restructuring. Nat Plants 6: 600–601 Funding note: Open Access funding enabled and organized by Projekt
DEAL.
Open Access: Dieser Artikel wird unter der Creative Commons
Namensnennung 4.0 International Lizenz veröffentlicht, welche die
Nutzung, Vervielfältigung, Bearbeitung, Verbreitung und Wiedergabe in
jeglichem Medium und Format erlaubt, sofern Sie den/die ursprünglichen
Autor(en) und die Quelle ordnungsgemäß nennen, einen Link zur Creative
Commons Lizenz beifügen und angeben, ob Änderungen vorgenommen
wurden. Die in diesem Artikel enthaltenen Bilder und sonstiges
Drittmaterial unterliegen ebenfalls der genannten Creative Commons
Lizenz, sofern sich aus der Abbildungslegende nichts anderes ergibt.
Sofern das betreffende Material nicht unter der genannten Creative
Commons Lizenz steht und die betreffende Handlung nicht nach
gesetzlichen Vorschriften erlaubt ist, ist für die oben aufgeführten
Weiterverwendungen des Materials die Einwilligung des jeweiligen
Rechteinhabers einzuholen. Weitere Details zur Lizenz entnehmen Sie bitte
der Lizenzinformation auf http://creativecommons.org/licenses/by/4.0/
deed.de. [1] Wolter F, Schindele P, Puchta H (2019) Plant breed-
ing at the speed of light: the power of CRISPR/Cas to gen-
erate directed genetic diversity at multiple sites. BMC
plant biology 19: 176
[2] Wolter F, Puchta H (2017) Genome Engineering mit
CRISPR/Cas – Revolution in der Pfl anzenzüchtung.
BIOspektrum 23: 159–161
[3] Puchta H (2005) The repair of double-strand breaks
in plants: mechanisms and consequences for genome
evolution. J Exp Bot 56: 1–14
[4] Schmidt C, Schindele P, Puchta H (2020) From gene
editing to genome engineering: restructuring plant chro-
mosomes via CRISPR/Cas. aBIOTECH 1: 21–31
[5] Schindele A, Dorn A, Puchta H (2020) CRISPR/Cas
brings plant biology and breeding into the fast lane. Curr
Opin Biotechnol 61: 7–14
[6] Rönspies M, Dorn A, Schindele P et al. (2021)
CRISPR-Cas-mediated chromosome engineering for crop
improvement and synthetic biology. Nat Plants 7:566–
573 Literatur [1] Wolter F, Schindele P, Puchta H (2019) Plant breed-
ing at the speed of light: the power of CRISPR/Cas to gen-
erate directed genetic diversity at multiple sites. BMC
plant biology 19: 176 [9] Schwartz C, Lenderts B, Feigenbutz L et al. (2020)
CRISPR-Cas9-mediated 75.5-Mb inversion in maize. Nat
Plants 6: 1427–1431 Holger Puchta Biochemiestudium an den Universitäten Tübingen und München. Promotion
am Max-Planck-Institut für Biochemie, Martinsried. 1989–1995 Postoc am
Friedrich-Miescher-Institut in Basel, Schweiz. 1995–2002 Gruppenleiter am
Institut für Pfl anzengenetik und Kulturpfl anzenforschung, Gatersleben. Seit
2002 Professor für Molekularbiologie und Biochemie am Karlsruher Institut
für Technologie (KIT). Rebecca Wetzel
2013–2018 Studium der Biologie am Karlsruher Institut für Technologie (KIT). Seit 2019 Promotion am Lehrstuhl für Molekularbiologie und Biochemie des
KIT. Patrick Schindele
2011–2016 Studium der Biologie am Karlsruher Institut für Technologie (KIT). 2016–2020 Promotion am Lehrstuhl für Molekularbiologie und Biochemie am
KIT. Seit 2020 Postdoc am Lehrstuhl für Molekularbiologie und Biochemie am
KIT. BIOspektrum | 06.21 | 27. Jahrgang BIOspektrum | 06.21 | 27. Jahrgang
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Fingerprinting the effects of hyperfine structure on CH and OH far infrared spectra using Wiener filter deconvolution
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Astronomy & astrophysics
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1. Introduction studies and abundance measurements, in particular in the sub-
millimetre and far-infrared (FIR) regions. Observations made
between 2010 and 2013 with the Herschel-High Frequency
Instrument for the Far-Infrared (HIFI, de Graauw et al. 2010),
later followed by the German REceiver at Terahertz frequencies
(GREAT, Heyminck et al. 2012) on board of the Stratospheric
Observatory for Infrared Astronomy (SOFIA, Young et al. 2012)
have resulted in high resolution (down to 10−7) absorption spec-
tra of a plethora of light hydride molecules of which the study
is important for various reasons (for an overview see Gerin et al. 2016). In many cases, the background sources reside far away in
the inner Galaxy. Thus the differential Galactic rotation causes
complex velocity crowding, which is further complicated by the
HFS of many of these molecules, including CH and OH, which
are studied here. All this requires advanced techniques of anal-
ysis of the observed line profiles. Several different approaches
have previously been used to fit the HFS of various spectral
lines through techniques ranging from simulated annealing algo-
rithms (Wiesemeyer et al. 2016) and Monte Carlo approaches
that use genetic algorithms (Estalella 2017) to direct deconvo-
lution (Gerin et al. 2010), and multi-Gaussian fitting techniques
(Neufeld et al. 2015; Persson et al. 2016). In this paper, we intro-
duce a technique that decomposes the contributions of HFS from
astrophysical spectra, based on the theory developed by Wiener
(1949). A linear and time-invariant method, the Wiener filter Hyperfine structure (HFS) splitting is a commonly observed phe-
nomenon in spectroscopy that arises from interactions between
unfilled electron shells and the nuclear spin magnetic moment of
atoms or molecules. It leaves a unique imprint on the spectrum
of a given chemical species and transition, much like finger-
prints. Over the years, knowledge of HFS in atoms and molecules
has served both atomic-, molecular-, and astro-physicists alike in
interpreting observations. The effects of such splitting, despite
being small, leads to energy differences that are typically of the
order of ∼0.03 K in terms of temperature. Since HFS interac-
tions broaden, shift, and even alter the shape of spectra (Booth &
Blackwell 1983), not taking them into consideration might lead
to erroneous interpretations of astrophysical quantities. ⋆The reduced observed spectra are only available at the CDS via
anonymous ftp to cdsarc.u-strasbg.fr (130.79.128.5) or via
http://cdsarc.u-strasbg.fr/viz-bin/cat/J/A+A/632/A60 ABSTRACT 2010),
later followed by the German REceiver at Terahertz frequencies
(GREAT, Heyminck et al. 2012) on board of the Stratospheric
Observatory for Infrared Astronomy (SOFIA, Young et al. 2012)
have resulted in high resolution (down to 10−7) absorption spec-
tra of a plethora of light hydride molecules of which the study
is important for various reasons (for an overview see Gerin et al. 2016). In many cases, the background sources reside far away in
the inner Galaxy. Thus the differential Galactic rotation causes
complex velocity crowding, which is further complicated by the
HFS of many of these molecules, including CH and OH, which
are studied here. All this requires advanced techniques of anal-
ysis of the observed line profiles. Several different approaches
have previously been used to fit the HFS of various spectral
lines through techniques ranging from simulated annealing algo-
rithms (Wiesemeyer et al. 2016) and Monte Carlo approaches
that use genetic algorithms (Estalella 2017) to direct deconvo-
lution (Gerin et al. 2010), and multi-Gaussian fitting techniques
(Neufeld et al. 2015; Persson et al. 2016). In this paper, we intro-
duce a technique that decomposes the contributions of HFS from
astrophysical spectra, based on the theory developed by Wiener
(1949). A linear and time-invariant method, the Wiener filter Fingerprinting the effects of hyperfine structure on CH and OH
far infrared spectra using Wiener filter deconvolution⋆
Arshia M. Jacob1, Karl M. Menten1, Helmut Wiesemeyer1, Min-Young Lee1,2, Rolf Güsten1, and Carlos A. Durán1 1 Max-Planck-Institut für Radioastronomie, Auf dem Hügel 69, 53121 Bonn, Germany
e-mail: ajacob@mpifr-bonn.mpg.de 2 Korea Astronomy and Space Science Institute, 776 Daedeokdae-ro, 34055 Daejeon, Republic of Korea Received 6 June 2019 / Accepted 17 October 2019 Astronomy
&
Astrophysics Astronomy
&
Astrophysics A&A 632, A60 (2019)
https://doi.org/10.1051/0004-6361/201936037
© A. M. Jacob et al. 2019 A&A 632, A60 (2019)
https://doi.org/10.1051/0004-6361/201936037
© A. M. Jacob et al. 2019 A60, page 1 of 14
Open Access article, published by EDP Sciences, under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Open Access funding provided by Max Planck Society. ABSTRACT Context. Despite being a commonly observed feature, the modification of the velocity structure in spectral line profiles by hyperfine
structure complicates the interpretation of spectroscopic data. This is particularly true for observations of simple molecules such as
CH and OH toward the inner Galaxy, which show a great deal of velocity crowding. Aims. In this paper, we investigate the influence of hyperfine splitting on complex spectral lines, with the aim of evaluating canonical
abundances by decomposing their dependence on hyperfine structures. This is achieved from first principles through deconvolution. Methods. We present high spectral resolution observations of the rotational ground state transitions of CH near 2 THz seen
in absorption toward the strong FIR-continuum sources AGAL010.62−00.384, AGAL034.258+00.154, AGAL327.293−00.579,
AGAL330.954−00.182, AGAL332.826−00.549, AGAL351.581−00.352 and SgrB2(M). These were observed with the GREAT instru-
ment on board SOFIA. The observed line profiles of CH were deconvolved from the imprint left by the lines’ hyperfine structures using
the Wiener filter deconvolution, an optimised kernel acting on direct deconvolution. p
g
Results. The quantitative analysis of the deconvolved spectra first entails the computation of CH column densities. Reliable N(CH)
values are of importance owing to the status of CH as a powerful tracer for H2 in the diffuse regions of the interstellar medium. The
N(OH)/N(CH) column density ratio is found to vary within an order of magnitude with values ranging from one to 10, for the individual
sources that are located outside the Galactic centre. Using CH as a surrogate for H2, we determined the abundance of the OH molecule
to be X(OH) = 1.09 × 10−7 with respect to H2. The radial distribution of CH column densities along the sightlines probed in this study,
excluding SgrB2(M), showcase a dual peaked distribution peaking between 5 and 7 kpc. The similarity between the correspondingly
derived column density profile of H2 with that of the CO-dark H2 gas traced by the cold neutral medium component of [CII] 158 µm
emission across the Galactic plane, further emphasises the use of CH as a tracer for H2. Key words. ISM: abundances – ISM: clouds – ISM: lines and bands – ISM: molecules – methods: data analysis studies and abundance measurements, in particular in the sub-
millimetre and far-infrared (FIR) regions. Observations made
between 2010 and 2013 with the Herschel-High Frequency
Instrument for the Far-Infrared (HIFI, de Graauw et al. Open Access article, published by EDP Sciences, under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Open Access funding provided by Max Planck Society. 2. Theory The convolution theorem
renders this task trivial, as it reduces the operations of both con-
volution and deconvolution into those of simple multiplication
and division, in Fourier space, given a priori knowledge of g(ν)
and h(ν). Hence, in Fourier space, Eq. (1) becomes G(˜ν) = F(˜ν) · H(˜ν)
Convolution;
F(˜ν) = G(˜ν)
H(˜ν)
Deconvolution,
(2) G(˜ν) = F(˜ν) · H(˜ν)
Convolution; (2) p
Concentrating for most of this paper on the transitions of
a single molecule, we illustrate the working of the Wiener fil-
ter algorithm on spectra that are of astrophysical importance,
through the spectroscopy of the N, J = 2, 3/2 →1, 1/2 ground
state rotational transitions of CH, the methylidene radical. CH
was amongst the first molecules detected in the interstellar
medium (ISM) by Dunham (1937), and identified by Swings &
Rosenfeld (1937). Being the simplest organic carbyne and one
of the lightest hydrides, CH initiates the formation of a large
fraction of the molecules present in the ISM, thereby playing a
pivotal role in both the physics and chemistry of interstellar gas
clouds. CH has been widely studied in various spectral regimes
to infer the thermal, chemical and dynamical evolution of diffuse
and translucent cloud regions (Rydbeck et al. 1973; Turner &
Zuckerman 1974; Stacey et al. 1987). Moreover, the advent of
space- and air-borne telescopes like Herschel and SOFIA have
established the use of CH alongside other interstellar hydrides as
tracers for H2 in the diffuse regions of the ISM (Gerin et al. 2010;
Wiesemeyer et al. 2018). Figure 1 shows the low-lying energy
level structure of the ground electronic state of the CH molecule,
abiding by Hund’s case b. The total angular momentum states
labelled J splits into a pair of Λ-doublet levels of opposing parity
(±Λ). Each of these are further split into hyperfine levels given
by F = J ± I, where I is the nuclear spin of the H atom, I(H) =
1/2. where G(˜ν), F(˜ν) and H(˜ν) represent the Fourier transforms
(FTs) of g(ν), f(ν) and h(ν), respectively. However, such a point-
wise division of the Fourier transforms is sufficient to obtain an
estimate of f(ν), only as long as the transform of the response
function remains non-zero over all frequencies. Moreover, realis-
tic systems are influenced by noise, n(ν), which further degrades
the observed spectrum, d(ν). 1. Introduction Overall,
taking HFS splitting into account makes it possible to precisely
model and resolve the underlying spectral line shapes, since
the observed integral line profile is a superposition of the var-
ious HFS components. In recent years, the onset of improved
observational techniques with greater sensitivity and higher
spectral resolution have paved the way for rigorous spectroscopic ⋆The reduced observed spectra are only available at the CDS via
anonymous ftp to cdsarc.u-strasbg.fr (130.79.128.5) or via
http://cdsarc.u-strasbg.fr/viz-bin/cat/J/A+A/632/A60 A&A 632, A60 (2019) in Sect. 2.2, we detail our procedure for spectral line analysis:
the Wiener filter algorithm. Having motivated our astronomical
case study on the N, J = 2, 3/2 →1, 1/2 HFS transitions of CH,
in Sect. 3, we describe our observations. In Sect. 4, we present
the implementation of the Wiener filter on the absorption spec-
tra and its analysis, while the results obtained are discussed in
Sect. 5, in which we also compare the distribution of CH with
that of OH, and explore the usefulness of CH as a tracer for H2. We conclude this work in Sect. 6. CH
N = 2
J = 3/2
N = 1
J = 1/2
N = 1
Parity
+
–
Parity
–
+
F
2
1
2
1
0
1
1
0
F
1
2
1
2
Parity
+
–
J = 3/2
2006 GHz
2006 GHz
532 GHz
532 GHz
Fig. 1. Low-lying energy level structure of CH. The N, J = 2, 3/2 →
1, 1/2 HFS transitions of CH near 2006 GHz that were observed using
SOFIA are represented in red while the N, J = 1, 3/2 →1, 1/2 HFS
transitions near 532 GHz observed with Herschel/HIFI are displayed in
blue. CH
N = 2
J = 3/2
N = 1
J = 1/2
N = 1
Parity
+
–
Parity
–
+
F
2
1
2
1
0
1
1
0
F
1
2
1
2
Parity
+
–
J = 3/2
2006 GHz
2006 GHz
532 GHz
532 GHz Parity
+ Parity 2. Theory This additive noise is often ampli-
fied when directly deconvolved, because it acts as a low-pass
filter and gives rise to faux features in the reconstructed signal. Such issues of sensitivity are often tackled by using filters, and in
the following sections, we introduce and employ one such filter,
namely the Wiener filter. where G(˜ν), F(˜ν) and H(˜ν) represent the Fourier transforms
(FTs) of g(ν), f(ν) and h(ν), respectively. However, such a point-
wise division of the Fourier transforms is sufficient to obtain an
estimate of f(ν), only as long as the transform of the response
function remains non-zero over all frequencies. Moreover, realis-
tic systems are influenced by noise, n(ν), which further degrades
the observed spectrum, d(ν). This additive noise is often ampli-
fied when directly deconvolved, because it acts as a low-pass
filter and gives rise to faux features in the reconstructed signal. Such issues of sensitivity are often tackled by using filters, and in
the following sections, we introduce and employ one such filter,
namely the Wiener filter. Time invariant in first and second order statistics. 2. Theory Parity Convolution is a formal mathematical operation used to describe
the interaction between an input signal, f(ν), and an impulse
response function, h(ν), to produce an output signal, g(ν), in a
linear system. In spectroscopic terms, the input and output sig-
nals are analogous to the original and observed spectra while
the impulse response in this case arises from the HFS split-
ting. For a discrete sample, such a convolution model can be
mathematically formulated as follows: Fig. 1. Low-lying energy level structure of CH. The N, J = 2, 3/2 →
1, 1/2 HFS transitions of CH near 2006 GHz that were observed using
SOFIA are represented in red while the N, J = 1, 3/2 →1, 1/2 HFS
transitions near 532 GHz observed with Herschel/HIFI are displayed in
blue. g(ν) = f(ν) ⊛h(ν) =
+∞
X
ν′=−∞
f(ν′)h(ν −ν′). (1) (1) theory has found its application in practical signal reconstruction
scenarios. It stands out from other filtering techniques because it
provides the maximum a posteriori estimate of the unknown sig-
nal through a simple least squares analysis. Consequently, the
Wiener filter has also found its application in astronomy as a
tool that aids in cleaning contaminated images retrieved by aper-
ture synthesis techniques (Caprari et al. 2000). In cosmological
studies, deconvolution using the Wiener filter has been employed
to accurately decompose the observed polarization of the cosmic
microwave background (CMB) radiation into maps of pure polar-
ization for its two components: E and B (Bunn & Wandelt 2017). Given the proven capability of the Wiener filter deconvolution as
a method of choice for decomposition, we decided to adapt it for
a novel application, the deconvolution of HFS from astrophysical
spectra. With astrophysical observations, one is posed with the inverse
problem of convolution, which is deconvolution, wherein the
output spectrum is known, while its main constituent, the orig-
inal spectrum, remains unknown. The convolution theorem
renders this task trivial, as it reduces the operations of both con-
volution and deconvolution into those of simple multiplication
and division, in Fourier space, given a priori knowledge of g(ν)
and h(ν). Hence, in Fourier space, Eq. (1) becomes With astrophysical observations, one is posed with the inverse
problem of convolution, which is deconvolution, wherein the
output spectrum is known, while its main constituent, the orig-
inal spectrum, remains unknown. ˆF(˜ν) = D(˜ν)W(˜ν)
⇒
ˆF(˜ν) = [F(˜ν)H(˜ν) + N(˜ν)] W(˜ν).
(3) ˆF(˜ν) = D(˜ν)W(˜ν)
⇒
ˆF(˜ν) = [F(˜ν)H(˜ν) + N(˜ν)] W(˜ν).
(3
1 Time invariant in first and second order statistics. 2.1. Wiener filter deconvolution Wiener’s theory formulated a linear tool, the Wiener filter (WF)
for additive noise reduction aimed to resolve the signal restora-
tion problems (singularities) faced by direct deconvolution. The
WF model assumes all signals to be stationary1, and modelled
by linear stochastic processes with a signal-independent noise. The WF output in the Fourier or inverse frequency domain, ˆF(˜ν)
is the product of the (noise) degraded observed spectrum, D(˜ν),
and the frequency response of the filter, W(˜ν): (3) The outline of this paper is as follows: in Sect. 2.1, we
develop the theory behind the working of the Wiener filter, and A60, page 2 of 14 A. M. Jacob et al.: Fingerprinting the effects of hyperfine structure t-
he
4)
5)
6)
f(ν)
⊛
h(ν)
+
n(ν)
w(ν)
ˆf(ν)
g(ν)
d(ν)
Fig. 2. Schematic diagram showcasing working principle of the Wiener
filter in frequency space. The filter further models the input signal by implement-
ing a minimum mean square error (MSE) ϵ2 constraint on the
deconvolution: The filter further models the input signal by implement-
ing a minimum mean square error (MSE) ϵ2 constraint on the
deconvolution:
h( )
( ) ϵ2 =
X
˜ν
F(˜ν) −ˆF(˜ν)
2
(4)
=
X
˜ν
|F(˜ν) −[F(˜ν)H(˜ν) + N(˜ν)] W(˜ν)|2
(5)
=
X
˜ν
|F(˜ν)[1 −H(˜ν)W(˜ν)] −N(˜ν)W(˜ν)|2
(6)
f(ν)
⊛
h(ν)
+
n(ν)
g(ν) ϵ2 =
X
˜ν
F(˜ν) −ˆF(˜ν)
2
(4)
=
X
˜ν
|F(˜ν) −[F(˜ν)H(˜ν) + N(˜ν)] W(˜ν)|2
(5)
=
X
˜ν
|F(˜ν)[1 −H(˜ν)W(˜ν)] −N(˜ν)W(˜ν)|2
(6)
f(ν)
⊛
h(ν)
+
n(ν)
w(ν)
ˆf(ν)
g(ν)
d( ) Expanding the quadratic term in Eq. (6), Expanding the quadratic term in Eq. (6), Fig. 2. Schematic diagram showcasing working principle of the Wiener
filter in frequency space. ϵ2 =
X
˜ν
([1 −H(˜ν)W(˜ν)] [1 −H(˜ν)W(˜ν)]∗) |F(˜ν)|2 (7)
2.2. WF algorithm using ∂(zz∗)
∂z
= 2z∗(property of conjugates), where the asterisk
represents the complex conjugate. Minimising ∂ϵ2/∂W(˜ν) by
equating Eq. (9) to zero and solving for W(˜ν) results in the
general form of the Wiener filter, using ∂(zz∗)
∂z
= 2z∗(property of conjugates), where the asterisk
represents the complex conjugate. Minimising ∂ϵ2/∂W(˜ν) by
equating Eq. (9) to zero and solving for W(˜ν) results in the
general form of the Wiener filter, W(˜ν) =
H∗(˜ν)
|H(˜ν)|2 +
N(˜ν)
F(˜ν)
2 . W(˜ν) =
H∗(˜ν)
|H(˜ν)|2 +
N(˜ν)
F(˜ν)
2 . Since the exact forms of both N(˜ν) and F(˜ν) are model-
dependent, they remain as unknowns for most practical systems. However, for additive white noise that is independent of the sig-
nal, the F(˜ν)
N(˜ν) ratio can be approximated by the signal-to-noise
ratio (S/N), or some form of normalized noise variance, σ˜ν, char-
acterised by the system noise. Rearranging the terms to fit the
inverse filter formulation, the Wiener filter and restored spectra
are given as: The next step of the algorithm determines the kernel term,
which constitutes the hyperfine response and the noise term. Hence, the quality of the restored spectrum relies both on the
impulse response of the HFS and an accurate representation
of the system noise. The effects of HFS splitting are charac-
terised by using weighted impulses in the frequency domain. The
impulse response of each hyperfine component is represented
by a Dirac-δ function, weighted by the relevant spectroscopic
parameters. The weighting function, Ω(HFS), accounts for the
fraction of the specified species in the upper energy level, Eu (in
Kelvin) of a given transition that has an upper-level degeneracy,
gu and Einstein A coefficient, AE as follows: W(˜ν) =
1
H(˜ν)
|H(˜ν)|2
|H(˜ν)|2 +
1
S/N
2
and
(10)
ˆF(˜ν) =
D(˜ν)
H(˜ν)
|{z}
Inverse filter
|H(˜ν)|2
|H(˜ν)|2 +
1
S/N
2
| {z }
Kernel
. (11) W(˜ν) =
1
H(˜ν)
|H(˜ν)|2
|H(˜ν)|2 +
1
S/N
2
and
(10)
ˆF(˜ν) =
D(˜ν)
H(˜ν)
|{z}
Inverse filter
|H(˜ν)|2
|H(˜ν)|2 +
1
S/N
2
| {z }
Kernel
. (11) W(˜ν) =
1
H(˜ν)
|H(˜ν)|2
|H(˜ν)|2 +
1
S/N
2
and
(10) (10) (11) Ω(HFS) =
guAE
Q(Trot)exp
−Eu
Tex
! . (13) (13) Inverse filter Kernel Figure 2 illustrates the idea behind the WF deconvolution algo-
rithm as expressed in Eq. (11). (7)
2.2. WF algorithm −[1 −H(˜ν)W(˜ν)] W(˜ν)∗(F(˜ν)N(˜ν)∗)
−W(˜ν) [1 −H(˜ν)W(˜ν)]∗(N(˜ν)F(˜ν)∗) + |W(˜ν)|2|N(˜ν)|2. This section features a brief description of our WF fitting and
deconvolution algorithm written in python, based on the math-
ematical formulation discussed in Sect. 2.1. The WF algorithm
uses the radiative transfer equation to express the observed line
profile, Tl, of a given molecular transition containing nHFS hyper-
fine components in terms of optical depths, τ. This is done
under the assumption of local thermodynamic equilibrium (LTE)
and unsaturated absorption. The formal solution of the radiative
transfer equation for a constant excitation temperature reads: We assume that N(˜ν) is independent of F(˜ν), therefore
(F(˜ν) · N(˜ν)∗) = (N(˜ν) · F(˜ν)∗) = 0. This reduces Eq. (7) to, ϵ2 =
X
˜ν
|F(˜ν)|2 |1 −H(˜ν)W(˜ν)|2 + |N(˜ν)|2 |W(˜ν)|2 . (8) (8) (8) The derivative of the MSE, ϵ2 with respect to W(˜ν) yields Tl = ΦlTex(1 −e−τ) + ΦcTce−τ,
(12) ∂ϵ2
∂W(˜ν) = |F(˜ν)|2 2(1 −W∗H∗)(−H) + |N(˜ν)|2 2W∗ ,
(9) (12) (9) where the excitation temperature, Tex and the background con-
tinuum, Tc are expressed as Rayleigh–Jeans equivalent temper-
atures, and Φl and Φc are the beam filling factors of the line
and continuum emission, respectively. For the specific case of
absorption spectroscopy of far-infrared ground state transitions
in low-excitation gas (hν » kTex), we can neglect the first term
and recast the radiative transfer equation displayed in Eq. (12)
as Tl = ΦcTce−τ. In cases where we cannot neglect the emis-
sion term, particularly in the immediate environment of the
continuum source, a realistic description needs to account for
an excitation gradient, and therefore requires a full non-LTE
solution, which is outside the scope of this paper. where the excitation temperature, Tex and the background con-
tinuum, Tc are expressed as Rayleigh–Jeans equivalent temper-
atures, and Φl and Φc are the beam filling factors of the line
and continuum emission, respectively. For the specific case of
absorption spectroscopy of far-infrared ground state transitions
in low-excitation gas (hν » kTex), we can neglect the first term
and recast the radiative transfer equation displayed in Eq. (12)
as Tl = ΦcTce−τ. In cases where we cannot neglect the emis-
sion term, particularly in the immediate environment of the
continuum source, a realistic description needs to account for
an excitation gradient, and therefore requires a full non-LTE
solution, which is outside the scope of this paper. (7)
2.2. WF algorithm Therefore, a reliable estimation
of the “original”, underlying spectrum using the WF deconvo-
lution is dependent upon the assumptions made in determining
the kernel term, W(˜ν), of the filter (Eq. (11)). The kernel term is
dependent on the noise of the system and on the response func-
tion, which in this case is tailored to address the effects of HFS
splitting. The following section details the kernel estimation, as
well as the adopted algorithm. For a given hyperfine transition, all of the above spectroscopic
terms remain constant, except for the partition function, Q,
which itself is a function of rotation temperature, Trot, which in
our case (LTE) is equal to the excitation temperature, Tex. When
deriving the formulation of the WF, in Sect. 2.1, the noise term
was approximated to be the inverse of the S/N. While this serves
as an acceptable choice for the noise contribution, it can be fur-
ther refined and expressed explicitly as a function of the receiver
system, which we will not discuss here in this paper. For a given hyperfine transition, all of the above spectroscopic
terms remain constant, except for the partition function, Q,
which itself is a function of rotation temperature, Trot, which in
our case (LTE) is equal to the excitation temperature, Tex. When
deriving the formulation of the WF, in Sect. 2.1, the noise term
was approximated to be the inverse of the S/N. While this serves
as an acceptable choice for the noise contribution, it can be fur-
ther refined and expressed explicitly as a function of the receiver
system, which we will not discuss here in this paper. A60, page 3 of 14 A60, page 3 of 14 A&A 632, A60 (2019) Table 1. Continuum source parameters. Continuum
RA (J2000)
Dec (J2000)
l
b
D
3LSR
Tc
source
[h m s]
[◦′ ′′]
[◦]
[◦]
[kpc]
[km s−1]
[K]
AGAL010.624−00.384 (G10.62)
18:10:28.69
−19:55:50.0
10.624
−0.383
4.9
−2.9
8.2
AGAL034.258+00.154 (G34.26)
18:53:18.49
+01:14:58.7
34.257
+0.153
1.6
+58.5
7.5
AGAL327.293−00.579 (G327.29)
15:53:08.55
−54:37:05.1
327.292
−0.578
3.1
−44.7
2.5
AGAL330.954−00.182 (G330.95)
16:09:53.01
−51:54:55.0
330.954
−0.183
9.3
−91.2
11.7
AGAL332.826−00.549 (G332.83)
16:20:10.65
−50:53:17.6
332.825
−0.549
3.6
−57.1
7.7
AGAL351.581−00.352 (G351.58)
17:25:25.03
−36:12:45.3
351.580
−0.352
6.8
−95.9
5.2
Sgr B2(M)
17:47:20.16
−28:23:04.5
0.667
−0.036
8.3
+64.0
15.1
Notes. 4 Software package developed by IRAM, see https://www.iram.
fr/IRAMFR/GILDAS/ for more information regarding GILDAS pack-
ages. (7)
2.2. WF algorithm Columns are, left to right, source designation, equatorial coordinates, Galactic coordinates, heliocentric distance, LSR velocity, and signal
band continuum brightness temperature derived by means of a DSB calibration. References. Heliocentric distance references: AGAL010.624−00.384: Sanna et al. (2014), AGAL034.258+00.154: Zhang et al. (2009),
AGAL327.293−00.579: Urquhart et al. (2013), AGAL330.954−00.182: Urquhart et al. (2012), AGAL332.826−00.549: Moisés et al. (2011),
AGAL351.581−00.352: Green & McClure-Griffiths (2011), Sgr B2(M): Reid et al. (2014). g
p
y
References. Heliocentric distance references: AGAL010.624−00.384: Sanna et al. (2014), AGAL034.258+00.154: Zhang et al. (2009),
AGAL327.293−00.579: Urquhart et al. (2013), AGAL330.954−00.182: Urquhart et al. (2012), AGAL332.826−00.549: Moisés et al. (2011),
AGAL351.581−00.352: Green & McClure-Griffiths (2011), Sgr B2(M): Reid et al. (2014). g
p
y
References. Heliocentric distance references: AGAL010.624−00.384: Sanna et al. (2014), AGAL034.258+00.154: Zhang et al. (2009),
AGAL327.293−00.579: Urquhart et al. (2013), AGAL330.954−00.182: Urquhart et al. (2012), AGAL332.826−00.549: Moisés et al. (2011),
AGAL351.581−00.352: Green & McClure-Griffiths (2011), Sgr B2(M): Reid et al. (2014). Table 2. Spectroscopic parameters for N, J = 2, 3/2 →1, 1/2 hyperfine
transitions of CH. The WF kernel is then determined following Eq. (10), and the
algorithm then fits the spectrum. The standard deviation of the
residual distribution is used to measure the quality of the spectral
fit, and based on this, the algorithm carries out deconvolution
following Eq. (11). In the last step, the WF algorithm applies an
inverse FT to convert the modelled spectrum ˆF(˜ν) into ˆf(ν) in
frequency space. The deconvolved optical depth signature, τdecon
is then converted to molecular column density values using transitions of CH. Transition
Frequency
AE
Eu
Parity
F
[GHz]
10−2 × [s−1]
[K]
−→+
1 →1
2006.74892
1.117
96.31
1 →0
2006.76263
2.234
2 →1
2006.79912
3.350
+ →−(∗)
1 →1
2010.73859
1.128
96.66
1 →0
2010.81046
2.257
2 →1
2010.81192
3.385
Notes. Columns are quantum number information, frequency, Ein-
stein A coefficient and upper level energy; all taken from the Cologne
Database for Molecular Spectroscopy (Müller et al. 2005). The fre-
quencies were determined by Davidson et al. (2001). (∗) – indicate the
transitions that are not observed in this experiment. Transition
Frequency
AE
Eu
Parity
F
[GHz]
10−2 × [s−1]
[K]
−→+
1 →1
2006.74892
1.117
96.31
1 →0
2006.76263
2.234
2 →1
2006.79912
3.350
+ →−(∗)
1 →1
2010.73859
1.128
96.66
1 →0
2010.81046
2.257
2 →1
2010.81192
3.385 dN
d3
! i
= 8πν3
i
c3Ω
"
exp
hνi
kBTex
! 2 upGREAT, an extension of the German REceiver for Astronomy
at Terahertz frequencies, is a development by the MPI für Radioas-
tronomie and KOSMA/Universität zu Köln, in cooperation with the
MPI für Sonnensystemforschung, and the DLR Institut für Optische
Sensorsysteme.
3 APEX Telescope Large Area Survey of the GALaxy (ATLASGAL) (7)
2.2. WF algorithm −1
#−1
(τi)decon,
(14) (14) for each velocity channel, i, since, the backend provides the
frequencies (and the velocities) as a discrete sequence of data,
which was further smoothed by us to a useful spectral resolution
as explained in Sect. 3 and where Ωrepresents the mean HFS
weight. Having established the general scheme of the WF algo-
rithm, the following sections detail its functioning on spectra that
are of astrophysical importance. for each velocity channel, i, since, the backend provides the
frequencies (and the velocities) as a discrete sequence of data,
which was further smoothed by us to a useful spectral resolution
as explained in Sect. 3 and where Ωrepresents the mean HFS
weight. Having established the general scheme of the WF algo-
rithm, the following sections detail its functioning on spectra that
are of astrophysical importance. Notes. Columns are quantum number information, frequency, Ein-
stein A coefficient and upper level energy; all taken from the Cologne
Database for Molecular Spectroscopy (Müller et al. 2005). The fre-
quencies were determined by Davidson et al. (2001). (∗) – indicate the
transitions that are not observed in this experiment. Notes. Columns are quantum number information, frequency, Ein-
stein A coefficient and upper level energy; all taken from the Cologne
Database for Molecular Spectroscopy (Müller et al. 2005). The fre-
quencies were determined by Davidson et al. (2001). (∗) – indicate the
transitions that are not observed in this experiment. the CH hyperfine transitions at 2010 GHz. These ozone lines,
despite having relatively narrow spectral features, contaminate
the CH absorption measurements made toward the strong contin-
uum sources. Therefore, the receivers were tuned to 2006.7 GHz
in the upper side band mode. 3 APEX Telescope Large Area Survey of the GALaxy (ATLASGAL). 3. GREAT observations and data reduction The
observations
presented
here
were
performed
using
upGREAT2/LFA (Risacher et al. 2018), on board SOFIA over its
cycle 5 and 6 campaigns, in a number of the observatory’s flights
(on 26 June and 6 July in 2017, and 20 and 22 June in 2018). Our source selection consists of several far-infrared bright hot
cores from the ATLASGAL3 survey (Csengeri et al. 2017) and
SgrB2(M). The observed sources, whose strong far-infrared dust
continuum emission serves as background for our absorption
measurements, are listed in Table 1. Both atmospheric as well as systematic fluctuations arising
from the instrumental set-up were removed by using a double
beam chop-nod mode. The secondary mirror chopped at a rate
of 2.5 Hz with a chop amplitude of 60′′ and 105′′ for the
observations carried out in 2017 and 2018. An advanced ver-
sion of the MPIfR-built Fast Fourier transform spectrometer
(dFFTS4G; Klein 2017) was used as the backend to carry out
the spectral analysis of the procured data. A velocity resolution
of 0.036 km s−1 was achieved by using a 4 GHz bandwidth per
pixel, with a channel spacing of 244 kHz over 16384 channels for
almost all the 2006 GHz spectra. The spectra were further cali-
brated using the KALIBRATE program (Guan et al. 2012) with
an underlying forward efficiency of 0.97 and a main-beam effi-
ciency of 0.68. The fully calibrated spectra were subsequently
analysed using the GILDAS-CLASS software4. The spectra were
smoothed to 0.36 km s−1-wide velocity bins, and up to a second The receiver configuration is comprised of the (7+7) pixel
low frequency array (LFA) receiver on upGREAT, in dual
polarization. Only one set of HFS transitions near 2006 GHz
(Table 2) was observed, because the atmospheric transmission
along some sight-lines was affected by telluric ozone absorp-
tion lines. The wavelengths of the ozone features at 149.1558 µm
and 149.7208 µm originating from the signal band coincide with A60, page 4 of 14 A. M. Jacob et al.: Fingerprinting the effects of hyperfine structure of CH increase by ∼15% at Tex = 10 K, and up to <50% at
Tex = 30 K. This further indicates that the uncertainties in our
column density estimates at velocity intervals corresponding to
those of the molecular cloud components predominantly arise
from our assumption of a single excitation temperature. 3. GREAT observations and data reduction Hence,
the column densities derived over their associated velocity com-
ponents represent a lower limit on the N(CH) values. The derived
column densities for each sightline are summarised in Table 3. The CH column densities per velocity component are found to
vary between ∼1012 and 1.2 × 1014 cm−2 along inter-arm and
spiral-arm clouds along the different sightlines. We inspect the
reliability of the column densities derived using the WF by
comparing them with other techniques in Sect. 4.2. order polynomial baseline was removed. After baseline subtrac-
tion, the continuum level determined by using a double-sideband
(DSB) calibration, was added back to the spectra to obtain the
correct line-to-continuum ratio. Based on the instrumental per-
formance and atmospheric transmission, we assume a 5% error
in the calibration of the continuum level. The relative sideband
gain is calibrated by assuming a signal-to-image band gain ratio
of unity, as determined by Kester et al. (2017) for all bands of the
HIFI instrument, with an accuracy between 1 and 4%. Hence,
even for the upGREAT instrument sharing the same receiver
technology as that of HIFI bands 6 and 7, we assume that there
is no significant departure in the band gain ratio from unity. The CH spectra are detected in deep, yet unsaturated absorp-
tion toward each individual sightline, even at velocities corre-
sponding to star forming regions (SFRs) as shown in Fig. 3. This is because almost all of the CH molecules are expected to
remain in the ground state within dilute envelopes and diffuse
clouds, surrounding the denser hot-core regions. The observed
absorption features correspond to the radial velocities expected
for different spiral arms, and are broadened by the kinematic
structure of the intervening absorbing medium. p
g
q
As the optical depths are computed from the line-to-
continuum ratio, uncertainties in the continuum level give rise to
systematic errors in the derived column densities. The uncertain-
ties are partly due to the receiver-gain ratio between the signal
and the image band, possibly deviating from unity. For the mixer
technology (hot-electron bolometers) used by upGREAT, this
quantity is difficult to measure without re-tuning the receiver. For the HIFI bands employing the same technology and clos-
est to our tuning, a dedicated study (Kester et al. 2017) provides
typical deviations of 1 to 4%. 5 The error in the derived column densities (per velocity interval) scale
with the deconvolved optical depths by a constant value. This constant
is a function of the spectroscopic parameters that govern the transition
and the excitation temperature. 3. GREAT observations and data reduction At this level, given the available
sensitivity and typical optical depths ranging from 0.1 to 1, the
impact of the continuum uncertainty on the derived column den-
sities is difficult to disentangle from that of the thermal noise in
the absorption profile. However, following the DSB error esti-
mation presented in Wiesemeyer et al. (2018), we account for
the uncertainties present in the continuum level that will subse-
quently lead to errors in the optical depths and derived column
densities. Further, the errors in the WF computed column den-
sities are determined by sampling a posterior distribution of the
deconvolved optical depths5. We sampled 5000 artificial spectra,
each generated by adding a pseudo-random noise contribution
with the same standard deviation as the line free part of the con-
tinuum, to the absorption spectra prior to applying the WF fit
and deconvolution, over each iteration. The deconvolved optical
depths and subsequently derived column densities (per veloc-
ity interval) sample a point in the channel-wise distribution of
the column densities across all spectra. The empirical spread
of these distributions yield the asymmetric errors in the com-
puted column densities. However, the column densities are not
always normally distributed, and often showcase skewed distri-
butions, as normality is not imposed. For this reason, the profiles
of the distributions are best described by using the median and
inter-quartile range from which the sample mean and standard
deviation are derived following Wan et al. (2014). This is done
so as to remove any biases introduced in the determination of the
mean due to the asymmetry of the distribution. The new mean
and standard deviation are then used to determine the asym-
metric errors through positive and negative deviations. While
errors in the deep absorption features corresponding to spiral-
arm velocities arise from imperfections in the fit, the errors in
the inter-arm gas features are dominated by uncertainties in the
true continuum. 4.1. WF deconvolution In Fig. 3, we display how the WF fits to the CH spectra
observed towards the different lines of sight (LOS), following
the algorithm described in Sect. 2.2. In Appendix A, we illus-
trate how the observed degraded spectrum is the convolution
product of the deconvolved spectrum and the hyperfine weights. For two sources, we compare the line profiles of the observed
spectrum with those of the deconvolved one in Fig. 4. The col-
umn densities determined from the former will be systematically
larger over velocity intervals corresponding to the molecular
clouds due to the line broadening effect of the HFS splitting,
as evidenced from the differences between the two line profiles. While the extent of these variations are sightline-dependent, we
find the column densities of CH determined from the observed
spectra (prior to deconvolution) to be on average 24.8% larger
than those determined post deconvolution, for the molecular
cloud features. The quality of the WF fit toward the varied
sightlines are assessed prior to deconvolution by means of the
fit residuals (see Appendix B). Furthermore, we discuss the
efficiency of the WF fit and deconvolution in Appendix C. The column densities are derived following Eq. (14) by
assuming an excitation temperature of ∼3.1 K, which is close
to the temperature of the cosmological blackbody radiation
(2.738 K), and was found to represent contributions of the Galac-
tic interstellar radiation field (Gerin et al. 2010), and integrated
over velocity intervals thought to be characteristic of spiral-
arm and inter-arm features, based on the spiral-arm structure
of the Milky Way presented by Reid et al. (2014), and consis-
tent with those used by Godard et al. (2012) and Wiesemeyer
et al. (2016). The ground state occupation assumption at LTE
(Tex = Trot = 3.1 K) is valid for the physical conditions that pre-
vail over diffuse and translucent regions, and is questionable
only within the molecular cloud components themselves. The
dense envelopes of these molecular clouds can result in higher
values of Tex as a consequence of collisional excitation of CH. The consequences of determining column densities by assum-
ing a single excitation temperature component along each LOS
is briefly discussed in Appendix D. Upon modelling the absorp-
tion features associated with the dense molecular environments
of our continuum sources, we find the derived column densities 4.2. Comparison of WF algorithm with other procedures Example illustrating effects of HFS splitting. The grey-filled area
represents the observed spectra toward AGAL327.293−00.579 (top) and
AGAL332.826−00.549 (bottom) in terms of optical depth, while their
WF deconvolved spectra are displayed in red. The green hashed regions
indicate velocity intervals where we observe line broadening due to HFS
splitting. from other commonly used techniques. To carry out an unbiased
analysis, we performed the WF algorithm on the N = 1, J =
3/2 →1/2 HFS spectra of CH near 532 GHz, obtained using
Herschel/HIFI toward SgrB2(M) as a part of the HEXOS6 sur-
vey toward SgrB2(M). The spectroscopic parameters of these
HFS transitions are summarised in Table E.1. The LOS toward
SgrB2(M) was chosen because of its complex spectral line
profile which contains an amalgamation of narrow and broad fea-
tures, alike. Qin et al. (2010) modelled the CH spectra by using
XCLASS (Möller et al. 2017) and the automated fitting routine
provided by MAGIX7 for an excitation temperature of 2.73 K. Assuming LTE, XCLASS solves the radiative transfer equa-
tion with an underlying Gaussian brightness profile to model
the observed spectral line profile. Using the WF algorithm, we
derived the column densities of the CH HFS transitions near
532 GHz, integrated over the same velocity intervals as those
specified in Table 1 of Qin et al. (2010). The errors were com-
puted as before, but with the assumption of a 20% error in the
continuum level. A comparison between the CH column densities derived
using the WF algorithm and the XCLASS fits is displayed in
Fig. 5 for all absorption features along the LOS except, for the
6 The guaranteed time key project, Herschel/HIFI observations of
EXtra-Ordinary Sources (HEXOS; Bergin et al. 2010) was aimed to
investigate the chemical composition of several sources in the Orion
and SgrB2 star-forming regions. 7 See https://magix.astro.uni-koeln.de/ for more informa-
tion about the MAGIX software. 0.0
2.0
4.0
AGAL327.293
00.579
100
50
0
VLSR (km/s)
0.0
2.0
obs
AGAL332.826
00.549
0.0
2.0
4.0
0.0
2.0
decon
Fig. 4. Example illustrating effects of HFS splitting. The grey-filled area
represents the observed spectra toward AGAL327.293−00.579 (top) and
AGAL332.826−00.549 (bottom) in terms of optical depth, while their
WF deconvolved spectra are displayed in red. The green hashed regions
indicate velocity intervals where we observe line broadening due to HFS
splitting. 6 The guaranteed time key project, Herschel/HIFI observations of
EXtra-Ordinary Sources (HEXOS; Bergin et al. 2010) was aimed to
investigate the chemical composition of several sources in the Orion
and SgrB2 star-forming regions.
7 See https://magix.astro.uni-koeln.de/ for more informa-
tion about the MAGIX software. 6 The guaranteed time key project, Herschel/HIFI observations of
EXtra-Ordinary Sources (HEXOS; Bergin et al. 2010) was aimed to
investigate the chemical composition of several sources in the Orion
and SgrB2 star-forming regions. 7 See https://magix.astro.uni-koeln.de/ for more informa-
tion about the MAGIX software. 4.2. Comparison of WF algorithm with other procedures In this section, we briefly compare column densities of the CH
molecule inferred using the WF algorithm with those obtained A60, page 5 of 14 A&A 632, A60 (2019) 0.0
0.5
1.0
AGAL010.624
00.384
0
50
100
VLSR (km/s)
0.0
0.5
1.0
Tl / Tc
AGAL034.258 + 00.154
0.0
0.5
1.0
AGAL327.293
00.579
100
50
0
VLSR (km/s)
0.0
0.5
1.0
Tl / Tc
AGAL332.826
00.549
0.0
0.5
1.0
AGAL330.954
00.182
150
100
50
0
50
VLSR (km/s)
0.0
0.5
1.0
Tl / Tc
AGAL351.581
00.352
150
100
50
0
50
100
VLSR (km/s)
0.0
0.5
1.0
Tl / Tc
SgrB2(M)
Fig. 3. CH (N, J = 2, 3/2 →1, 1/2) hyperfine transition spectra observed toward (from top) AGAL010.624−00.384, AGAL034.258+00.154,
AGAL327.293−00.579, AGAL332.826−00.549, AGAL330.954−00.182, AGAL351.581−00.352, and SgrB2(M) using GREAT/SOFIA. Each spec-
trum is normalized with respect to its continuum level. The WF fits are overlaid in red and include the HFS structure. The positions and relative
intensities of the HFS structure are displayed in black. Sources with similar velocity distributions were grouped to display the absorption features
along the LOS, in detail. The systemic velocities of the sources are represented by the dotted blue lines. 0.0
0.5
1.0
AGAL327.293
00.579
100
50
0
VLSR (km/s)
0.0
0.5
1.0
Tl / Tc
AGAL332.826
00.549 0.0
0.5
1.0
AGAL010.624
00.384
0
50
100
VLSR (km/s)
0.0
0.5
1.0
Tl / Tc
AGAL034.258 + 00.154 50
VLSR (km/s) 150
100
50
0
50
100
VLSR (km/s)
0.0
0.5
1.0
Tl / Tc
SgrB2(M) 0.0
0.5
1.0
AGAL330.954
00.182
150
100
50
0
50
VLSR (km/s)
0.0
0.5
1.0
Tl / Tc
AGAL351.581
00.352 Fig. 3. CH (N, J = 2, 3/2 →1, 1/2) hyperfine transition spectra observed toward (from top) AGAL010.624−00.384, AGAL034.258+00.154,
AGAL327.293−00.579, AGAL332.826−00.549, AGAL330.954−00.182, AGAL351.581−00.352, and SgrB2(M) using GREAT/SOFIA. Each spec-
trum is normalized with respect to its continuum level. The WF fits are overlaid in red and include the HFS structure. The positions and relative
intensities of the HFS structure are displayed in black. Sources with similar velocity distributions were grouped to display the absorption features
along the LOS, in detail. The systemic velocities of the sources are represented by the dotted blue lines. 0.0
2.0
4.0
AGAL327.293
00.579
100
50
0
VLSR (km/s)
0.0
2.0
obs
AGAL332.826
00.549
0.0
2.0
4.0
0.0
2.0
decon
arm gas features are dominated by uncertainties in the true con-
Fig. 4. A. M. Jacob et al.: Fingerprinting the effects of hyperfine structure A. M. Jacob et al.: Fingerprinting the effects of hyperfine structure
= 2, 3/2 →1, 1/2 absorption line analysis using WF deconvolution algorithm and comparison of column densi
3min
3max
Rg,min (a) Rg,max (a) Remark (b)
N(CH)
N(H2) (c)
N(OH) (d)
[km s−1] [km s−1] [kpc]
[kpc]
[1013 cm−2]
[1021 cm−2]
[1014 cm−2]
AGAL010.624−00.384
−11
−2
8.9
11.6
MC
>4.82
–
>3.42
−2
+2
8.1
8.9
MC
>4.84
–
>10.21
+2
+7
7.2
8.1
Norma
7.78+0.22
−0.05
2.22
5.68+0.17
−0.03
+7
+15
6.2
7.2
Sgr
15.70+0.05
−0.01
4.48
6.54+0.09
−0.02
+15
+20
5.7
6.2
25.26+0.17
−0.08
7.21
14.45+0.10
−0.05
+20
+31
4.8
5.7
Scu/Per
27.31+0.06
−0.02
7.80
17.67+0.14
−0.06
+31
+36
4.5
4.8
22.63+0.10
−0.00
6.46
S
+36
+51
3.7
4.5
Sgr
5.31+0.19
−0.01
1.51
S
AGAL034.258+00.154
+0
+16
7.5
8.5
1.65+0.28
−0.33
0.47
2.71+0.11
−0.03
+16
+32
6.8
7.5
Sgr
1.51+0.20
−0.17
0.43
1.81+0.08
−0.11
+32
+38
6.5
6.8
0.76+1.37
−0.84
0.22
0.48+0.82
−0.61
+38
+68
5.5
6.5
MC
>9.35
–
S
AGAL327.293−00.579
−61
−32
7.4
9.5
Scu, MC
>9.68
–
S
−32
−25
9.5
10.2
0.22+0.38
−0.41
0.06
0.05+0.71
−0.38
−25
−14
10.2
11.5
1.10+0.50
−0.09
0.31
0.52+0.25
−0.15
−14
−6
11.5
12.6
0.82+1.10
−0.05
0.23
0.98+0.24
−0.18
−6
+3
12.6
14.2
0.22+0.85
−1.08
0.06
0.42+0.30
−0.15
AGAL330.954−00.182
−115
−75
4.1
5.0
MC
>2.96
–
S
−75
−55
5.0
5.6
3.80+0.05
−0.12
1.09
1.23+0.05
−0.02
−55
−30
5.6
6.6
Norma
3.66+0.01
−0.02
1.04
S
−30
−10
6.6
7.7
Scu
0.19+1.31
−1.07
0.05
0.04+0.70
−0.51
−10
+4
7.7
8.8
Norma
0.45+0.36
−0.31
0.12
0.09+0.03
−0.02
AGAL332.826−00.549
−81
−50
4.7
5.6
Norma, MC
>5.00
–
S
−50
−40
5.6
6.0
1.12+0.02
−0.02
0.32
5.20+0.09
−0.02
−40
−32
6.0
6.4
0.34+0.60
−0.52
0.09
3.94+0.13
−0.01
−32
−8
6.4
7.8
Sgr
0.72+0.75
−0.67
0.20
2.57+0.11
−0.01
AGAL351.581−00.352
−109
−88
2.0
2.3
MC
>3.65
–
S
−88
−68
2.3
2.7
0.45+0.30
−0.25
0.13
0.20+0.10
−0.03
−68
−42
2.7
3.7
Sgr
0.81+0.31
−0.23
0.23
0.94+0.05
−0.01
−42
−14
3.7
6.0
Norma
2.69+0.06
−0.04
0.76
S
actocentric distances for each velocity interval is computed using, RG = R0
Θ(RG)sin(l)cos(b)
vlsr+Θ0sin(l)cos(b), with R0 = 8.3 kpc, Θ
flat Galactic rotation curve, i.e, Θ(RG) = Θ0. 4.2. Comparison of WF algorithm with other procedures 0.0
2.0
4.0
AGAL327.293
00.579
100
50
0
VLSR (km/s)
0.0
2.0
obs
AGAL332.826
00.549
0.0
2.0
4.0
0.0
2.0
decon arm gas features are dominated by uncertainties in the true con-
Herschel/HIFI toward SgrB2(M) as a part of the HEXOS6 sur-
vey toward SgrB2(M). The spectroscopic parameters of these
HFS transitions are summarised in Table E.1. The LOS toward
SgrB2(M) was chosen because of its complex spectral line
profile which contains an amalgamation of narrow and broad fea-
tures, alike. Qin et al. (2010) modelled the CH spectra by using
XCLASS (Möller et al. 2017) and the automated fitting routine
provided by MAGIX7 for an excitation temperature of 2.73 K. Assuming LTE, XCLASS solves the radiative transfer equa-
tion with an underlying Gaussian brightness profile to model
the observed spectral line profile. Using the WF algorithm, we
derived the column densities of the CH HFS transitions near
532 GHz, integrated over the same velocity intervals as those
specified in Table 1 of Qin et al. (2010). The errors were com-
puted as before, but with the assumption of a 20% error in the
continuum level. Fig. 4. Example illustrating effects of HFS splitting. The grey-filled area
represents the observed spectra toward AGAL327.293−00.579 (top) and
AGAL332.826−00.549 (bottom) in terms of optical depth, while their
WF deconvolved spectra are displayed in red. The green hashed regions
indicate velocity intervals where we observe line broadening due to HFS
splitting. A comparison between the CH column densities derived
using the WF algorithm and the XCLASS fits is displayed in
Fig. 5 for all absorption features along the LOS except, for the from other commonly used techniques. To carry out an unbiased
analysis, we performed the WF algorithm on the N = 1, J =
3/2 →1/2 HFS spectra of CH near 532 GHz, obtained using A60, page 6 of 14 A. M. Jacob et al.: Fingerprinting the effects of hyperfine structure Notes. (a)The galactocentric distances for each velocity interval is computed using, RG = R0
Θ(RG)sin(l)cos(b)
vlsr+Θ0sin(l)cos(b), with R 5. Results In Sect. 5.1, we investigate the correlation between the column
densities of CH with values determined for another widely stud-
ied light hydride, hydroxyl (OH), and evaluate its abundance
using CH as a proxy for H2. To further appreciate the global
characteristics of the CH molecule as a surrogate for H2 within
the Milky Way, in Sect. 5.2, we study its radial distribution across
the Galactic plane. In addition, we also compare the column densities obtained
using the WF deconvolution with those obtained by Wiesemeyer
et al. (2018) when using simulated annealing (SA) to the N, J =
2, 3/2 →1, 1/2 hyperfine transitions of CH toward W49 N. The
column densities derived using both methods have a tight rela-
tion with a slope of, N(CH)WFD/N(CH)SA = 1.08 ± 0.08. The A. M. Jacob et al.: Fingerprinting the effects of hyperfine structure The weighted regression at lower
column densities systematically follows a one-to-one correlation
while at larger column densities the values derived using the WF
algorithm are almost always smaller than those computed using
XCLASS, with an overall ratio of N(CH)WFD/N(CH)XCLASS =
0.76 ± 0.06. The deviations may arise due to the intrinsically
different means by which the two methods account for HFS
splitting, an effect that is particularly prominent at the larger
column density values. Additionally, inconsistencies in the post-
processing of the Herschel/HIFI data (baseline fitting, etc) also
contribute to the observed deviations. Nonetheless, the CH abun-
dances determined using both methods are in agreement within
the systematic uncertainties. strong absorption from the envelope of the SgrB2(M) molecular
cloud, which traces denser gas. The weighted regression at lower
column densities systematically follows a one-to-one correlation
while at larger column densities the values derived using the WF
algorithm are almost always smaller than those computed using
XCLASS, with an overall ratio of N(CH)WFD/N(CH)XCLASS =
0.76 ± 0.06. The deviations may arise due to the intrinsically
different means by which the two methods account for HFS
splitting, an effect that is particularly prominent at the larger
column density values. Additionally, inconsistencies in the post-
processing of the Herschel/HIFI data (baseline fitting, etc) also
contribute to the observed deviations. Nonetheless, the CH abun-
dances determined using both methods are in agreement within
the systematic uncertainties. column densities derived using the two methods deviate in value
only within the inter-arm gas components and the overall con-
cordances between the two methods further validates the use of
the non-iterative WF deconvolution scheme. A. M. Jacob et al.: Fingerprinting the effects of hyperfine structure Note that we do not account for the non-circular motion of the 3 kp
(b)The positions of the four main spiral arms and the local arms have been inferred from the spiral arm characte
14) and the velocities corresponding to the molecular cloud envelopes are indicated by MC. (c)N(H2) is derived fr
by Sheffer et al. (2008). (d)S denotes velocity components with saturated absorption line profiles. →1, 1/2 absorption line analysis using WF deconvolution algorithm and comparison of column densities with OH. Table 3. CH N, J = 2, 3/2 →1, 1/2 absorption line analysis using WF deconvolution algorithm and comparison of col Notes. (a)The galactocentric distances for each velocity interval is computed using, RG = R0
Θ(RG)sin(l)cos(b)
vlsr+Θ0sin(l)cos(b), with R0 = 8.3 kpc, Θ0 = 240 km s−1
and assuming a flat Galactic rotation curve, i.e, Θ(RG) = Θ0. Note that we do not account for the non-circular motion of the 3 kpc arm (Dame &
Thaddeus 2008). (b)The positions of the four main spiral arms and the local arms have been inferred from the spiral arm characteristics presented
in Reid et al. (2014) and the velocities corresponding to the molecular cloud envelopes are indicated by MC. (c)N(H2) is derived from [CH]/[H2] =
3.5 × 10−8 given by Sheffer et al. (2008). (d)S denotes velocity components with saturated absorption line profiles. A60, page 7 of 14 A&A 632, A60 (2019) Table 3. continued. Table 3. continued. 3min
3max
Rg,min (a) Rg,max (a) Remark (b)
N(CH)
N(H2) (c)
N(OH) (d)
[km s−1] [km s−1] [kpc]
[kpc]
[1013 cm−2]
[1021 cm−2]
[1014 cm−2]
AGAL351.581−00.352
−14
−12
6.0
6.2
0.90+0.24
−0.20
0.25
0.15+1.06
−0.08
−12
−3
6.2
7.7
Sgr
1.80+0.11
−0.07
5.14
0.18+0.26
−0.08
−3
+3
7.7
9.4
Scu
2.99+0.06
−0.04
0.85
S
+3
+12
9.4
13.5
0.44+0.40
−0.24
0.12
0.28+0.02
−0.01
SgrB2(M)
−124
−67
27.81+0.01
−0.02
7.94
–
−67
−38
3.16+0.10
−0.13
0.90
–
−38
−12
3 kpc
3.60+0.09
−0.08
1.30
–
−12
+8
GC
6.28+0.01
−0.02
1.79
–
+8
+29
Sgr
4.57+0.11
−0.11
1.31
–
+29
+40
Scu
0.89+0.61
−0.49
0.25
–
+40
+90
MC
>73.02
–
–
0.0
0.5
1.0
1.5
N(CH)(XCLASS) [ 1014 cm
2]
0.0
0.5
1.0
1.5
N(CH)(WFD) [ 1014 cm
2]
Sgr B2(M)
0
1
2
3
N(CH)(SA) [ 1014 cm
2]
0
1
2
3
N(CH)(WFD) [ 1014 cm
2]
W49 N
Fig. 5. A. M. Jacob et al.: Fingerprinting the effects of hyperfine structure Left: comparison of N = 1, J = 3/2 →
1/2 CH column densities derived using WF
deconvolution and the XCLASS software pre-
sented in Qin et al. (2010). Right: compar-
ison of N, J = 2, 3/2 →1, 1/2 CH column
densities derived using WF deconvolution and
the simulated annealing algorithm presented in
Wiesemeyer et al. (2018). The black solid line rep-
resents the weighted fit to the data, while the red
dashed line represents a ratio of one. The data
points that lie within 1σ intervals of the weighted
linear regression are indicated by the grey-shaded
regions. strong absorption from the envelope of the SgrB2(M) molecular
cloud, which traces denser gas. The weighted regression at lower
column densities systematically follows a one to one correlation
column densities derived using the two methods deviate in value
only within the inter-arm gas components and the overall con-
cordances between the two methods further validates the use of 3min
3max
Rg,min (a) Rg,max (a) Remark (b)
N(CH)
N(H2) (c)
N(OH) (d)
[km s−1] [km s−1] [kpc]
[kpc]
[1013 cm−2]
[1021 cm−2]
[1014 cm−2]
AGAL351.581−00.352
−14
−12
6.0
6.2
0.90+0.24
−0.20
0.25
0.15+1.06
−0.08
−12
−3
6.2
7.7
Sgr
1.80+0.11
−0.07
5.14
0.18+0.26
−0.08
−3
+3
7.7
9.4
Scu
2.99+0.06
−0.04
0.85
S
+3
+12
9.4
13.5
0.44+0.40
−0.24
0.12
0.28+0.02
−0.01
SgrB2(M)
−124
−67
27.81+0.01
−0.02
7.94
–
−67
−38
3.16+0.10
−0.13
0.90
–
−38
−12
3 kpc
3.60+0.09
−0.08
1.30
–
−12
+8
GC
6.28+0.01
−0.02
1.79
–
+8
+29
Sgr
4.57+0.11
−0.11
1.31
–
+29
+40
Scu
0.89+0.61
−0.49
0.25
–
+40
+90
MC
>73.02
–
–
0.0
0.5
1.0
1.5
N(CH)(XCLASS) [ 1014 cm
2]
0.0
0.5
1.0
1.5
N(CH)(WFD) [ 1014 cm
2]
Sgr B2(M)
0
1
2
3
N(CH)(SA) [ 1014 cm
2]
0
1
2
3
N(CH)(WFD) [ 1014 cm
2]
W49 N
Fig. 5. Left: comparison of N = 1, J = 3/2 →
1/2 CH column densities derived using WF
deconvolution and the XCLASS software pre-
sented in Qin et al. (2010). Right: compar-
ison of N, J = 2, 3/2 →1, 1/2 CH column
densities derived using WF deconvolution and
the simulated annealing algorithm presented in
Wiesemeyer et al. (2018). The black solid line rep-
resents the weighted fit to the data, while the red
dashed line represents a ratio of one. A60, page 8 of 14 A. M. Jacob et al.: Fingerprinting the effects of hyperfine structure The data
points that lie within 1σ intervals of the weighted
linear regression are indicated by the grey-shaded
regions. strong absorption from the envelope of the SgrB2(M) molecular
cloud, which traces denser gas. The weighted regression at lower
column densities systematically follows a one-to-one correlation
while at larger column densities the values derived using the WF
algorithm are almost always smaller than those computed using
XCLASS, with an overall ratio of N(CH)WFD/N(CH)XCLASS =
0.76 ± 0.06. The deviations may arise due to the intrinsically
column densities derived using the two methods deviate in value
only within the inter-arm gas components and the overall con-
cordances between the two methods further validates the use of
the non-iterative WF deconvolution scheme. 5. Results
5
i
i
h
l i
b
h
l 0.0
0.5
1.0
1.5
N(CH)(XCLASS) [ 1014 cm
2]
0.0
0.5
1.0
1.5
N(CH)(WFD) [ 1014 cm
2]
Sgr B2(M) 0
1
2
3
N(CH)(SA) [ 1014 cm
2]
0
1
2
3
N(CH)(WFD) [ 1014 cm
2]
W49 N 0
1
N(CH)(WFD) [ 1014 cm
2] Fig. 5. Left: comparison of N = 1, J = 3/2 →
1/2 CH column densities derived using WF
deconvolution and the XCLASS software pre-
sented in Qin et al. (2010). Right: compar-
ison of N, J = 2, 3/2 →1, 1/2 CH column
densities derived using WF deconvolution and
the simulated annealing algorithm presented in
Wiesemeyer et al. (2018). The black solid line rep-
resents the weighted fit to the data, while the red
dashed line represents a ratio of one. The data
points that lie within 1σ intervals of the weighted
linear regression are indicated by the grey-shaded
regions. Fig. 5. Left: comparison of N = 1, J = 3/2 →
1/2 CH column densities derived using WF
deconvolution and the XCLASS software pre-
sented in Qin et al. (2010). Right: compar-
ison of N, J = 2, 3/2 →1, 1/2 CH column
densities derived using WF deconvolution and
the simulated annealing algorithm presented in
Wiesemeyer et al. (2018). The black solid line rep-
resents the weighted fit to the data, while the red
dashed line represents a ratio of one. The data
points that lie within 1σ intervals of the weighted
linear regression are indicated by the grey-shaded
regions. strong absorption from the envelope of the SgrB2(M) molecular
cloud, which traces denser gas. A. M. Jacob et al.: Fingerprinting the effects of hyperfine structure The WF faces singularities
when Tl tends to zero and thus avoids the OH features arising
from these regions because they showcase saturated absorption. Therefore, the use of a lower excitation temperature does not
affect our analysis. Sheffer et al. 2008). As a precursor to the formation of CO,
OH has also gained recognition as a promising tracer for the
“CO-dark” component (Grenier et al. 2005) of molecular gas
in the ISM (Tang et al. 2017; Li et al. 2018; Engelke & Allen
2018; Rugel et al. 2018). In Table 3, we present column densities
of H2 estimated using the N(CH)/N(H2) ∼3.5+2.1
−1.4 × 10−8 relation
derived by Sheffer et al. (2008). This correlation was established
using optical observations of the A2∆– X2Π system of CH at
4300 Å and the (2–0), (3–0), and (4–0) bands of the Lyman B–X
transitions of H2 toward 37 bright stars. Akin to the CH obser-
vations presented in our study, the optical CH and UV H2 lines
presented by Sheffer et al. (2008) are seen in absorption toward
sightlines that probe molecular clouds in the local diffuse ISM
(1019 < N(H2) < 1021 cm−2). The derived CH and OH column densities are presented in
Fig. 7. The used data correspond to column densities based on
values computed within the velocity intervals used in Table 3. The plotted data points represent values re-sampled to 1 km s−1
wide velocity bins (except towards the velocity intervals cor-
responding to the molecular clouds, as well as those contami-
nated by outflows). The N(CH)-N(OH) correlation plots for the
individual sources are presented in Appendix F. Across the dif-
ferent sightlines, we obtained a range of N(OH)/N(CH) values
between one and 10. Moreover, almost all the sources, except
AGAL351.581−00.352 and AGAL332.826−00.549, have signif-
icant correlation coefficients between 0.46 and 0.75 with false-
alarm probabilities below 5% (summarised in Table F.1) within
the spiral arm data, while there is no remarkable correlation
observed in the inter-arm regions. The lack of, or weak corre-
lations present in the above mentioned sources can be attributed
to the fact that the OH spectra toward these two sources showcase
more saturated features along the LOS than in the remainder of
the sources. (
(
2)
)
In order to carry out a comparison between N(OH) and
N(CH), we use data published in Wiesemeyer et al. A. M. Jacob et al.: Fingerprinting the effects of hyperfine structure 1010
1011
1012
1013
1014
1015
Nv(CH) [cm
2 (km /s)
1]
1010
1011
1012
1013
1014
1015
Nv(OH) [cm
2 (km /s)
1]
r = 0.83
N(OH) = (3.85 ± 0.15) N(CH)
AGAL010.624
00.384
AGAL034.258 + 00.154
AGAL327.293
00.579
AGAL330.954
00.182
AGAL332.826
00.549
AGAL351.581
00.352
1018
1019
1020
1021
1022
Nv(H2) [cm
2 (km /s)
1]
Fig. 7. N(OH) vs. N(CH), per unit velocity interval derived using WF
algorithm. The different coloured data points represent contributions
from the different sources. The black dashed, solid, and dotted-dashed
lines indicate N(OH)/N(CH) ratios of 10, one, and 0.1, respectively
while the linear regression fit to the data, N(OH) = (3.85±0.15) N(CH)
is displayed in green. 1010
1011
1012
1013
1014
1015
Nv(CH) [cm
2 (km /s)
1]
1010
1011
1012
1013
1014
1015
Nv(OH) [cm
2 (km /s)
1]
r = 0.83
N(OH) = (3.85 ± 0.15) N(CH)
AGAL010.624
00.384
AGAL034.258 + 00.154
AGAL327.293
00.579
AGAL330.954
00.182
AGAL332.826
00.549
AGAL351.581
00.352
1018
1019
1020
1021
1022
Nv(H2) [cm
2 (km /s)
1] 0.0
0.5
1.0
Tl / Tc
WF
fit OH
CH spectrum
50
0
VLSR (km/s)
0.0
0.5
1.0
Tl / Tc
AGAL332.826
00.549
Deconvolution OH
Deconvolution CH 50
VLSR (km/s) Fig. 6. Top: normalised line profile and HFS structure of OH
near 2514 GHz (in black) overlaid with WF fit in red toward
AGAL332.826−00.549. The WF fit avoids velocity intervals of the OH
spectrum that show saturation between −65 < 3lsr <-56 km s−1, bounded
by the blue-shaded regions. The grey shaded and hatched region dis-
plays the CH absorption profile toward the same source for comparison. Bottom: normalised WF deconvolved line profile of OH (avoiding the
saturated region) in blue, and corresponding deconvolved spectrum of
CH displayed by the shaded and hatched grey region. Fig. 7. N(OH) vs. N(CH), per unit velocity interval derived using WF
algorithm. The different coloured data points represent contributions
from the different sources. The black dashed, solid, and dotted-dashed
lines indicate N(OH)/N(CH) ratios of 10, one, and 0.1, respectively
while the linear regression fit to the data, N(OH) = (3.85±0.15) N(CH)
is displayed in green. those used for CH. Our assumption of a complete ground-state
occupation for OH is broken down for spectral features associ-
ated with the dense molecular clouds. A. M. Jacob et al.: Fingerprinting the effects of hyperfine structure A. M. Jacob et al.: Fingerprinting the effects of hyperfine structure
0.0
0.5
1.0
Tl / Tc
WF
fit OH
CH spectrum
50
0
VLSR (km/s)
0.0
0.5
1.0
Tl / Tc
AGAL332.826
00.549
Deconvolution OH
Deconvolution CH
Fig. 6. Top: normalised line profile and HFS structure of OH
near 2514 GHz (in black) overlaid with WF fit in red toward
AGAL332.826−00.549. The WF fit avoids velocity intervals of the OH
spectrum that show saturation between −65 < 3lsr <-56 km s−1, bounded
by the blue-shaded regions. The grey shaded and hatched region dis-
plays the CH absorption profile toward the same source for comparison. Bottom: normalised WF deconvolved line profile of OH (avoiding the
saturated region) in blue, and corresponding deconvolved spectrum of
CH displayed by the shaded and hatched grey region. 1010
1011
1012
1013
1014
1015
Nv(CH) [cm
2 (km /s)
1]
1010
1011
1012
1013
1014
1015
Nv(OH) [cm
2 (km /s)
1]
r = 0.83
N(OH) = (3.85 ± 0.15) N(CH)
AGAL010.624
00.384
AGAL034.258 + 00.154
AGAL327.293
00.579
AGAL330.954
00.182
AGAL332.826
00.549
AGAL351.581
00.352
1018
1019
1020
1021
1022
Nv(H2) [cm
2 (km /s)
1]
Fig. 7. N(OH) vs. N(CH), per unit velocity interval derived using WF
algorithm. The different coloured data points represent contributions
from the different sources. The black dashed, solid, and dotted-dashed
lines indicate N(OH)/N(CH) ratios of 10, one, and 0.1, respectively
while the linear regression fit to the data, N(OH) = (3.85±0.15) N(CH)
is displayed in green. those used for CH. Our assumption of a complete ground-state 0.0
0.5
1.0
Tl / Tc
WF
fit OH
CH spectrum
50
0
VLSR (km/s)
0.0
0.5
1.0
Tl / Tc
AGAL332.826
00.549
Deconvolution OH
Deconvolution CH
Fig. 6. Top: normalised line profile and HFS structure of OH
near 2514 GHz (in black) overlaid with WF fit in red toward
AGAL332.826−00.549. The WF fit avoids velocity intervals of the OH
spectrum that show saturation between −65 < 3lsr <-56 km s−1, bounded
by the blue-shaded regions. The grey shaded and hatched region dis-
plays the CH absorption profile toward the same source for comparison. Bottom: normalised WF deconvolved line profile of OH (avoiding the
saturated region) in blue, and corresponding deconvolved spectrum of
CH displayed by the shaded and hatched grey region. 5.1. CH versus OH column density For quite some time, CH has been used as a tracer for H2
in the diffuse regions of the ISM based on its observed cor-
relation with H2 (Federman & Willson 1982; Mattila 1986; A60, page 8 of 14 A. M. Jacob et al.: Fingerprinting the effects of hyperfine structure A. M. Jacob et al.: Fingerprinting the effects of hyperfine structure This result is con-
sistent with the column density ratios presented by Liszt & Lucas
(2002), and is in the range of theoretical predictions presented
by Albertsson et al. (2014). When using a N(CH)/N(H2) ratio
of ∼4.97 × 10−8 as derived by Weselak (2019), we find the
corresponding OH abundance to be X(OH) = 1.56 × 10−7. with N(OH)/N(CH) > 10 correspond to the broad line wings
of saturated OH features, while those with N(OH)/N(CH) < 1
have larger errors in the determined N(OH) arising from their
continuum-level uncertainties. Since the spectral features aris-
ing from star-forming regions are unsaturated in the case of
the CH molecule, its effective broadening into the inter-arm
regions is less. We further extend this analysis by combining
our data set with optical observations8 of the CH B–X transi-
tions at 3886 and 3890 Å, and OH A–X transitions at 3078 and
3082 Å presented toward 20 bright OB-type stars at low Galactic
latitudes (−17◦< b < +23◦) from Weselak et al. (2010). More-
over, Weselak et al. (2010) show that the LOS toward these stars
probe physical conditions (gas densities, n(H) = 250–600 cm−3
and temperatures, Tkin = 20−30 K) that are typical for diffuse
molecular clouds (Snow & McCall 2006) similar to those of
our data set, and report a N(OH)/N(CH) value of 2.52 ± 0.35. The revised abundance ratio (combining both sets of data) dis-
played in Fig. 8 follows N(OH)/N(CH) = (3.14 ± 0.49). Using
this abundance ratio and the Sheffer et al. (2008) relation, we
derive the OH abundance using CH as a surrogate for H2 to be
XOH = N(OH)/N(H2) = (1.09 ± 0.27) × 10−7. This result is con-
sistent with the column density ratios presented by Liszt & Lucas
(2002), and is in the range of theoretical predictions presented
by Albertsson et al. (2014). When using a N(CH)/N(H2) ratio
of ∼4.97 × 10−8 as derived by Weselak (2019), we find the
corresponding OH abundance to be X(OH) = 1.56 × 10−7. We find that the CH column densities peak at galactocen-
tric distances between 4 and 8 kpc. LOSs within the l = 0–180◦
range dominate contributions toward the first column density
peak observed around 5 kpc, while the second peak near 7 kpc
originates from LOSs in the l = 180–360◦range. A. M. Jacob et al.: Fingerprinting the effects of hyperfine structure computed assuming a flat rotation curve, with the distance of
the Sun from the Galactic centre given by R0 = 8.3 kpc, and an
orbital velocity of Θ0 = 240 km s−1 of the Sun, with respect 0
2
4
6
8
10
12
RG [kpc]
0.00
0.05
0.10
0.15
0.20
0.25
N(CH) × 1014 [cm
2 kpc
1]
Norma
Cygnus
Scutum
Crux
Sagittarius
Carina
Perseus
0.0
0.2
0.4
0.6
N(H2) × 1021 [cm
2 kpc
1]
Fig. 9. Radial distribution of CH column densities toward lines of sight
probed in this study except SgrB2(M). The column density values for
each distance interval are averages over the underlying distribution. The
secondary y-axis display N(H2) values computed using the Sheffer et al. (2008) relation. The dashed red vertical line indicates the galactocen-
tric distance to the Sun. The spiral arm locations typical for a fourth
quadrant source, are plotted with spiral arm widths of 0.85 kpc. 1011
1012
1013
1014
1015
N(CH) [cm
2]
1011
1012
1013
1014
1015
N(OH) [cm
2]
r = 0.72
N(OH) = (3.14 ± 0.49) N(CH)
This work
Weselak et al. 2010
1019
1020
1021
1022
N(H2) [cm
2]
Fig. 8. Correlation plot between column densities of the CH, and OH
molecules, integrated over broad velocity intervals as per Table 3. The
red triangles and blue squares represent column density measures from
Weselak et al. (2010) and this work, respectively. The black dashed,
solid, and dotted-dashed lines indicate N(OH)/N(CH) ratios of 10,
one, and 0.1, respectively while the linear regression fit to the data,
N(OH) = (3.15 ± 0.49) N(CH) is displayed by the red dotted line. 1011
1012
1013
1014
1015
N(CH) [cm
2]
1011
1012
1013
1014
1015
N(OH) [cm
2]
r = 0.72
N(OH) = (3.14 ± 0.49) N(CH)
This work
Weselak et al. 2010
1019
1020
1021
1022
N(H2) [cm
2] Fig. 9. Radial distribution of CH column densities toward lines of sight
probed in this study except SgrB2(M). The column density values for
each distance interval are averages over the underlying distribution. The
secondary y-axis display N(H2) values computed using the Sheffer et al. (2008) relation. The dashed red vertical line indicates the galactocen-
tric distance to the Sun. The spiral arm locations typical for a fourth
quadrant source, are plotted with spiral arm widths of 0.85 kpc. Fig. 8. 8 These observations were made using the UVES spectrograph at the
European Southern Observatory (ESO) Paranal in Chile. A. M. Jacob et al.: Fingerprinting the effects of hyperfine structure The column
density peak near 5 kpc is associated with absorption arising
from a combination of the Perseus and Norma spiral-arm cross-
ings, and the 7 kpc peak may be attributed to the intersection of
the Scutum-Crux spiral-arm crossing along the sightlines. The
higher observed column densities peaking near these spiral arms
can be attributed to the larger (mass) molecular content that is
present at these galactocentric radii. Owing to the larger error
bars, the significance of a third peak near 9 kpc is unclear. How-
ever, it is in line with contributions that may arise from the
Scutum- and Sagittarius-arm far side crossings. Due to a lack
of data points at galactocentric distances smaller than 2 kpc,
the nature of the CH molecular gas distribution there remains
unknown. We do not include contributions from the sightline
toward SgrB2(M) in this analysis, because in the Galactic centre
direction, it is difficult to assign the CH absorption to different
components based on their LSR velocities. A similar dual-peaked radial distribution profile has been
reported by both Pineda et al. (2013), and Velusamy & Langer
(2014) for the cold neutral medium (CNM) component of [CII]
emission. [CII] is a proficient tracer that is sensitive to various
evolutionary stages of the ISM, but by taking advantage of dif-
fering densities, Pineda et al. (2013) were able to decompose
contributions of the [CII] emissivity from the different phases of
the ISM, excluding the warm ionised medium (WIM) for sources
in the Galactic plane, b = 0◦. Despite the smaller sample size, A. M. Jacob et al.: Fingerprinting the effects of hyperfine structure Correlation plot between column densities of the CH, and OH
molecules, integrated over broad velocity intervals as per Table 3. The
red triangles and blue squares represent column density measures from
Weselak et al. (2010) and this work, respectively. The black dashed,
solid, and dotted-dashed lines indicate N(OH)/N(CH) ratios of 10,
one, and 0.1, respectively while the linear regression fit to the data,
N(OH) = (3.15 ± 0.49) N(CH) is displayed by the red dotted line. computed assuming a flat rotation curve, with the distance of
the Sun from the Galactic centre given by R0 = 8.3 kpc, and an
orbital velocity of Θ0 = 240 km s−1 of the Sun, with respect
to the Galactic centre (Reid et al. 2014). The uncertainties in
the CH column densities are typically of the order of a few
1012 cm−2(km s−1)−1, and are too small to be visible in some
parts of the plot. This analysis is limited to a small number
of strong background continuum sources, distributed at low
Galactic latitudes, which lie close to the Galactic plane. with N(OH)/N(CH) > 10 correspond to the broad line wings
of saturated OH features, while those with N(OH)/N(CH) < 1
have larger errors in the determined N(OH) arising from their
continuum-level uncertainties. Since the spectral features aris-
ing from star-forming regions are unsaturated in the case of
the CH molecule, its effective broadening into the inter-arm
regions is less. We further extend this analysis by combining
our data set with optical observations8 of the CH B–X transi-
tions at 3886 and 3890 Å, and OH A–X transitions at 3078 and
3082 Å presented toward 20 bright OB-type stars at low Galactic
latitudes (−17◦< b < +23◦) from Weselak et al. (2010). More-
over, Weselak et al. (2010) show that the LOS toward these stars
probe physical conditions (gas densities, n(H) = 250–600 cm−3
and temperatures, Tkin = 20−30 K) that are typical for diffuse
molecular clouds (Snow & McCall 2006) similar to those of
our data set, and report a N(OH)/N(CH) value of 2.52 ± 0.35. The revised abundance ratio (combining both sets of data) dis-
played in Fig. 8 follows N(OH)/N(CH) = (3.14 ± 0.49). Using
this abundance ratio and the Sheffer et al. (2008) relation, we
derive the OH abundance using CH as a surrogate for H2 to be
XOH = N(OH)/N(H2) = (1.09 ± 0.27) × 10−7. A. M. Jacob et al.: Fingerprinting the effects of hyperfine structure (2016) of the
2Π3/2, J = 5/2 →3/2 ground-state transitions of OH, procured
using GREAT/SOFIA as a part of the observatory’s cycle 1 cam-
paign. The spectroscopic parameters of these OH transitions are
summarised in Table E.1. For this preliminary search, obser-
vations were carried out toward all the sightlines discussed in
this paper, except for SgrB2(M). The OH spectra are saturated
at velocities corresponding to the SFR-related molecular cloud
components (that provide the background continuum emission)
themselves, as shown in Fig. 6. The saturated absorption fea-
tures of the OH spectra arising in the envelopes of the hot cores
of these star-forming regions are indicative of the fact that a
complete ground-state occupation assumption is no longer valid
(Csengeri et al. 2012). Moreover, from Fig. 6, it can be seen
that most of the observed OH absorption features have corre-
sponding features in the CH spectrum with notable differences
at velocity intervals between −13 and −21 km s−1, and from
−36 to −42 km s−1, in this example. The WF fit and decon-
volution is applied to the OH spectra with a Tex = 3.1 K (for
consistency), and integrated over the same velocity intervals as Fitting a linear regression to the combination of all the sight-
lines yields Nv(OH)/Nv(CH) = (3.85 ± 0.15), with a Pearson’s
correlation coefficient of 0.83 (false-alarm probability below
1%) for velocity intervals of 1 km s−1. The functional form of
the regression was chosen after comparing the standard error
of a regression fit through the origin with that of an ordinary
least square regression, both of which yielded best fit slopes
that are consistent with one another (within a 5% error). From
Fig. 7, we find that this relation spans roughly two orders of
magnitude between 0.1 and 10, with a majority of the ratios
lying between one and 10. We noted that those data points A60, page 9 of 14 A60, page 9 of 14 A60, page 9 of 14 A60 (2019)
0
2
4
6
8
10
12
RG [kpc]
0.00
0.05
0.10
0.15
0.20
0.25
N(CH) × 1014 [cm
2 kpc
1]
Norma
Cygnus
Scutum
Crux
Sagittarius
Carina
Perseus
0.0
0.2
0.4
0.6
N(H2) × 1021 [cm
2 kpc
1]
Fig. 9. Radial distribution of CH column densities toward lines of sight
probed in this study except SgrB2(M). A. M. Jacob et al.: Fingerprinting the effects of hyperfine structure The column density values for
each distance interval are averages over the underlying distribution. The
secondary y-axis display N(H2) values computed using the Sheffer et al. (2008) relation. The dashed red vertical line indicates the galactocen-
tric distance to the Sun. The spiral arm locations typical for a fourth
quadrant source, are plotted with spiral arm widths of 0.85 kpc. A&A 632, A60 (2019) 0
2
4
6
8
10
12
RG [kpc]
0.00
0.05
0.10
0.15
0.20
0.25
N(CH) × 1014 [cm
2 kpc
1]
Norma
Cygnus
Scutum
Crux
Sagittarius
Carina
Perseus
0.0
0.2
0.4
0.6
N(H2) × 1021 [cm
2 kpc
1] 1011
1012
1013
1014
1015
N(CH) [cm
2]
1011
1012
1013
1014
1015
N(OH) [cm
2]
r = 0.72
N(OH) = (3.14 ± 0.49) N(CH)
This work
Weselak et al. 2010
1019
1020
1021
1022
N(H2) [cm
2]
Fig. 8. Correlation plot between column densities of the CH, and OH
molecules, integrated over broad velocity intervals as per Table 3. The
red triangles and blue squares represent column density measures from
Weselak et al. (2010) and this work, respectively. The black dashed,
solid, and dotted-dashed lines indicate N(OH)/N(CH) ratios of 10,
one, and 0.1, respectively while the linear regression fit to the data,
N(OH) = (3.15 ± 0.49) N(CH) is displayed by the red dotted line. 0
2
4
6
8
10
12
RG [kpc]
0.00
0.05
0.10
0.15
0.20
0.25
N(CH) × 1014 [cm
2 kpc
1]
Norma
Cygnus
Scutum
Crux
Sagittarius
Carina
Perseus
0.0
0.2
0.4
0.6
N(H2) × 1021 [cm
2 kpc
1]
Fig. 9. Radial distribution of CH column densities toward lines of sight
probed in this study except SgrB2(M). The column density values for
each distance interval are averages over the underlying distribution. The
secondary y-axis display N(H2) values computed using the Sheffer et al. (2008) relation. The dashed red vertical line indicates the galactocen-
tric distance to the Sun. The spiral arm locations typical for a fourth
quadrant source, are plotted with spiral arm widths of 0.85 kpc. Acknowledgements. SOFIA Science Mission Operations is jointly operated
by the Universities Space Research Association, Inc., under NASA contract References A., Evenson, K. M., & Brown, J. M. 2001, ApJ, 546, 330
de Graauw, T., Helmich, F., Phillips, T., et al. 2010, A&A, 518, L6
Dunham, T. 1937, PASP, 49, 26 The general agreement between the column density of the
CO-dark gas traced by [CII] and the H2 column densities traced
by CH at galactocentric distances <8 kpc, is a good indicator of
the usefulness of CH as a tracer for the CO-dark component of
molecular gas. Engelke, P. D., & Allen, R. J. 2018, ApJ, 858, 57 Federman, S., & Willson, R. 1982, ApJ, 260, 124 Grenier, I. A., Casandjian, J.-M., & Terrier, R. 2005, Science, 307, 1292 Grenier, I. A., Casandjian, J.-M., & Terrier, R. 2005, Science, 307, A. M. Jacob et al.: Fingerprinting the effects of hyperfine structure A. M. Jacob et al.: Fingerprinting the effects of hyperfine structure our results broadly resemble the shape of the [CII] integrated
intensity distribution profile. NAS2-97001, and the Deutsches SOFIA Institut under DLR contract 50 OK 0901
and 50 OK 1301 to the University of Stuttgart. We are GREATful to the SOFIA
operations team for their help and support through out the course of the obser-
vations and after. A.M.J. is a fellow of the International Max-Planck-Research
School (IMPRS) for A&A at the Universities of Bonn and Cologne. We also
thank Carsten König and Dario Colombo for their helpful discussions on identi-
fying the different spiral-arm and inter-arm LOS crossings. M.-Y.L. was partially
funded through the sub-project A6 of the Collaborative Research Council 956,
funded by the Deutsche Forschungsgemeinschaft (DFG). The framework of carbon chemistry in the diffuse regions,
which is initiated by the radiative association of the ground
vibrational state of H2 with ionised carbon, explains the observed
correspondence between CH and [CII] radial profiles (Gerin
et al. 2016). Peaking at the same RG values as those of HI (see
Fig. 12 in Pineda et al. 2013), suggests that CH likely traces the
more diffuse regions of the CNM between [CII] and the extended
HI emission. Upon comparing the distribution of H2 column den-
sities derived from CH (using the Sheffer et al. 2008 relation)
with those derived from both CO (CO-traced H2 gas) and [CII]
(CO-dark H2 gas) by Pineda et al. (2013, their Fig. 18) we find
that: (1) at RG < 2 kpc, we cannot comment on the behaviour
of the distribution due to the lack of data points, (2) 2 kpc <
RG < 8 kpc, the CH-traced H2 column density is comparable to
the [CII]-traced H2 column density, while being smaller than the
CO-traced H2 column density and, (3) RG > 8 kpc, despite attain-
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H2 gas traced by [CII], the column density distribution of H2
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all the sightlines in this study, except SgrB2(M), are plotted
as a function of galactocentric distances, RG, in Fig. 9. The
galactocentric distances to the different spiral arm features were A60, page 10 of 14 6. Conclusions Guan, X., Stutzki, J., Graf, U. U., et al. 2012, A&A, 542, L4 Heyminck, S., Graf, U., Güsten, R., et al. 2012, A&A, 542, L1 In this paper, we presented the analysis of SOFIA/GREAT obser-
vations of the N, J = 2, 3/2 →1, 1/2 transitions of CH detected
in absorption toward strong, FIR-bright continuum sources by
using the Wiener filter algorithm. Although it has previously not
been used as a conventional tool for spectral line analysis, we
find the WF deconvolution algorithm to be a self-consistent con-
cept that sufficiently alleviates biases manifested in spectra with
close hyperfine spacing, provided that the transitions are opti-
cally thin. The absorption features along each LOS are broadly
classified into contributions arising from different spiral arm and
inter-arm regions based on the spiral arm model of the Milky
Way presented by Reid et al. (2014). Using the WF as the basis
for our spectral analysis, we derived a linear relationship between
CH and OH, N(OH)/N(CH) = 3.85 ± 0.15 that is consistent with
previous work. Upon increasing the number of sightlines in this
analysis by including those studied by Weselak et al. (2010), we
derived a revised relationship with a N(OH)/N(CH) = 3.14 ±
0.49. By subsequently using CH as a surrogate for tracing H2,
we derived an OH abundance of X(OH) = (1.09 ± 0.27) ×10−7. It is important to note that this result pivots on the validity of the
N(CH)-N(H2) relation used. We find the radial distribution of
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and H2 established by Sheffer et al. (2008), the likeness of the
CH abundance distribution to that of [CII], a well known tracer,
of the CO-dark H2 gas, lends credence to its use as tracer for
H2 in these regions. Furthermore, this work may initiate follow-
up observational studies, investigating a larger sample of sources
covering a wide range of physical conditions at varying galacto-
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20
0
20
40
60
VLSR (km/s)
0.0
0.5
1.0
Tl / Tc
f( ) + n( )
20
0
20
40
60
VLSR (km/s)
0.0
0.5
1.0
h( )
20
0
20
40
60
VLSR (km/s)
0.0
0.5
1.0
d( )
Fig. A.1. Convolution model illustrated for LOS toward AGAL010.624−00.384. The input signal with additive noise or the original (deconvolved)
spectrum (left) is convolved with the normalized HFS weights (center) to produce the resulting convolved spectrum or the observed noise-degraded
spectrum (right). Appendix A: Convolution model Appendix A: Convolution model 20
0
20
40
60
VLSR (km/s)
0.0
0.5
1.0
h( ) Appendix A: Convolution model
20
0
20
40
60
VLSR (km/s)
0.0
0.5
1.0
Tl / Tc
f( ) + n( ) 20
0
20
40
60
VLSR (km/s)
0.0
0.5
1.0
d( ) Fig. A.1. Convolution model illustrated for LOS toward AGAL010.624−00.384. The input signal with additive noise or the original (deconvolved)
spectrum (left) is convolved with the normalized HFS weights (center) to produce the resulting convolved spectrum or the observed noise-degraded
spectrum (right). Figure A.1 presents an example that showcases the effects of
HFS splitting on astrophysical spectra. The deviations between
the observed spectrum and noise-degraded spectrum (resulting
from the convolution model) computed using residuals, vary on
average between −0.03 and +0.02. Figure A.1 presents an example that showcases the effects of
HFS splitting on astrophysical spectra. The deviations between
the observed spectrum and noise-degraded spectrum (resulting
from the convolution model) computed using residuals, vary on
average between −0.03 and +0.02. not expect large discrepancies. Figure B.1 displays the combined
results of the residuals obtained for each source. The left panel shows no clear pattern or structure, with the
residuals randomly scattered around zero. The lack of any uni-
form structure reveals that our assumptions of a linear system
with an independent noise signal is maintained. 6. Conclusions From the right
panel, we see that the residuals are normally distributed for
each source, with a mean of 0.001, and standard deviation of
0.024. Only 2.4 and 3.7% of all the residual points lie out-
side the 99 and 95% confidence intervals. These outliers can
be attributed to small variations in the power spectrum of the
additive noise that lead to an over-(positive-valued residual) or
under-(negative-valued residual) estimation of the true noise
level. Such inaccuracies in the estimated noise are more pro-
nounced at the spectral peaks, and correspond to the outliers
seen in Fig. B.1. Overall, the residuals validate our fit and con-
firm our assumptions by reproducing independent errors that are
not serially-correlated with a near constant variance and normal
distribution. Appendix B: Assessment of the WF fit Appendix B: Assessment of the WF fit
0
1
2
3
fit
0.30
0.15
0.00
0.15
0.30
Residuals
0
20
40
Nr. of channels
AGAL010.624
00.384
AGAL034.258 + 00.154
AGAL327.293
00.579
AGAL332.826
00.549
AGAL351.581
00.352
AGAL330.954
00.182
SgrB2(M)
Fig. B.1. Left panel: optical depth residuals vs. WF fit. The different
coloured markers correspond to residuals computed from the WF fits to
the different sources. Right panel: distribution of residuals correspond-
ing to different sources. They follow approximate normal distributions
centred slightly off-zero around +0.001 for all seven sources. The grey
(hatched) and pink regions bound the 99 and 95% confidence intervals,
respectively. Appendix B: Assessment of the WF fit
0
1
2
3
fit
0.30
0.15
0.00
0.15
0.30
Residuals
0
20
40
Nr. of channels
AGAL010.624
00.384
AGAL034.258 + 00.154
AGAL327.293
00.579
AGAL332.826
00.549
AGAL351.581
00.352
AGAL330.954
00.182
SgrB2(M) Residuals Residuals Appendix C: Computational efficiency of the WF
algorithm From the mathematical formulation presented in Sect. 2.1, the
WF can be interpreted as a kernel acting on the inverse filter. If the signal happens to be much stronger than the noise, then
(S/N)−1 ≈0, in which case the WF kernel reduces to H−1(˜ν)
or the inverse filter. Alternatively, if the signal is weak in
comparison to the noise, then (S/N)−1 →∞and W(˜ν) →0 and
the WF attenuates contributions from higher noise levels which
characterises it as a bandpass filter. Fig. B.1. Left panel: optical depth residuals vs. WF fit. The different
coloured markers correspond to residuals computed from the WF fits to
the different sources. Right panel: distribution of residuals correspond-
ing to different sources. They follow approximate normal distributions
centred slightly off-zero around +0.001 for all seven sources. The grey
(hatched) and pink regions bound the 99 and 95% confidence intervals,
respectively. p
WF deconvolution, while efficient in its performance, is still
an approximate method, in the sense that its main drawbacks
arise from the MSE constraint, which is also, ironically, what
sets it apart form other filters. The process of aptly minimis-
ing the overall MSE carries out inverse filtering by accounting
for additive noise. However, since the human eye is willing to
accept more noise than allowed for by the filter (provided that
it is spatially associated with the spectral profile), the restored
spectra might seem unpleasantly smooth. This is because the
trade-off between filtering and noise smoothing holds true only
up to a second order in precision, meaning the required levels
of degradation are not always achieved when characterising the
signal. This leads to deviations between the obtained results and
the true optimum. Hence the main performance limitation arises
form the finite variance in the noise at any given channel and its In this appendix, we discuss the quality of the WF fit prior
to deconvolution. This analysis aims to curb the propagation of
biases introduced from an inadequate fit into any derived quan-
tities. For this reason, we carry out a residual analysis to test the
validity of the WF fitting model and its underlying assumptions. The “residuals” of a fit may be viewed as deviations in the val-
ues predicted by the model from what is observed. Given that
the Wiener filter itself is derived by minimising the mean square
error between the data and the model (Sect. A60, page 12 of 14 Appendix D: Impact of using a single excitation
temperature on derived column densities Appendix D: Impact of using a single excitation
temperature on derived column densities
100
50
0
VLSR (km/s)
0
2
4
6
Nv (CH) × 1014 [cm
2 (km/s)
1]
AGAL327.293
00.579
Tex [K]
26.00
18.75
15.00
9.37
3.10
Fig. D.1. Variation in WF deconvolved column density profile toward
AGAL327.293−00.579 at varying values of excitation temperature
between 3.1 and 26 K. 100
50
0
VLSR (km/s)
0
2
4
6
Nv (CH) × 1014 [cm
2 (km/s)
1]
AGAL327.293
00.579
Tex [K]
26.00
18.75
15.00
9.37
3.10 Fig. C.1. Left: scatter plots of residuals obtained from the WF fit and
WF + WT (ForWARD) algorithm against their fits in dark blue and
magenta, respectively. Right: weighted distributions of residuals from
the WF fit alone (blue hatched region) and the WF + WT (magenta). treatment. These effects are seen in the form of small fluctua-
tions in the WF fit (as discussed in Appendix B), particularly in
regions of the spectra where the S/N is very large. One could then
turn toward using this response as a respectable initial guess,
and repeating the same filtering process over multiple iterations
in order to improve the restored spectrum. This iterative line of
thought is futile, because the scheme converges as H(˜ν) →0. The non-iterative nature of the WF is in fact one of its biggest
advantages, as it is less time-consuming, and, additionally, aids
in any subsequent Monte Carlo-based error estimates. Fig. D.1. Variation in WF deconvolved column density profile toward
AGAL327.293−00.579 at varying values of excitation temperature
between 3.1 and 26 K. y
q
The wavelet transform (WT) is another method used to quan-
tify the degree of noise attenuation. Based on the quantization of
a signal in the form of wavelets, the WT has the advantage that
it characterises the local behaviour of a signal while maintain-
ing all temporal information, unlike the FT. The Fourier-wavelet
regularisation deconvolution (ForWARD) algorithm developed
by Neelamani et al. (2004) is one such technique that combines
deconvolution in the Fourier domain with noise suppression
in the wavelet domain. In this approach, the WF deconvolu-
tion product is first decomposed in the wavelet domain into a
scaling function, which acts as a low-pass filter and a wavelet
function or a band-pass filter, which in turn constitutes each
wavelet level. 9 The critical densities for CH are computed using rate coefficients
calculated by Dagdigian (2018) and Einstein A coefficients as given in
Table 2. Appendix C: Computational efficiency of the WF
algorithm 2.1), one would A60, page 12 of 14 A. M. Jacob et al.: Fingerprinting the effects of hyperfine structure 0
1
fit
0.1
0.0
0.1
Residuals
WF + WT
WF
0
20
40
Nr. of channels
AGAL010.624
00.384
WF + WT
WF
Fig. C.1. Left: scatter plots of residuals obtained from the WF fit and
WF + WT (ForWARD) algorithm against their fits in dark blue and
magenta, respectively. Right: weighted distributions of residuals from
the WF fit alone (blue hatched region) and the WF + WT (magenta). the wavelet decomposition of the spectral signal, and remains
sparse over consecutive iterations. This makes it difficult to con-
clude whether the WT actually takes into account the outliers, or
whether their effects are averaged out in the process of forming
wavelets. While the addition of the WT to the WF deconvolution
makes it more robust and rigorous, it has a longer computa-
tional time, and application to time-invariant systems makes it
non-ideal. 0
1
fit
0.1
0.0
0.1
Residuals
WF + WT
WF
0
20
40
Nr. of channels
AGAL010.624
00.384
WF + WT
WF Appendix D: Impact of using a single excitation
temperature on derived column densities A subtle problem
that this creates in a time-invariant system is that the wavelet
domain is sparse (reduced number of data points) because of A60, page 13 of 14 A60, page 13 of 14 A&A 632, A60 (2019) Appendix F: Correlation plots for individual
sources Appendix E: Additional table Appendix E: Additional table
Table E.1. Spectroscopic parameters of other studied transitions. Species
Transition
Frequency
AE
Eu
J
Parity, F
[GHz]
[s−1]
[K]
CH
3/2 →1/2
−1 →1+
532.7216
0.020
25.76
−1 →0+
532.7239
0.062
−2 →1+
532.7933
0.041
OH
5/2 →3/2
+2 →2−
2514.2987
1.367
120.75
+2 →3−
2514.3167
13.678
+1 →2−
2514.3532
12.310
Notes. The spectroscopic data for other studied CH and OH transitions–
quantum numbers, frequencies, Einstein-A coefficients and upper level
energies were taken from the Cologne Database for Molecular Spec-
troscopy (Müller et al. 2001). Source
N(OH)/N(CH)
Pearson’s
N(OH)/N(H2)
r-value
(×10−7)
AGAL010.624−00.384
5.66 ± 0.06
0.75
1.98
AGAL034.258+00.154
2.48 ± 0.17
0.67
0.87
AGAL327.293−00.579
4.13 ± 0.11
0.46
1.44
AGAL330.954−00.182
2.78 ± 0.07
0.68
0.97
AGAL332.826−00.549
9.70 ± 0.48
0.32
3.40 Source
N(OH)/N(CH)
Pearson’s
N(OH)/N(H2)
r-value
(×10−7) Notes. The spectroscopic data for other studied CH and OH transitions–
quantum numbers, frequencies, Einstein-A coefficients and upper level
energies were taken from the Cologne Database for Molecular Spec-
troscopy (Müller et al. 2001). Notes. The spectroscopic data for other studied CH and OH transitions–
quantum numbers, frequencies, Einstein-A coefficients and upper level
energies were taken from the Cologne Database for Molecular Spec-
troscopy (Müller et al. 2001). 1011
1012
1013
1014
1015
Nv(OH) [cm
2 (km /s)
1]
AGAL010.624
00.384
r = 0.75
1011
1012
1013
1014
1015
AGAL034.258 + 00.154
r = 0.67
1011
1012
1013
1014
1015
AGAL327.293
00.579
r = 0.46
Nv(OH)/Nv(CH) = 10.0
Nv(OH)/Nv(CH) = 1.0
Nv(OH)/Nv(CH) = 0.1
spiral-arm
inter-arm
1010
1011
1012
1013
1014
1011
1012
1013
1014
1015
AGAL330.954
00.182
r = 0.68
1010
1011
1012
1013
1014
Nv(CH) [cm
2 (km /s)
1]
1011
1012
1013
1014
1015
AGAL332.826
00.549
r = 0.32
1010
1011
1012
1013
1014
1011
1012
1013
1014
1015
AGAL351.581
00.352
r
0
1018
1019
1020
1021
1018
1019
1020
1021
Nv(H2) [cm
2 (km /s)
1]
1018
1019
1020
1021
Fig. F.1. Correlation between CH and OH column densities derived using the WF deconvolution algorithm toward (clockwise
from top left) AGAL010.624−00.384, AGAL034.258+00.154, AGAL327.293−00.579, AGAL330.954−00.182, AGAL332.826−00.549 and
AGAL351.58−00.352. Appendix D: Impact of using a single excitation
temperature on derived column densities A second step of wavelet de-noising was added
to our existing WF algorithm, in order to improve the robustness
and efficiency of this method. Once again, we visualise the fit
through its residuals as shown in Fig. C.1 toward a single source
AGAL010.624−00.384. The column densities presented in Table 3 are derived using
the WF deconvolution by assuming a single excitation tempera-
ture of 3.1 K for all components. Since the critical densities, ncrit9
of the CH transitions presented in this work (∼2.4 × 109 cm−3,
assuming a gas temperature value of 50 K) are several orders of
magnitude higher than the typical conditions that prevail over
the foreground LOS clouds, almost all the CH molecules are
expected to be in the ground state. Furthermore, the large val-
ues of ncrit imply that population of any rotational level above the
ground state of CH must arise from radiative excitation processes
rather than collisional excitation, as shown by Black (1994). As mentioned in Sect. 4.1, radiative excitation is unlikely for
CH molecules residing in locations that correspond to velocity
intervals associated with the diffuse and the translucent regions. Using higher values for the excitation temperature, we model
the absorption features associated with the molecular environ-
ment of the bright FIR continuum sources (Fig. D.1). Changes
in the excitation temperature between 3.1 and 10 K result in
uncertainties in the calculated column densities that are less than
15%, while this margin increases to nearly 50% as Tex →30 K. While a proper estimation of the excitation temperature requires
a thorough radiative transfer description across the envelope of
the continuum source, here, we merely illustrate how the calcu-
lated column densities are affected by the assumption of a single
excitation temperature. In general, both sets of residuals are scattered randomly
and centred around zero. The outliers in the WF fit are curbed
when it is followed by the use of the WT, giving rise to a less
variant or narrower distribution. However, the wavelet domain
analysis is more suited to transient or time-varying signals,
whereas our spectra are time independent. Appendix D: Impact of using a single excitation
temperature on derived column densities The different markers indicate contributions from spiral-arm (in blue), and inter-arm gas (in red). The linear regression
is shown by the blue dashed line along with N(OH)/N(CH) ratios of 0.1, one and 10. Secondary x-axis represents H2 column densities obtained
using the CH/H2 abundance ratio given by Sheffer et al. (2008). The corresponding Pearson’s correlation coefficients are given in the lower-right
corners. 1011
1012
1013
1014
1015
m /s)
1]
AGAL010.624
00.384
r = 0.75
1011
1012
1013
1014
1015
AGAL034.258 + 00.154
r = 0.67
1011
1012
1013
1014
1015
AGAL327.293
00.579
r = 0.46
Nv(OH)/Nv(CH) = 10.0
Nv(OH)/Nv(CH) = 1.0
Nv(OH)/Nv(CH) = 0.1
spiral-arm
inter-arm
1018
1019
1020
1021
1018
1019
1020
1021
Nv(H2) [cm
2 (km /s)
1]
1018
1019
1020
1021 1011
1012
1013
1014
1015
AGAL034.258 + 00.154
r = 0.67
1011
1012
1013
1014
1015
AGAL327.293
00.579
r = 0.46
Nv(OH)/Nv(
Nv(OH)/Nv(
Nv(OH)/Nv(
spiral-arm
inter-arm
1018
1019
1020
1021
Nv(H2) [cm
2 (km /s)
1]
1018
1019
1020
1021 1011
1012
1013
1014
1015
m /s)
]
AGAL010.624
00.384
r = 0.75
1011
1012
1013
1014
1015
AGAL034.258 + 00.154
r = 0.67
1011
1012
1013
1014
1015
AGAL327.293
1018
1019
1020
1021
1018
1019
1020
1021
Nv(H2) [cm
2 (km /s)
1]
1018
1019 Nv(OH) [cm
2 (km /s)
1] 1014
68
1010
1011
1012
1013
1014
Nv(CH) [cm
2 (km /s)
1]
1011
1012
1013
1014
1015
AGAL332.826
00.549
r = 0.32
1010
1011
1012
1013
1014
1011
1012
1013
1014
1015
AGAL351.581
00.352
r
0 Nv(OH) [cm
2 (
1010
1011
1012
1013
1014
1011
1012
1013
1014
1015
AGAL330.954
00.182
r = 0.68 Fig. F.1. Correlation between CH and OH column densities derived using the WF deconvolution algorithm toward (clockwise
from top left) AGAL010.624−00.384, AGAL034.258+00.154, AGAL327.293−00.579, AGAL330.954−00.182, AGAL332.826−00.549 and
AGAL351.58−00.352. The different markers indicate contributions from spiral-arm (in blue), and inter-arm gas (in red). The linear regression
is shown by the blue dashed line along with N(OH)/N(CH) ratios of 0.1, one and 10. Secondary x-axis represents H2 column densities obtained
using the CH/H2 abundance ratio given by Sheffer et al. (2008). The corresponding Pearson’s correlation coefficients are given in the lower-right
corners. A60, page 14 of 14
|
https://openalex.org/W3129313809
|
https://www.nature.com/articles/s41598-021-83740-w.pdf
|
English
| null |
Human induced pluripotent stem cell-based platform for modeling cardiac ischemia
|
Scientific reports
| 2,021
|
cc-by
| 12,388
|
Human induced pluripotent stem
cell‑based platform for modeling
cardiac ischemia
OPEN Martta Häkli
1*, Joose Kreutzer2, Antti‑Juhana Mäki2, Hannu Välimäki2, Henna Lappi1
Heini Huhtala3, Pasi Kallio2, Katriina Aalto‑Setälä1 & Mari Pekkanen‑Mattila1 Ischemic heart disease is a major cause of death worldwide, and the only available therapy to
salvage the tissue is reperfusion, which can initially cause further damage. Many therapeutics that
have been promising in animal models have failed in human trials. Thus, functional human based
cardiac ischemia models are required. In this study, a human induced pluripotent stem cell derived-
cardiomyocyte (hiPSC-CM)-based platform for modeling ischemia–reperfusion was developed
utilizing a system enabling precise control over oxygen concentration and real-time monitoring of
the oxygen dynamics as well as iPS-CM functionality. In addition, morphology and expression of
hypoxia-related genes and proteins were evaluated as hiPSC-CM response to 8 or 24 h hypoxia and
24 h reoxygenation. During hypoxia, initial decrease in hiPSC-CM beating frequency was observed,
after which the CMs adapted to the conditions and the beating frequency gradually increased already
before reoxygenation. During reoxygenation, the beating frequency typically first surpassed the
baseline before settling down to the values close the baseline. Furthermore, slowing on the field
potential propagation throughout the hiPSC-CM sheet as well as increase in depolarization time
and decrease in overall field potential duration were observed during hypoxia. These changes were
reversed during reoxygenation. Disorganization of sarcomere structures was observed after hypoxia
and reoxygenation, supported by decrease in the expression of sarcomeric proteins. Furthermore,
increase in the expression of gene encoding glucose transporter 1 was observed. These findings
indicate, that despite their immature phenotype, hiPSC-CMs can be utilized in modeling ischemia–
reperfusion injury. Ischemic heart disease (IHD) is the most common cardiovascular disease and a major cause of death worldwide1. In IHD, blood flow to myocardium is reduced or blocked leading to oxygen and nutrient deprivation, and accu-
mulation of metabolic waste in the tissue. This causes damage and death to the cells in the myocardium, including
cardiomyocytes (CMs) that are responsible for contraction of heart2. While a lot of research resources has been
invested to the study of the disease, reperfusion, i.e. restoring blood flow to the ischemic tissue, is currently the
only available therapy to reduce damage to the ischemic area in addition to prevention of arrhythmias by anti-
arrhythmic medication. However, reperfusion itself causes further damage to the tissue. www.nature.com/scientificreports www.nature.com/scientificreports Results
hi SC C During 7–15 h
hypoxia, the normalized beating frequency was 0.358 ± 0.336 (p = 2.4*10–8 compared to baseline) and during
15–24 h hypoxia, it increased to 0.656 ± 0.402 (p = 8.8*10–7 compared to baseline and p = 2.6*10–5 compared to
7–15 h hypoxia). During 0–6 h reoxygenation, the normalized beating frequency increased to 1.274 ± 0.396
(p = 0.015 compared to baseline and p = 2.8*10–8 compared to 15–24 h hypoxia) and then decreased close to the
baseline level during 6–24 h reoxygenation, to 0.981 ± 0.333 (p = 3.0*10–6 compared to 0–6 h reoxygenation). Figure 1b presents the mean normalized beating frequency at the baseline, 7–15 h hypoxia, 15–24 h hypoxia,
0–6 h reoxygenation and 6–24 h reoxygenation.i yg
yg
Depolarization time and field potential duration (FPD) were evaluated from three samples at two timepoints
of baseline measurement (nspikes = 19 for both time points for all three samples), as well as from one sample after
6.5 h (nspikes = 4) and from two samples after 8 h hypoxia (nspikes = 4 for both samples), depending on whether
beating had stopped already at 8 h. Depolarization time and FPD were also evaluated from the three samples
after 24 h of hypoxia (nspikes = 13 for all three samples), and 6 h (nspikes = 17 for all three samples) and 24 h
(nspikes = 14 for all three samples) of reoxygenation. Depolarization time was determined as the time from the
first peak to the second peak, whereas FPD was determined as the time from the first peak to the flat peak as
shown in Fig. S4. Both parameters were normalized for each sample to the mean of their own baseline values. As
a response to hypoxia, depolarization time increased while the total FPD decreased, whereas during reoxygena-
tion, both values returned close to the baseline level again, as demonstrated in Fig. 1c,d. Compared to baseline,
the mean depolarization time increased 1.26 ± 0.09 -fold at 6.5 h hypoxia (p = 0.0044), 1.65 ± 0.43 -fold at 8 h
hypoxia (p = 1.3*10–5) and 1.49 ± 0.27 -fold at 24 h hypoxia (p = 8.67*10–17). At 6 h reoxygenation, the increase
was 1.15 ± 0.24 -fold (p = 0.00033 compared to baseline, p = 2.64*10–7 compared to 24 h hypoxia) whereas at 24 h
reoxygenation it was 1.52 ± 0.92 -fold (not significant) compared to baseline. Results
hi SC C hiPSC‑CM functionality during hypoxia and reoxygenation. Functionality of the hiPSC-CMs was
studied with MEA to evaluate changes in the beating characteristics of the hiPSC-CMs during hypoxia and
reoxygenation. For beating frequency, signals from 41 electrodes were analyzed (4–6 electrodes from 9 samples,
3 parallel samples from 3 differentiation batches). The luminescence-based oxygen measurement was incor-
porated to one sample to evaluate the oxygen dynamics of the platform. It was observed that after initiation
of hypoxia or reoxygenation, the oxygen level in the culture stabilized within four hours. Clear and repeatable
changes were observed in the beating frequency of the hiPSC-CMs during hypoxia and reoxygenation, which
together with the observed oxygen dynamics were used to divide hypoxia and reoxygenation to periods for
statistical analysis. y
For beating frequency analysis, hypoxia was divided into two periods, 7–15 h hypoxia and 15–24 h hypoxia,
as during 7–15 h hypoxia the oxygen level had already stabilized and the beating frequency of the hiPSC-CMs
was lowest, while it gradually started to increase during 15–24 h of hypoxia. Reoxygenation was divided into
two groups as well; 0–6 h reoxygenation to observe the immediate increase of the beating frequency during the
increase of the pO2, and 6–24 h reoxygenation, during which the beating of the hiPSC-CMs stabilized. Figure 1a
presents a representative measurement of pO2 and a normalized beating frequency from a single electrode of
one MEA measurement, while Fig. S1 presents examples of a normalized beating rate from other experiments. Fig. S2 presents a representative MEA signal during different time points of hypoxia and reoxygenation and Fig. S3 presents the post-calibration of the oxygen measurement. Beating frequency (beats per minute, BPM) was normalized for each electrode to a median baseline BPM
value so that the results are comparable despite the differences in the baseline BPM. Median baseline BPM value
was chosen for normalization instead of mean so that individual time points that deviate much from the baseline
do not affect the normalization. The baseline was determined from measurements of 20 h before initiation of
hypoxia and it was relatively stable throughout the time period. Mean of the normalized beating frequency of all
the chosen electrodes at baseline was 1.081 ± 0.173 and started to decrease after hypoxia started. www.nature.com/scientificreports/ cell death8–10 and disruption of the sarcomere structure9,10. Furthermore, changes in the functionality and elec-
trophysiology have been observed, including decrease in hiPSC-CM beating frequency11,12,15,16,24, contractility,
calcium overload and arrhythmias14. In these models, hypoxic conditions have been induced to the hiPSC-CMs
by multiple methods, such as by using hypoxic gas8–10,13–15,24,25 or causing oxidative stress via peroxide treatment16. In addition, cell culture media composition has been modified to better recapitulate the ischemic conditions in
several studies. The modifications include removal of glucose23 or serum11, or both8–10,13,14,22, as well as acidosis
to better mimic the physiological ischemic event8–10,13,22.h death8–10 and disruption of the sarcomere structure9,10. Furthermore, changes in the functionality and elec-
hysiology have been observed, including decrease in hiPSC-CM beating frequency11,12,15,16,24, contractility, y
gy
g
g
q
y
y
um overload and arrhythmias14. In these models, hypoxic conditions have been induced to the hiPSC-CMs
ultiple methods, such as by using hypoxic gas8–10,13–15,24,25 or causing oxidative stress via peroxide treatment16. i p y
g
In this study, we present a hiPSC-CM based platform for modeling cardiac ischemia–reperfusion. The base of
the platform is an OxyGenie mini-incubator26 combined with a microelectrode array (MEA) and a luminescence-
based oxygen sensor12,24. The system allows precise control over oxygen concentration, real-time monitoring
of the cardiomyocyte functionality as well as the measurement of oxygen level in the cell culture area during
hypoxia and reoxygenation without disturbing or interrupting the experiment. The main objective of the pre-
sent study was develop a platform to assess the functional, structural and molecular responses of hiPSC-CMs to
ischemia and reperfusion. The hiPSC-CMs were combined with an extensive set of analysis methods including
a microelectrode array and oxygen measurement. In addition, the platform is compatible with collecting and
analyzing samples for immunocytochemistry, qPCR and western blot. As the studies on the electrophysiology
and functionality of hiPSC-CMs under hypoxia and reoxygenation are very limited in their number, this study
strongly contributes to the hiPSC-CM based ischemia–reperfusion modeling. Human induced pluripotent stem
cell‑based platform for modeling
cardiac ischemia
OPEN Animal experiments
have provided promising results for medical interventions at the time of reperfusion, but they have failed in
human clinical trials3. As the difference between species is considered to be one reason behind the failure4,5, it is
important to establish functional human based models to evaluate and develop new therapies.fif p
p
p
Human induced pluripotent stem cells (hiPSCs) can be endlessly produced and efficiently differentiated into
cardiomyocytes (hiPSC-CMs) enabling development of human-based cell models for the research of cardiac
diseases and therapies6. However, hiPSC-CMs are developmentally immature and structurally, functionally and
metabolically resemble more fetal than adult CMs7. Especially, the metabolic immaturity affects the use of the
hiPSC-CMs in ischemia modeling, as their metabolism relies on glucose making them more resistant to hypoxia
and reperfusion. Adult CMs mainly utilize fatty acid oxidation in energy production and thus are more vulner-
able to oxidative stress than fetal CMs8–10. Despite their drawbacks, hiPSC-CM based ischemia–reperfusion models have lately emerged8–23 and these
models have been reported to recapitulate the typical cardiac ischemia responses in the cells, such as increased 1Heart Group, Faculty of Medicine and Health Technology, Tampere University, Arvo Ylpön katu 34,
33520 Tampere, Finland. 2Micro‑ and Nanosystems Research Group, Faculty of Medicine and Health Technology,
Tampere University, Tampere, Finland. 3Faculty of Social Sciences, Tampere University, Tampere, Finland. *email:
martta.hakli@tuni.fi Scientific Reports | (2021) 11:4153 | https://doi.org/10.1038/s41598-021-83740-w www.nature.com/scientificreports/ Results
hi SC C On the other hand, compared to
baseline, the total field potential duration decreased to 0.75 ± 0.01 at 6.5 h hypoxia (p = 0.0007), 0.87 ± 0.25 at
8 h hypoxia (not significant), and then increased to 0.96 ± 0.15 at 24 h hypoxia (p = 0.0085), 0.92 ± 0.14 at 6 h
reoxygenation (p = 1.0*10–5), and 0.97 ± 0.19 at 24 h reoxygenation (not significant). https://doi.org/10.1038/s41598-021-83740-w Scientific Reports | (2021) 11:4153 | www.nature.com/scientificreports/ Figure 1. (a) Representative measurement of the partial pressure of oxygen and normalized beating frequency
from a single experiment. Black line marks the initiation of hypoxia, after which the oxygen level of the culture
starts to decrease and reaches 0 kPa approximately within 4 h. Green line marks the initiation of reoxygenation,
after which the oxygen level starts to increase and reaches 19 kPa approximately within 4 h of reoxygenation. The beating frequency of the hiPSC-CMs clearly decreases during hypoxia and increases after reoxygenation. (b) The mean normalized beating frequency extracted from MEA recordings for hiPSC-CMs during baseline
measurement, 7–15 h hypoxia, 15–24 h hypoxia, 0–6 h reoxygenation and 6–24 h reoxygenation presented
as mean + standard deviation (nelectrodes = 41). Normalization is done to each electrode separately with regard
to median baseline value, due to the variation observed in the beating frequency between different electrodes
during baseline measurements. The beating frequency starts to decrease after hypoxia is initiated and was
observed to be on its lowest during 7–15 h of hypoxia. The beating frequency started to recover already before
reoxygenation was initiated and was statistically significantly greater during 15–24 h hypoxia compared to
7–15 h hypoxia. Furthermore, overcompensation in the beating frequency was observed during 0–6 h of
reoxygenation, when the mean normalized beating frequency exceeded the baseline. After 6 h reoxygenation,
the beating frequency returned close to the baseline level. (c) Normalized depolarization time of hiPSC-CMs at
baseline, 6.5 h, 8 h and 24 h hypoxia as well as 6 h and 24 h reoxygenation. Depolarization time increased during
h
i
d
d l
h b
li
l
l d
i
i
(d) N
li
d fi ld
i l d
i Figure 1. (a) Representative measurement of the partial pressure of oxygen and normalized beating frequency
from a single experiment. Results
hi SC C Black line marks the initiation of hypoxia, after which the oxygen level of the culture
starts to decrease and reaches 0 kPa approximately within 4 h. Green line marks the initiation of reoxygenation,
after which the oxygen level starts to increase and reaches 19 kPa approximately within 4 h of reoxygenation. The beating frequency of the hiPSC-CMs clearly decreases during hypoxia and increases after reoxygenation. (b) The mean normalized beating frequency extracted from MEA recordings for hiPSC-CMs during baseline
measurement, 7–15 h hypoxia, 15–24 h hypoxia, 0–6 h reoxygenation and 6–24 h reoxygenation presented
as mean + standard deviation (nelectrodes = 41). Normalization is done to each electrode separately with regard
to median baseline value, due to the variation observed in the beating frequency between different electrodes
during baseline measurements. The beating frequency starts to decrease after hypoxia is initiated and was
observed to be on its lowest during 7–15 h of hypoxia. The beating frequency started to recover already before
reoxygenation was initiated and was statistically significantly greater during 15–24 h hypoxia compared to
7–15 h hypoxia. Furthermore, overcompensation in the beating frequency was observed during 0–6 h of
reoxygenation, when the mean normalized beating frequency exceeded the baseline. After 6 h reoxygenation,
the beating frequency returned close to the baseline level. (c) Normalized depolarization time of hiPSC-CMs at
baseline, 6.5 h, 8 h and 24 h hypoxia as well as 6 h and 24 h reoxygenation. Depolarization time increased during
hypoxia and returned close to the baseline level during reoxygenation. (d) Normalized field potential duration
(FPD) of hiPSC-CMs at baseline, 6.5 h, 8 h and 24 h hypoxia as well as 6 h and 24 h reoxygenation. FPD
decreased during hypoxia and returned close to the baseline level during reoxygenation *p < 0.05, **p < 0.01,
***p < 0.001. Figure 1. (a) Representative measurement of the partial pressure of oxygen and normalized beating frequency
from a single experiment. Black line marks the initiation of hypoxia, after which the oxygen level of the culture
starts to decrease and reaches 0 kPa approximately within 4 h. Green line marks the initiation of reoxygenation,
after which the oxygen level starts to increase and reaches 19 kPa approximately within 4 h of reoxygenation. The beating frequency of the hiPSC-CMs clearly decreases during hypoxia and increases after reoxygenation. Results
hi SC C (b) The mean normalized beating frequency extracted from MEA recordings for hiPSC-CMs during baseline
measurement, 7–15 h hypoxia, 15–24 h hypoxia, 0–6 h reoxygenation and 6–24 h reoxygenation presented
as mean + standard deviation (nelectrodes = 41). Normalization is done to each electrode separately with regard
to median baseline value, due to the variation observed in the beating frequency between different electrodes
during baseline measurements. The beating frequency starts to decrease after hypoxia is initiated and was
observed to be on its lowest during 7–15 h of hypoxia. The beating frequency started to recover already before
reoxygenation was initiated and was statistically significantly greater during 15–24 h hypoxia compared to
7–15 h hypoxia. Furthermore, overcompensation in the beating frequency was observed during 0–6 h of
reoxygenation, when the mean normalized beating frequency exceeded the baseline. After 6 h reoxygenation,
the beating frequency returned close to the baseline level. (c) Normalized depolarization time of hiPSC-CMs at
baseline, 6.5 h, 8 h and 24 h hypoxia as well as 6 h and 24 h reoxygenation. Depolarization time increased during
hypoxia and returned close to the baseline level during reoxygenation. (d) Normalized field potential duration
(FPD) of hiPSC-CMs at baseline, 6.5 h, 8 h and 24 h hypoxia as well as 6 h and 24 h reoxygenation. FPD
decreased during hypoxia and returned close to the baseline level during reoxygenation *p < 0.05, **p < 0.01,
***p < 0.001. Figure 1. (a) Representative measurement of the partial pressure of oxygen and normalized beating frequency
from a single experiment. Black line marks the initiation of hypoxia, after which the oxygen level of the culture
starts to decrease and reaches 0 kPa approximately within 4 h. Green line marks the initiation of reoxygenation,
after which the oxygen level starts to increase and reaches 19 kPa approximately within 4 h of reoxygenation. The beating frequency of the hiPSC-CMs clearly decreases during hypoxia and increases after reoxygenation. (b) The mean normalized beating frequency extracted from MEA recordings for hiPSC-CMs during baseline
measurement, 7–15 h hypoxia, 15–24 h hypoxia, 0–6 h reoxygenation and 6–24 h reoxygenation presented
as mean + standard deviation (nelectrodes = 41). Normalization is done to each electrode separately with regard
to median baseline value, due to the variation observed in the beating frequency between different electrodes
during baseline measurements. Results
hi SC C f)
Field potential conduction time from electrode to electrode at baseline, 8 h and 24 h hypoxia, and 6 h and 24 h
reoxygenation (mean + standard deviation). Conduction time increased during hypoxia compared to baseline,
whereas it was restored close to baseline level during reoxygenation. ***p < 0.001. Signal propagation was evaluated at two baseline timepoints, as well as after 8 h and 24 h hypoxia and 6 h
and 24 h reoxygenation from all signals of all electrodes of two samples that were showing field potential signals. The conduction velocity was observed to slow down during hypoxia and increase close to the baseline level
during reoxygenation, as shown in Fig. 2a–f. At baseline, the mean duration of field potential propagation from
one electrode to the adjacent (in x or y direction) was observed to be 0.45 ± 0.75 ms, whereas it was 0.72 ± 1.26
at 8 h hypoxia (p = 6.4*10–18), 0.86 ± 1.29 ms at 24 h hypoxia (p = 5.2*10–35). At 6 h reoxygenation, the time was
observed to be 0.71 ± 1.15 ms (p = 1.1*10–40 compared to baseline) and at 24 h reoxygenation it was 0.56 ± 0.74 ms
(p = 3.8*10–5 compared to 6 h reoxygenation). hiPSC‑CM morphology, sarcomere structure and nucleus size. The morphology, sarcomere struc-
ture, nucleus size and HIF1α expression of the hiPSC-CMs was studied with immunocytochemistry. After expo-
sure to hypoxia or hypoxia-reoxygenation, hiPSC-CMs were immunolabeled against MyBPC3 and HIF1α and
cell nuclei were visualized with DAPI (Fig. 3a). 6 hypoxia and control samples (2 parallel samples from 3 dif-
ferentiation batches) from both 8 and 24 h experiments as well as 4 hypoxia-reoxygenation samples (2 parallel
samples from 2 differentiation batches) from both 8 and 24 h experiments were stained and imaged. Qualitative
analysis revealed changes in the cell morphology, expression of sarcomeres and the size of the nuclei, which were
then quantitated and determined statistically significant. However, the expression of HIF1α was not found to
increase in any of the used time points.i Quantitative analysis on sarcomere expression and nuclei size of the cells revealed statistically significant
differences in these parameters between hypoxia or hypoxia-reoxygenation and control samples (Fig. 3b,c). Sarcomere coverage was analyzed from 124 images of control samples, 47 images of 8 h hypoxia samples and
50 images of 24 h hypoxia samples. Results
hi SC C The beating frequency starts to decrease after hypoxia is initiated and was
observed to be on its lowest during 7–15 h of hypoxia. The beating frequency started to recover already before
reoxygenation was initiated and was statistically significantly greater during 15–24 h hypoxia compared to
7–15 h hypoxia. Furthermore, overcompensation in the beating frequency was observed during 0–6 h of
reoxygenation, when the mean normalized beating frequency exceeded the baseline. After 6 h reoxygenation,
the beating frequency returned close to the baseline level. (c) Normalized depolarization time of hiPSC-CMs at
baseline, 6.5 h, 8 h and 24 h hypoxia as well as 6 h and 24 h reoxygenation. Depolarization time increased during
hypoxia and returned close to the baseline level during reoxygenation. (d) Normalized field potential duration
(FPD) of hiPSC-CMs at baseline, 6.5 h, 8 h and 24 h hypoxia as well as 6 h and 24 h reoxygenation. FPD
decreased during hypoxia and returned close to the baseline level during reoxygenation *p < 0.05, **p < 0.01,
***p < 0.001. https://doi.org/10.1038/s41598-021-83740-w Scientific Reports | (2021) 11:4153 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ Signal propagation was evaluated at two baseline timepoints, as well as after 8 h and 24 h hypoxia and 6 h
and 24 h reoxygenation from all signals of all electrodes of two samples that were showing field potential signals. Th
d
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Figure 2. (a–e) Field potential propagation over hiPSC-CM sheet on microelectrode array at baseline before
hypoxia, at 8 h and 24 h hypoxia and at 6 h and 24 h reoxygenation. The heatmaps clearly indicate slowing of
the field potential conduction during hypoxia, but the conduction velocity is restored during reoxygenation. f)
Field potential conduction time from electrode to electrode at baseline, 8 h and 24 h hypoxia, and 6 h and 24 h
reoxygenation (mean + standard deviation). Conduction time increased during hypoxia compared to baseline,
whereas it was restored close to baseline level during reoxygenation. ***p < 0.001. Figure 2. (a–e) Field potential propagation over hiPSC-CM sheet on microelectrode array at baseline before
hypoxia, at 8 h and 24 h hypoxia and at 6 h and 24 h reoxygenation. The heatmaps clearly indicate slowing of
the field potential conduction during hypoxia, but the conduction velocity is restored during reoxygenation. Results
hi SC C The mean coverage of clear and distinguishable sarcomeres of the total area
of the MyBPC3 staining in control samples was 69.4 ± 24.0%, whereas for 8 and 24 h hypoxia samples it was
30.1 ± 36.0% (p = 4.3*10–10) and 16.1 ± 24.8% (p = 6.1*10–18), respectively. Nucleus area was analyzed from the
fluorescent microscopy images (n8h hypoxia-reox = 18, n24h hypoxia-reox = 21) using CellProfiler. The mean nucleus area
was 35.7 ± 16.4 µm2 for control samples (nnuclei = 3632), whereas for 8 and 24 h hypoxia samples it was respec-
tively 27.2 ± 15.2 µm2 (nnuclei = 685, p = 8.9*10–43) and 22.3 ± 13.5 µm2 (nnuclei = 833, p = 1.5*10–113), and 8 and 24 h https://doi.org/10.1038/s41598-021-83740-w Scientific Reports | (2021) 11:4153 | www.nature.com/scientificreports/ Figure 3. (a) The structure, sarcomere expression and nucleus size changes during hypoxia. Sarcomeres
(MyBPC3, yellow) are clearly visible and the cells are spread and properly attached to the plate in control
(nimages = 124) samples, whereas the cell structure seems to deteriorate after 8 h (nimages = 47) and 24 h (nimages = 50)
hypoxia. Furthermore, the nucleus (DAPI, blue) size seems to decrease. On the other hand, no significant
changes are seen in the expression of hypoxia marker HIF1α (magneta). (b) Sarcomere coverage was quantified
from the fluorescent images and calculated as the ratio between the area of visible sarcomeres to the total cell
area and is expressed as percentage. The data are presented as mean + standard deviation. The expression of
clear and visible sarcomeres decreases as the length of the hypoxia increases. (c) Area of the nuclei in control
(nnuclei = 3632), 8 h hypoxia (nnuclei = 685), 24 h hypoxia (nnuclei = 833), 8 h hypoxia-reoxygenation (nnuclei = 329)
and 24 h hypoxia-reoxygenation (nnuclei = 782) samples presented as mean + standard deviation. The area of the
nuclei decreases with hypoxia and reoxygenation compared to control. **p < 0.01; ***p < 0.001. Figure 3. (a) The structure, sarcomere expression and nucleus size changes during hypoxia. Sarcomeres
(MyBPC3, yellow) are clearly visible and the cells are spread and properly attached to the plate in control
(nimages = 124) samples, whereas the cell structure seems to deteriorate after 8 h (nimages = 47) and 24 h (nimages = 50)
hypoxia. Furthermore, the nucleus (DAPI, blue) size seems to decrease. Results
hi SC C On the other hand, no significant
changes are seen in the expression of hypoxia marker HIF1α (magneta). (b) Sarcomere coverage was quantified
from the fluorescent images and calculated as the ratio between the area of visible sarcomeres to the total cell
area and is expressed as percentage. The data are presented as mean + standard deviation. The expression of
clear and visible sarcomeres decreases as the length of the hypoxia increases. (c) Area of the nuclei in control
(nnuclei = 3632), 8 h hypoxia (nnuclei = 685), 24 h hypoxia (nnuclei = 833), 8 h hypoxia-reoxygenation (nnuclei = 329)
and 24 h hypoxia-reoxygenation (nnuclei = 782) samples presented as mean + standard deviation. The area of the
nuclei decreases with hypoxia and reoxygenation compared to control. **p < 0.01; ***p < 0.001. Figure 3. (a) The structure, sarcomere expression and nucleus size changes during hypoxia. Sarcomeres
(MyBPC3, yellow) are clearly visible and the cells are spread and properly attached to the plate in control
(nimages = 124) samples, whereas the cell structure seems to deteriorate after 8 h (nimages = 47) and 24 h (nimages = 50)
hypoxia. Furthermore, the nucleus (DAPI, blue) size seems to decrease. On the other hand, no significant
changes are seen in the expression of hypoxia marker HIF1α (magneta). (b) Sarcomere coverage was quantified
from the fluorescent images and calculated as the ratio between the area of visible sarcomeres to the total cell
area and is expressed as percentage. The data are presented as mean + standard deviation. The expression of
clear and visible sarcomeres decreases as the length of the hypoxia increases. (c) Area of the nuclei in control
(nnuclei = 3632), 8 h hypoxia (nnuclei = 685), 24 h hypoxia (nnuclei = 833), 8 h hypoxia-reoxygenation (nnuclei = 329)
and 24 h hypoxia-reoxygenation (nnuclei = 782) samples presented as mean + standard deviation. The area of the
nuclei decreases with hypoxia and reoxygenation compared to control. **p < 0.01; ***p < 0.001. hypoxia-reoxygenation samples it was respectively 19.8 ± 11.1 µm2 (nnuclei = 329, p = 5.1*10–74) and 25.4 ± 14.6 µm2
(nnuclei = 782, p = 8.5*10–74). hypoxia-reoxygenation samples it was respectively 19.8 ± 11.1 µm2 (nnuclei = 329, p = 5.1*10–74) and 25.4 ± 14.6 µm2
(nnuclei = 782, p = 8.5*10–74). www.nature.com/scientificreports/ Western blot of HIF1α, MyBPC3 and Troponin T. Western blot was used to quantitate the expression
of HIF1α, MyBPC3 and Troponin T after hypoxia or hypoxia-reoxygenation. After 6 h (n = 4, 1 differentiation
batch), 8 h (n = 6, 2 parallel samples from 3 differentiation batches), 10 h (n = 3, 1 differentiation batch) and 12 h
(n = 3, 1 differentiation batch) hypoxia, the HIF1α expression remained close to control (n = 26, 4–5 parallel sam-
ples from 6 differentiation batches), but after 24 h hypoxia as well as 8 and 24 h hypoxia-reoxygenation (n = 4 for
both time points, 2 parallel samples from 2 differentiation batches), there was a statistically significant decrease
in the HIF1α expression (Fig. 4a). The mean expression of HIF1α in control samples was 1.39 ± 0.68, whereas
for 6, 8, 10, 12 and 24 h hypoxia samples it was respectively 1.29 ± 0.32, 1.05 ± 0.20, 1.06 ± 0.28, 1.34 ± 0.18 and
0.80 ± 0.39 (p = 0.006). For 8 and 24 h hypoxia-reoxygenation samples, the expression of HIF1α was 0.77 ± 0.08
(p = 0.001) and 0.84 ± 0.23 (p = 0.031), respectively.hit There were statistically significant decreases in the expressions of MyBPC3 and Troponin T after hypoxia and
hypoxia-reoxygenation (Fig. 4b). The mean MyBPC3 expression in the control samples was 1.11 ± 0.76, whereas
for 8 and 24 h hypoxia samples it was 0.53 ± 0.18 (p = 0.006) and 0.28 ± 0.32 (p = 0.000009), respectively. For 8
and 24 h hypoxia-reoxygenation samples the expression of MyBCP3 was 0.52 ± 0.22 (p = 0.023) and 0.11 ± 0.02
(p = 0.0002), respectively. As for Troponin T, the mean expression in the control samples was 0.96 ± 0.55. For 8
and 24 h hypoxia samples, the expression was respectively 0.47 ± 0.14 and 0.32 ± 0.27 (p = 0.007), while for 8 and
24 h hypoxia-reoxygenation samples the expression was 0.37 ± 0.26 (p = 0.037) and 0 (p = 0.0002), respectively. In
24 h hypoxia-reoxygenation samples, the Troponin T band did not become visible with exposure times suitable
for control samples. Examples of the western blot bands are presented in Fig. 4c, whereas full length blots of the
examples are presented in Supplementary Figures S5–S6. Gene expression of hiPSC‑CMs. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 4. (a) HIF1α expression in control (n = 26) samples and in 6 h (n = 4), 8 h (n = 6), 10 h (n = 3), 12 h
(n = 3) and 24 h (n = 6) hypoxia or 8 h (n = 4) and 24 h (n = 4) hypoxia-reoxygenation time points from western
blot analysis presented as mean + standard deviation. During the first 12 h of hypoxia, there seems to be no
significant changes in the expression of the protein, although in 6 h and 12 h samples there is a slight increase. On the other hand, after 24 h of hypoxia, as well as after 8 h hypoxia-reoxygenation there is a statistically
significant decrease in the HIF1α expression. (b) Expression of MyBPC3 and Troponin T decreases during 8 h
(n = 6) and 24 h (n = 6) hypoxia, and 8 h (n = 4) and 24 h (n = 4) hypoxia-reoxygenation compared to control
(n = 20) samples. The expression is especially low in 24 h hypoxia-reoxygenation samples but has decreased also
in other hypoxia samples. (c) Examples of the western blot bands. *p < 0.05; **p < 0.01; ***p < 0.001. Figure 4. (a) HIF1α expression in control (n = 26) samples and in 6 h (n = 4), 8 h (n = 6), 10 h (n = 3), 12 h
(n = 3) and 24 h (n = 6) hypoxia or 8 h (n = 4) and 24 h (n = 4) hypoxia-reoxygenation time points from western
blot analysis presented as mean + standard deviation. During the first 12 h of hypoxia, there seems to be no
significant changes in the expression of the protein, although in 6 h and 12 h samples there is a slight increase. On the other hand, after 24 h of hypoxia, as well as after 8 h hypoxia-reoxygenation there is a statistically
significant decrease in the HIF1α expression. (b) Expression of MyBPC3 and Troponin T decreases during 8 h
(n = 6) and 24 h (n = 6) hypoxia, and 8 h (n = 4) and 24 h (n = 4) hypoxia-reoxygenation compared to control
(n = 20) samples. The expression is especially low in 24 h hypoxia-reoxygenation samples but has decreased also
in other hypoxia samples. (c) Examples of the western blot bands. *p < 0.05; **p < 0.01; ***p < 0.001. Results
hi SC C hypoxia-reoxygenation samples it was respectively 19.8 ± 11.1 µm2 (nnuclei = 329, p = 5.1*10–74) and 25.4 ± 14.6 µm2
(nnuclei = 782, p = 8.5*10–74). https://doi.org/10.1038/s41598-021-83740-w Scientific Reports | (2021) 11:4153 | Discussion In the present study, a human based cardiac ischemia–reperfusion model which enables precise control and real
time monitoring of oxygen combined with simultaneous measurement of the electrophysiological parameters of
hiPS-CMs is presented. Contrary to our previous studies, in which the similar system was used with hiPS-CM
aggregates11,12,24, here the hiPS-CMs were sorted and re-plated as a monolayer on top of the MEA-plates provid-
ing more homogenous iPS-CM population and enabling a detailed assessment of the structural characteristics of
the cells. The more homogenous iPS-CM population also enables more precise qPCR analysis of cardiac markers
while the samples contain approximately similar percentage of cardiomyocytes.i p
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According to the results, hypoxia as well as reoxygenation alters the function of hiPSC-CMs significantly. The lowest beating frequency in the hiPSC-CMs was observed during 7–15 h hypoxia, whereas during 15–24 h
hypoxia, the beating frequency gradually increased, suggesting that the hiPSC-CMs adapt to the low oxygen envi-
ronment. During 0–6 h reoxygenation, the mean normalized beating frequency further increased and surpassed
the baseline level but returned close to the baseline during 6–24 h reoxygenation. However, it is noteworthy that
majority of the hiPSC-CMs retained their functionality after an initial drop in the beating frequency, during the
late phase of hypoxia.h p
yp
The decrease in cardiomyocyte beating frequency during ischemic stress has been earlier reported in pri-
mary CMs from rat27–29, in perfused whole mouse heart30. We and others have also reported the decrease
with pluripotent stem cell derived-CMs 11,12,24,31, as well as the increase in the beating frequency upon
reoxygenation11,12,24,27,29–31.h yg
The decrease in the beating rate might occur due to decrease in the ATP content in the CMs27,29 and due
to the conduction block affecting the propagation of the action potential from pacemaker cells to other cells31. Reoxygenation is thought to reverse these changes, restoring the beating frequency close to the baseline27,29,31. However, to our knowledge, this is the first time that an increase in the CM beating frequency already during a
late phase of hypoxia has been reported. The reason behind the hiPSC-CM adaptation is likely balancing of the
ATP supply and demand32, although the mechanisms for achieving the balance can only be speculated.h The immature metabolic phenotype of the hiPSC-CMs can be partially responsible for the observed adapta-
tion of the cells and increase of the beating frequency with prolonged hypoxia. www.nature.com/scientificreports/ Expression of several genes related to glucose and fatty acid metabolism,
calcium handling, sarcomeric proteins, hypoxia and apoptosis was analyzed from samples from 8 h (nhypoxia = 6,
ncontrol = 5, 1–2 parallel samples from 3 differentiation batches) and 24 h (nhypoxia = 5, ncontrol = 6, 1–2 parallel sam-
ples from 3 differentiation batches) hypoxia and 8 and 24 h hypoxia-reoxygenation experiments (nhypoxia = 4,
ncontrol = 4 for both time points, 2 parallel samples from 2 differentiation batches) with qPCR. There were no
statistically significant differences in the expression of most genes after 8 or 24 h hypoxia. However, after 24 h
hypoxia-reoxygenation, there was a statistically significant decrease in several genes. A statistically significant
increase was observed in the expression of SLC2A1 encoding glucose transporter 1 in all but 8 h hypoxia-
reoxygenation samples (Fig. 5a). For 8 h control and hypoxia samples, the mean expressions of SLC2A1 were
1.70 ± 1.65 and 28.32 ± 24.13 (p = 0.03), respectively. For 24 h control and hypoxia samples, the expressions were
1.88 ± 1.33 and 48.22 ± 42.47 (p = 0.004), respectively. For 8 h control and hypoxia-reoxygenation samples, the
expressions were 1.01 ± 0.16 and 0.85 ± 0.13, respectively. For 24 h control and hypoxia-reoxygenation samples,
the expressions were 1.77 ± 0.68 and 3.76 ± 0.77 (p = 0.029), respectively.i p
p
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In 8 and 24 h hypoxia-reoxygenation samples, there was also a statistically significant decrease in the expres-
sion of SLC8A1 encoding sodium/calcium exchanger 1 related to calcium handling of cardiomyocytes (Fig. 5b). Slight but not statistically significant decrease was also seen in 24 h hypoxia samples. For 8 h control and hypoxia
samples, the expressions were 1.38 ± 0.99 and 1.34 ± 0.95, while for 24 h control and hypoxia samples they were
1.64 ± 1.06 and 1.12 ± 0.57. For 8 h control and hypoxia-reoxygenation samples, the expressions were 1.01 ± 0.16
and 0.53 ± 0.12 (p = 0.029), respectively. For 24 h control and hypoxia-reoxygenation samples, the expressions
were 1.06 ± 0.23 and 0.26 ± 0.12 (p = 0.029), respectively. https://doi.org/10.1038/s41598-021-83740-w Scientific Reports | (2021) 11:4153 | www.nature.com/scientificreports/ Figure 5. www.nature.com/scientificreports/ (a) SLC2A1 expression in hiPSC-CMs after 8 and 24 h hypoxia in control (C, n8h = 6, n24h = 5) and
hypoxia (H, n8h = 5, n24h = 6) samples as well as 8 and 24 h hypoxia-reoxygenation (HR) in control (C, n8h = 4,
n24h = 4) and hypoxia (H, n8h = 4, n24h = 4) samples from qPCR analysis presented as mean + standard deviation. SLC2A1 encodes glucose transporter 1 and is related to glucose uptake of the cells. As can be seen, SLC2A1
expression increases during hypoxia, and decreases during reoxygenation. However, after 24 h hypoxia-
reoxygenation, the expression levels are still higher than in control. (b) SLC8A1 expression in hiPSC-CMs after
8 and 24 h hypoxia as well as 8 and 24 h hypoxia-reoxygenation. SLC8A1 encodes sodium/calcium exchanger
and is related to calcium handling of hiPSC-CMs. As can be seen from the figure, the expression decreases
after 8 and 24 h hypoxia-reoxygenation, but does not change significantly during hypoxia only. (c) Expression
of several genes after 24 h hypoxia-reoxygenation. The expression of metabolic genes (ACADM and PFKM),
sarcomeric genes (TNNT2 and MYBPC3), calcium handling genes (RYR2 and ATP2A2) and hypoxia marker
MAP4K4 are all decreasing when compared to control. *p < 0.05. Figure 5. (a) SLC2A1 expression in hiPSC-CMs after 8 and 24 h hypoxia in control (C, n8h = 6, n24h = 5) and
hypoxia (H, n8h = 5, n24h = 6) samples as well as 8 and 24 h hypoxia-reoxygenation (HR) in control (C, n8h = 4,
n24h = 4) and hypoxia (H, n8h = 4, n24h = 4) samples from qPCR analysis presented as mean + standard deviation. SLC2A1 encodes glucose transporter 1 and is related to glucose uptake of the cells. As can be seen, SLC2A1
expression increases during hypoxia, and decreases during reoxygenation. However, after 24 h hypoxia-
reoxygenation, the expression levels are still higher than in control. (b) SLC8A1 expression in hiPSC-CMs after
8 and 24 h hypoxia as well as 8 and 24 h hypoxia-reoxygenation. SLC8A1 encodes sodium/calcium exchanger
and is related to calcium handling of hiPSC-CMs. As can be seen from the figure, the expression decreases
after 8 and 24 h hypoxia-reoxygenation, but does not change significantly during hypoxia only. (c) Expression
of several genes after 24 h hypoxia-reoxygenation. www.nature.com/scientificreports/ The expression of metabolic genes (ACADM and PFKM),
sarcomeric genes (TNNT2 and MYBPC3), calcium handling genes (RYR2 and ATP2A2) and hypoxia marker
MAP4K4 are all decreasing when compared to control. *p < 0.05. Figure 5. (a) SLC2A1 expression in hiPSC-CMs after 8 and 24 h hypoxia in control (C, n8h = 6, n24h = 5) and
hypoxia (H n
5 n
6) samples as well as 8 and 24 h hypoxia reoxygenation (HR) in control (C n
4 In 24 h hypoxia-reoxygenation samples, there was also statistically significant decrease in the expression
of ACADM (c = 1.22 ± 0.13, 24 h HR = 0.60 ± 0.09, p = 0.029), PFKM (c = 1.52 ± 0.10, 24 h HR = 0.67 ± 0.13,
p = 0.029), TNNT2 (c = 1.48 ± 0.21, 24 h HR = 0.60 ± 0.11, p = 0.029), MYBPC3 (c = 1.47 ± 0.35, 24 h
HR = 0.38 ± 0.10, p = 0.029), MAP4K4 (c = 1.28 ± 0.10, 24 h HR = 0.59 ± 0.04, p = 0.029), RYR2 (c = 1.96 ± 0.31,
24 h HR = 0.56 ± 0.20, = 0.029) and ATP2A2 (c = 2.01 ± 0.28, 24 h HR = 0.44 ± 0.16, p = 0.029) (Fig. 5c). www.nature.com/scientificreports/ www.nature.com/scientificreports/ first committing step of glycolysis35 did not increase after 8 or 24 h hypoxia. Furthermore, the medium used in
the experiment did not contain either glucose or serum since presence of glucose attenuates the adverse effects
of hypoxia36. Thus, there must be also another mechanism for the hiPSC-CM functional adaptation. yph
p
In addition to changes in the beating frequency of the hiPSC-CMs as a response to hypoxia and reoxygenation,
changes in depolarization time and field potential duration were investigated. Hypoxia increased the depolariza-
tion time, whereas it decreased the overall FPD. During reoxygenation, both the depolarization time and the
FPD returned close to the baseline level. These findings are in line with other studies regarding prolongation of
CM action potential depolarization time as well as shortening of the overall action potential duration during
hypoxia and reoxygenation37 and indicate that the hiPSC-CMs in the model function as expected. yp
yg
p
Changes in the conduction velocity over the hiPSC-CM sheets were also investigated. During hypoxia, the
signal propagation speed slowed down, and it took longer for the action potential to travel across the cell sheet. This could be due to the conduction blocks that are known to occur in ischemic conditions38, which can prevent
the action potential from traveling the shortest route through the cell sheet. The slowing was observed to be
reversible, as the conduction velocity returned close the baseline level during reoxygenation. These findings are
in agreement with King et al. (2013), where delayed signal propagation has also been observed as response to
ischemia38.hi There were no significant changes in the expression of genes related to calcium handling and contractility
of the hiPSC-CMs after 8 or 24 h hypoxia. However, post-transcriptional changes in the proteins or changes in
their activity due to hypoxia could in part explain the increase in the beating frequency. Electrophysiological
alterations can include decrease in sodium and potassium currents, thus reducing the ATP demand of Na+/K+
ATPase. Decrease in calcium current could similarly reduce the ATP demand of Ca2+ ATPases as well as Na+/
K+ ATPases. Reduced potassium current would also lead to increased resting membrane potential making the
CMs more excitable, although the membrane potential must remain below the activation voltage of voltage-gated
sodium channels32. www.nature.com/scientificreports/ The initial decrease in the hiPSC-CM beating frequency can thus later be accompanied by
electrophysiological changes sufficiently reducing the ATP demand for the gradual increase in beating frequency
during the late phase of hypoxia. g
p
yp
A peak in the HIF1α expression was not found in this study. However, it is possible that the peak occurs
already earlier than the chosen time points, or that the peak is subtle and thus difficult to observe. Since there
was some variation in the expression between the samples from the same time points, it is possible that the subtle
increase in the expression of HIF1α was masked by the variation of the individual samples. It is also possible,
that oxygen independent HIF1α degradation pathways are activated39. Furthermore, nuclear translocation of
HIF1α was not seen in the immunocytochemical staining, although it is known to play a crucial role in activa-
tion of hypoxia pathways40.ft yp
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Differences in hiPSC-CM morphology and sarcomere structure were observed after hypoxia and hypoxia-
reoxygenation, which were supported by western blot analysis of the structural proteins. The morphological and
structural changes in the hiPSC-CMs included partial detachment from the culture plate as well as disruption of
the visible sarcomere structures. Furthermore, the area of the nuclei in the hiPSC-CMs decreased during hypoxia
and hypoxia-reoxygenation. However, cell death was not observed (data not shown). The structural disintegra-
tion and the decrease in the nucleus size are associated with apoptotic cell death41,42. However, the difference in
the nucleus area could also partially be due to detachment of the cells, because in attached cells the nucleus is
flat against the culture plate thus increasing the area in images, while in detached cells the nucleus has a more
3D spherical shape, which is not conveyed from 2D images. Furthermore, western blot analysis showed no band
for cleaved caspase-3 associated with apoptosis.h p
p p
The cellular damage in the hiPSC-CMs could also be indication of necrotic cell death, which is known to
be the primary mechanism of cell death in ischemia-reperfusion41,42. The decrease in MyBPC3 and Troponin T
protein expression further supports the structural changes seen in the hiPSC-CMs. The decrease in Troponin
T expression could be due to the release of Troponin T from the cells to the surroundings, although it was not
measured in this study. Discussion hiPSC-CMs are known to be more
resistant to hypoxia and reoxygenation because of the enhanced ability of immature CMs to increase glycolytic
flux33,34. In the current study, increase in the expression of SLC2A1 encoding glucose transporter 1 was observed
after 8 and 24 h hypoxia. During hypoxia, the CM metabolism relies on glycolysis, which is a relatively inefficient
way to produce energy requiring increase in glucose uptake and expression of glucose transporters35. However,
the expression of PFKM gene encoding muscle type ATP-dependent 6-phosphofructokinase responsible for the Scientific Reports | (2021) 11:4153 | https://doi.org/10.1038/s41598-021-83740-w www.nature.com/scientificreports/ However, Troponin T release from injured myocardium is a clinically used marker for
myocardial infarction43. Furthermore, necrotic cell death is associated with spillage of cellular contents into the
extracellular space. p
One limitation in this study was the immature phenotype of the hiPSC-CMs. As already mentioned, dif-
ferences in hiPSC-CM metabolism make these cells more resistant to hypoxia-reoxygenation injury. Another
limitation is the relatively constant pH throughout the hypoxia and reoxygenation treatments. Due to the large
volume of medium compared to the cell mass, the pH of the culture did not change significantly during the
hypoxia or reoxygenation. However, in heart, ischemia is accompanied by acidosis, a decrease in intracellular
pH, which is known to affect the CM response to ischemia–reperfusion in primary animal CM models28 and
hiPSC-CMs8,10. The third limitation is the slow speed in increase and decrease of the oxygen level in the culture,
which gives the cells time to adjust to the conditions, whereas acute ischemic events are typically sudden in the
clinical situation. Furthermore, electrical activity was recorded only in intervals, which could cause missing of
potentially interesting events. p
y
g
In summary, we conclude that hiPS-CM model presented in this study can be used in modeling cardiac
ischemia–reperfusion injury. With the iPS-CM based platform, we are able to monitor the temporal changes in
cardiomyocyte function as well as electrophysiological parameters during different stages of ischemia–reperfu-
sion event. There were significant differences in the beating frequency, depolarization time and field potential
duration as well as conduction velocity. In addition, hypoxia altered the cell morphology and caused changes in
the expression of structural proteins. With the presented hiPS-CM based platform, cardiac ischemia–reperfu-
sion can be modeled and these parameters can be utilized when evaluating the effects of putative ischemia drugs
and treatments in the future. https://doi.org/10.1038/s41598-021-83740-w Scientific Reports | (2021) 11:4153 | www.nature.com/scientificreports/ Figure 6. (a) Portable cell culture instrument include battery operated temperature controller and heat plate,
two gas cylinders and flow divider allowing six individual 1-well assemblies. 1-well assembly includes 1-well
chamber on plate (glass or MEA), the lid and the lid lock to seal the cell culture and to avoid evaporation, the
cover and the cover lock to create and maintain the gas environment around gas permeable 1-well chamber. 1-well assembly on MEA plate can be fitted also to MCS signal amplifier and thus use it for long-term recordings
outside the incubator. www.nature.com/scientificreports/ (b) Hypoxia and hypoxia-reoxygenation protocol. Figure 6. (a) Portable cell culture instrument include battery operated temperature controller and heat plate,
two gas cylinders and flow divider allowing six individual 1-well assemblies. 1-well assembly includes 1-well
chamber on plate (glass or MEA), the lid and the lid lock to seal the cell culture and to avoid evaporation, the
cover and the cover lock to create and maintain the gas environment around gas permeable 1-well chamber. 1-well assembly on MEA plate can be fitted also to MCS signal amplifier and thus use it for long-term recordings
outside the incubator. (b) Hypoxia and hypoxia-reoxygenation protocol. Figure 6. (a) Portable cell culture instrument include battery operated temperature controller and heat plate,
two gas cylinders and flow divider allowing six individual 1-well assemblies. 1-well assembly includes 1-well
chamber on plate (glass or MEA), the lid and the lid lock to seal the cell culture and to avoid evaporation, the
cover and the cover lock to create and maintain the gas environment around gas permeable 1-well chamber. 1-well assembly on MEA plate can be fitted also to MCS signal amplifier and thus use it for long-term recordings
outside the incubator. (b) Hypoxia and hypoxia-reoxygenation protocol. www.nature.com/scientificreports/ The pO2 was measured once a minute during the entire experiment,
while MEA signals were recorded for 1 min every 30 min throughout the experiment at 20 kHz sampling fre-
quency. After the experiment was finished, the cells were removed from the MEA plate and the oxygen measure-
ment was calibrated using the same MEA. Immunocytochemistry. Immunocytochemistry (full protocol in Supplementary Information) was per-
formed right after hypoxia or hypoxia-reoxygenation for 8 and 24 h hypoxia, and 8 and 24 h hypoxia-reoxygen-
ation samples. Mouse anti-MyBPC3 (1:500; Santa Cruz; sc-166081) and rabbit anti-HIF1α (1:1000; Invitrogen;
700505) were used as primary antibodies and donkey anti-mouse Alexa Fluor 568 and donkey anti-rabbit Alexa
Fluor 488 (1:800; Thermo Fisher Scientific) as secondary antibodies. Fluorescence was visualized with Zeiss LSM
800 Laser Scanning Confocal Microscope using Zeiss EC Plan-Neofluar 40x/0.75, WD 0.71 mm (Air) objective
and 2 channel spectral detection with high-sensitivity PMT detector. Analysis of hiPSC‑CM sarcomere coverage. The area of visible sarcomeres and the total cell area was
analyzed from the MyBPC3 channel of the fluorescent microscopy images using Paint.net (version 4.1.5, Dot-
PDN, LLC, USA) and ImageJ51. The wand tool in Paint.net was used to determine the total area of the cells
covering the image and the area covered by sarcomeres. ImageJ was then used to calculate the areas. Sarcomere
coverage was calculated as the ratio between the area of visible sarcomeres and the total cell area and is expressed
as percentage. Analysis of hiPSC‑CM nuclei area. The nucleus area was determined using Cell Profiler52. IdentifyPrima-
ryObjects was used to identify the nuclei in the DAPI channel of the fluorescent images. The minimum and max-
imum object size values were set as 50 and 250, respectively. Objects outside the set minimum and maximum
as well as objects touching borders were discarded. Threshold strategy was set as global and the thresholding
method used was two class Otsu. Shape was set for the method to distinguish clumped objects and MeasureOb-
jectSizeShape was used to determine the nuclei area from the objects identified in the IdentifyPrimaryObjects. Western blot. Protein samples were collected in 2 × Laemmli buffer (Bio-Rad) containing 5%
β-mercaptoethanol (Sigma) and run in 4–20% Mini PROTEAN TGX Precast Protein Gel with 10 50 µl wells
(Bio-Rad). The proteins were blotted from the gel to PVDF membrane using Trans-Blot Turbo Transfer System
(Bio-Rad) and Trans-Blot Turbo RTA Mini PVDF Transfer Kit (Bio-Rad). www.nature.com/scientificreports/ Hypoxia-reoxygenation experiments were performed similarly to the hypoxia experiments, but after 8 or 24 h
hypoxia, the gas was exchanged to 19% O2 and 5% CO2 (normoxic) gas for 24 h. The control cells were kept in
the 1-well chambers on the glass inside the incubator for the whole experiment. Samples were collected after the
hypoxia or hypoxia-reoxygenation one by one to minimize the exposure to ambient air. pH was measured from
cell culture medium during sample collection using Sentron SI600 pH meter with Sentron MicroFET pH probe.i g
g
With MEA plates, hypoxia-reoxygenation was performed so that first the samples were connected to normoxic
gas to measure a baseline overnight, after which, hypoxia was initiated using the hypoxic gas for 24 h. After the
hypoxia, reoxygenation was performed by connecting the samples again to the normoxic gas for another 24 h. Hypoxia and hypoxia-reoxygenation protocols are presented in Fig. 6b. Microelectrode array. Microelectrode array (MEA) plates (60MEA200/30iR-Ti, 8 × 8) were ordered from
MultiChannel Systems MCS GmbH (Reutlingen, Germany and PDMS 1-well chambers attached to the MEA
were manufactured inhouse50. MEA measurements were performed using MultiChannel Experimenter (version
2.14.0.19346, Multi Channel Systems, GmbH, Reutlingen, Germany). Sampling frequency was set to 25 kHz and
the baseline was recorded for 1 min every hour for 20 h. During hypoxia and reoxygenation, MEA signals were
recorded for 1 min every 30 min. The data were converted from MSRD into HDF5 format using MultiChannel
DataManager (version 1.12.0.20014, Multi Channel Systems, GmbH, Reutlingen, Germany). The HDF5 files
were analyzed with MATLAB (version R2018B, MathWorks, Inc., Natick, MA, USA) using scripts developed
inhouse for beating frequency, depolarization time and field potential duration. Multi Channel Analyzer (ver-
sion 2.14.0.19346, Multi Channel Systems, GmbH, Reutlingen, Germany) was used to detect peaks from the
recorded data. The analyzed data was converted into ASCII files using Multi Channel DataManager and these
files were analyzed for field potential propagation. Oxygen measurement. The partial pressure of oxygen (pO2) was measured from one sample to verify that
the oxygen dynamics in the cell culture were as expected. In the verification experiment, a luminescence-based
sensor together with a highly biocompatible sensing material, developed by Välimäki and coworkers24 was used
to monitor the oxygen level in the culture. Materials and methods
di
diff
i Cardiomyocyte differentiation and cell culture. UTA.04602.WT hiPSC line44 was used in the experi-
ments. hiPSCs were cultured on mouse embryonic fibroblast feeder cells (Applied StemCells, Inc) in KSR
medium (KnockOut DMEM (Gibco) containing 10% KnockOut Serum Replacement (Gibco), 1% MEM NEAA
(Gibco), 1% GlutaMAX (Gibco), 0.2% β-mercaptoethanol (Gibco) and 0.5% Penicillin/streptomycin (Lonza)). Embryoid body differentiation modified from Karakikes and coworkers45 and Lian and coworkers46 was used to
differentiate the hiPSCs into cardiomyocytes (full protocol in Supplementary Information). Magnetic activated cell sorting. Magnetic activated cell sorting (MACS) was performed on day 20
to enrich the cardiomyocyte concentration in the culture. MultiTissue Dissociation Kit (Miltenyi Biotec) was
used according to manufacturer’s instructions as described earlier47. PSC-Derived Cardiomyocyte Isolation Kit,
human (Miltenyi Biotec) was used to separate the cardiomyocytes from other cell types according to manufac-
turer’s instructions47. The CMs were suspended to 20% EB medium (KnockOut DMEM containing 20% FBS
(Gibco), 1% MEM NEAA, 1% GlutaMAX and 0.5% Penicillin/streptomycin) and seeded to 1-well chambers
on glass or MEA coated with 0.1% gelatin as cell sheets (density ~ 93,000 cells/cm2). The cells were cultured for
7–9 days before starting the hypoxia experiments. Half of the culture medium was exchanged three times a week. Hypoxia and hypoxia‑reoxygenation. Hypoxia and reoxygenation were performed using OxyGenie
mini-incubator (Baker, USA), which is based on our previous studies26,48,49. The portable cell culture incubator
includes a battery-operated temperature controller with a heat plate, two prefilled and replaceable gas cylinders
and a flow divider to hold six individual 1-well chamber assemblies on a glass plate (Fig. 6a). The 1-well assembly
on an MEA plate fits to a MultiChannel Systems signal amplifier allowing long-term recordings outside an incu-
bator. Portability of the OxyGenie and the individual 1-well chambers enable sample preparation and collection
one at a time, minimizing the cell culture exposure to ambient air. Day before exposing cells to hypoxia, serum- and glucose-free EB medium (glucose-free DMEM (Gibco)
containing 1% MEM NEAA, 1% GlutaMAX and 0.5% Penicillin/streptomycin) was changed. On the following
day, the samples were loaded into pre-warmed (37 °C) OxyGenie mini-incubator and hypoxia was initiated using
0% O2 and 5% CO2 (hypoxic) gas. The used time periods included 6, 8, 10, 12 and 24 h. Control samples were
cultured in serum- and glucose-free EB medium in a standard 5% CO2 incubator without the lids and covers
for the same time periods. Materials and methods
di
diff
i https://doi.org/10.1038/s41598-021-83740-w Scientific Reports | (2021) 11:4153 | www.nature.com/scientificreports/ Data availability y
All data are available from the authors by request. Received: 10 September 2020; Accepted: 5 February 2021 Received: 10 September 2020; Accepted: 5 February 2021 www.nature.com/scientificreports/ TaqMan 20 × Assays for TNNT2, MYBPC3, ACADM, ACAA1, SLC2A1,
PFKM, RYR2, ATP2A2, SLC8A1, MAP4K4, HIF1A, CASP3, GAPDH, EEF1A1 and TBP were used, details of
the assays are presented in Table 1. Master mixes were prepared as instructed in the kit. Three technical replicates
were used for each sample with each gene. 1 µl of diluted cDNA (diluted 2:1 to Milli-Q water) was pipetted into
9 µl of master mix (total reaction volume 10 µl). Plates were run in ABI7300 thermal cycler (Applied Biosystems)
with following protocol: 2 min at 50 °C, 10 min at 95 °C and 40 cycles of 15 s at 95 °C and 1 min at 60 °C. Statistical analysis. Statistical analyses were performed with IMB SPSS Statistics for Windows (version
25.0, IMB Corp., Armonk, NY, USA). Independent samples Mann–Whitney U test was used to test the statisti-
cal significance between control and hypoxia or hypoxia-reoxygenation groups for data extracted from qPCR,
western blot, immunostaining, depolarization time and field potential duration and field potential propagation,
while related-samples Wilcoxon Signed Rank Test was used for beating frequency. p < 0.05 was considered as
statistically significant. The data are presented as mean ± standard deviation. www.nature.com/scientificreports/ Mouse anti-β-actin (1:1000; Santa
Cruz; sc-47778), rabbit anti-HIF1α (1:1000), rabbit anti-cleaved caspase-3 (1:500; Abcam; ab32042), mouse anti-
MyBPC3 (1:500) and mouse anti-Troponin T (1:1000; Abcam; ab33589) were used as primary antibodies and
horseradish peroxidase-conjugated anti-mouse IgG (1:3000; Santa Cruz; sc-516102) and anti-rabbit IgG (1:2000;
Dako; P0217) as secondary antibodies (full protocol in Supplementary Information). The protein-antibody com-
plexes were detected using Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare Life
Sciences) and ChemiDoc MP Imaging System (Bio-Rad) was used for imaging. The images were analyzed using
Image Lab Software (Bio-Rad). RNA extraction, reverse transcription and qPCR. Samples for qPCR from hypoxia and hypoxia-
reoxygenation experiments were collected in QIAzol Lysis Reagent (Qiagen) and RNA was extracted using https://doi.org/10.1038/s41598-021-83740-w Scientific Reports | (2021) 11:4153 | www.nature.com/scientificreports/ Table 1. TaqMan 20 × Assays used in qPCR. Gene
Description
Function
Assay ID
ACADM
Acyl-CoA Dehydrogenase Medium Chain
Fatty acid metabolism
Hs00936584_m1
ACAA1
Acetyl-CoA acyltransferase 1
Fatty acid metabolism
Hs01576070_m1
PFKM
Phosphofructokinase, muscle
Glycolysis metabolism
Hs01075411_m1
SLC2A1
Solute carrier family 2, member 1/GLUT-1
Glycolysis metabolism
Hs00892681_m1
TNNT2
Cardiac type troponin T2
Sarcomeric
Hs00165960_m1
MYBPC3
Myosin binding protein C3, cardiac
Sarcomeric
Hs00165232_m1
RYR2
Ryanodine receptor 2, cardiac
Calcium handling
Hs00892883_m1
ATP2A2
ATPase, calcium transporting, cardiac muscle, slow twitch 2/ SERCA2a
Calcium handling
Hs00544877_m1
SLC8A1
Solute carrier family 8, member 1/NCX1
Calcium handling
Hs01062258_m1
HIF1A
Hypoxia inducible factor 1 alpha
Hypoxia marker
Hs00153153_m1
MAP4K4
Mitogen-activated protein kinase kinase kinase kinase 4
Hypoxia marker
Hs00377415_m1
CASP3
Caspase-3
Apoptosis
Hs00234387_m1
GAPDH
Glyceraldehyde-3-phosphate dehydrogenase
Housekeeping
Hs02758991_g1
TBP
TATA-box binding protein
Housekeeping
Hs00427620_m1
EEF1A1; EE +
Eukaryotic translation elongation factor 1 alpha 1
Housekeeping
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following manufacturer’s instructions. Shortly, 2X RT Master Mix was prepared based on instructions of the kit
and 10 µl of 2X RT Master Mix was mixed with 10 µl of sample. Samples were run in thermal cycler with follow-
ing protocol: 10 min at 25 °C, 120 min at 37 °C, 5 min at 85 °C and at 4 °C until samples were stored at − 20 °C. qPCR for mRNA samples was performed using TaqMan Gene Expression Master Mix (Applied Biosystems)
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C
t l C ll P
fil
3 0 N
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ti
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PL S Bi l 16 1 17 (2018) 51. Schindelin, J. et al. Fundingh g
This work was supported by grants from Academy of Finland, Finnish Foundation of Cardiovascular Research,
Biocenter Finland Stem Cell Platform, Juselius Foundation, Pirkanmaa Regional Foundation, Maud Kuistila
Memorial Foundation, and Inkeri and Mauri Vänskä Foundation. Author contributions
d d Conception and design: M.H. and M.P-M. Main manuscript text: M.H. and M.P-M. Conducting experiments:
M.H., J.K, H.V. and H.L. Data analysis and interpretation: M.H. and A-J.M. Formal analysis: M.H., A-J.M. and
H.H. Figure preparation: Figs. 1–5 and S1 M.H., Fig. 6 M.H and J.K., Supplementary Fig. S2 M.H and A-J.M.,
Supplementary Fig. S3 M.H. and H.V., supplementary Fig. S4 A-J.M. Resources: K.A-S. and P.K. Funding acquisi-
tion: K.A-S., P.K. and M.H. Supervision: M.P-M. and K.A-S. All authors reviewed and accepted the manuscript. di Conception and design: M.H. and M.P-M. Main manuscript text: M.H. and M.P-M. Conducting experiments:
M.H., J.K, H.V. and H.L. Data analysis and interpretation: M.H. and A-J.M. Formal analysis: M.H., A-J.M. and
H.H. Figure preparation: Figs. 1–5 and S1 M.H., Fig. 6 M.H and J.K., Supplementary Fig. S2 M.H and A-J.M.,
Supplementary Fig. S3 M.H. and H.V., supplementary Fig. S4 A-J.M. Resources: K.A-S. and P.K. Funding acquisi-
tion: K.A-S., P.K. and M.H. Supervision: M.P-M. and K.A-S. All authors reviewed and accepted the manuscript. Competing interests h p
g
The authors declare no competing interests. Acknowledgements Acknowledgements
We thank Tampere University Electrophysiology Core, Imaging Core and iPS Core. g
We thank Tampere University Electrophysiology Core, Imaging Core and iPS Core. https://doi.org/10.1038/s41598-021-83740-w Scientific Reports | (2021) 11:4153 | www.nature.com/scientificreports/ © The Author(s) 2021 Additional informationh Supplementary Information The online version contains supplementary material available at https://doi. org/10.1038/s41598-021-83740-w. Correspondence and requests for materials should be addressed to M.H. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International
License, which permits use, sharing, adaptation, distribution and reproduction in any medium or
format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the
Creative Commons licence, and indicate if changes were made. The images or other third party material in this
article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the
material. If material is not included in the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from
the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2021 https://doi.org/10.1038/s41598-021-83740-w Scientific Reports | (2021) 11:4153 |
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Proteomic analysis identifies subgroups of patients with active systemic lupus erythematosus
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Clinical proteomics
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cc-by
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Su et al. Clinical Proteomics (2023) 20:29
https://doi.org/10.1186/s12014-023-09420-1 Su et al. Clinical Proteomics (2023) 20:29
https://doi.org/10.1186/s12014-023-09420-1 Clinical Proteomics Open Access © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Objective Systemic lupus erythematosus (SLE) is a clinically and biologically heterogenous autoimmune disease. We aimed to investigate the plasma proteome of patients with active SLE to identify novel subgroups, or endotypes,
of patients. Method Plasma was collected from patients with active SLE who were enrolled in the British Isles Lupus Assess‑
ment Group Biologics Registry (BILAG-BR). The plasma proteome was analysed using a data-independent acquisition
method, Sequential Window Acquisition of All theoretical mass spectra mass spectrometry (SWATH-MS). Unsuper‑
vised, data-driven clustering algorithms were used to delineate groups of patients with a shared proteomic profile. Results In 223 patients, six clusters were identified based on quantification of 581 proteins. Between the clusters,
there were significant differences in age (p = 0.012) and ethnicity (p = 0.003). There was increased musculoskeletal
disease activity in cluster 1 (C1), 19/27 (70.4%) (p = 0.002) and renal activity in cluster 6 (C6) 15/24 (62.5%) (p = 0.051). Anti-SSa/Ro was the only autoantibody that significantly differed between clusters (p = 0.017). C1 was associated
with p21-activated kinases (PAK) and Phospholipase C (PLC) signalling. Within C1 there were two sub-clusters (C1A
and C1B) defined by 49 proteins related to cytoskeletal protein binding. C2 and C6 demonstrated opposite Rho fam‑
ily GTPase and Rho GDI signalling. Three proteins (MZB1, SND1 and AGL) identified in C6 increased the classification
of active renal disease although this did not reach statistical significance (p = 0.0617). Conclusions Unsupervised proteomic analysis identifies clusters of patients with active SLE, that are associated
with clinical and serological features, which may facilitate biomarker discovery. The observed proteomic heterogene‑
ity further supports the need for a personalised approach to treatment in SLE. Keywords SLE (systemic lupus erythematosus), SWATH, Proteomics, Cluster, Arthritis, Nephritis †Kevin Y. C. Su and John A. Reynolds have contributed equally to this work. John A. Reynolds
j.a.reynolds.1@bham.ac.uk
Full list of author information is available at the end of the article John A. Reynolds
j.a.reynolds.1@bham.ac.uk
Full list of author information is available at the end of the article John A. Reynolds
j.a.reynolds.1@bham.ac.uk
Full list of author information is available at the end of the article Proteomic analysis identifies subgroups
of patients with active systemic lupus
erythematosus Kevin Y. C. Su1,2†, John A. Reynolds1,2*† , Rachel Reed3, Rachael Da Silva3, Janet Kelsall3, Ivona Baricevic‑Jones3,
David Lee3, Anthony D. Whetton3,4, Nophar Geifman4, Neil McHugh5 and Ian N. Bruce6,7 on behalf of the
MASTERPLANS and BILAG-BR consortia © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 17 Su et al. Clinical Proteomics (2023) 20:29 Su et al. Clinical Proteomics (2023) 20:29 Su et al. Clinical Proteomics Introduction to a spectral library of protein fragments to deconvolve
the complex signal into relative quantities of peptides
and proteins across hundreds of samples [11]. DIA has
high reproducibility and can detect peptides in the order
of tens of thousands compared to TA, where typically
hundreds of peptides are measured in a directed non-
discovery approach [12]. Due to its unbiased nature and
ability to identify and quantify peptides at the proteome-
scale; SWATH-MS is a powerful method for biomarker
discovery.h Systemic lupus erythematosus (SLE) is an inflammatory
autoimmune rheumatic disease with significant morbid-
ity and mortality [1]; it is heterogeneous in both clini-
cal features and treatment response. Although several
new treatments for SLE have been developed, there is an
important unmet need for disease biomarkers to measure
active disease and predict treatment response. p
p
The clinical heterogeneity of SLE may reflect differ-
ent underlying cellular and molecular processes. Despite
a better understanding of mechanisms that contribute
to disease such as increased B cell activity and type 1
interferon production, which have led to more targeted
novel therapies, the overall response rates to these thera-
pies in clinical trials are typically around 40–60% [2, 3]. A more personalised approach to therapy may increase
response rates. For example, a post hoc analysis of ran-
domised controlled trials using belimumab (BEL), an
anti-BAFF monoclonal antibody, identified increased
efficacy in patients with elevated anti-dsDNA antibody or
low C3/4 complement, suggesting different patient sub-
groups may have different treatment response [4]. Com-
mon biomarkers have variable sensitivity and specificity
for active disease (approximately 62% and 93% for anti-
dsDNA antibodies, 75% and 71% for C3 complements,
and 48% and 71% for C4 complements) [5]. The UK Med-
ical Research Council (MRC) Precision Medicine Con-
sortium ‘Maximising SLE Therapeutic Potential by the
Application of Novel and Stratified Approaches’ (MAS-
TERPLANS) aimed to identify novel markers to predict
treatment response. An important first step was to iden-
tify biomarkers which may associate with specific clinical
features of SLE. The aim of the study was to use SWATH-MS to deter-
mine whether analysis of the plasma proteome can iden-
tify discrete subgroups in a large cohort of SLE patients
with highly active disease. Study population We included patients with SLE fulfilling the 1997
Updated American College of Rheumatology (ACR) or
the Systemic Lupus International Collaborating Clin-
ics (SLICC) 2012 classification criteria registered with
the BILAG-Biologics Registry (BILAG-BR) [13]. All had
active disease and due to commence rituximab (RTX),
belimumab (BEL) or mycophenolate mofetil (MMF). Patients commencing RTX or BEL needed to have suf-
ficiently active disease to satisfy the NHS England 2013
Interim Clinical Commissioning Policy and National
Institute for Health and Care Excellence (NICE) criteria
respectively [14, 15]. Baseline plasma samples were obtained from study
patients within a window of maximum 30 days prior to
and a maximum of five days after, receiving escalating
treatment with BEL, RTX or MMF. Proteomic analysis using mass-spectrometry (MS) has
been applied in numerous immune-mediated inflam-
matory diseases including Rheumatoid arthritis (RA)
[6], Sjogren’s syndrome [7] and Systemic sclerosis [8]. Although proteomic MS studies in SLE to date have been
modest in size, a systematic review by Nicolaou et al. identified 241 potential biomarkers from 25 studies using
gel electrophoresis or liquid chromatography tandem MS
across numerous sample types [9, 10] within different
manifestations of SLE such as nephritis, neuropsychiatric
and cutaneous disease. Of note, Annexin A2 which was
observed in three studies included in the review, was not
detected at a high level in our study and thus excluded
from analysis. Baseline disease activity was measured using BILAG-
2004 index [16] and SLEDAI-2000 (SLEDAI-2 K) [17]. Active disease in each domain was defined as BILAG-
2004 A or B score. Lupus-related damage was measured
using the ACR/SLICC damage index (SDI) [18]. Meas-
urement of C3 and C4 levels were conducted at recruiting
sites with low C3/C4 status determined by local labora-
tory reference ranges. Proteinuria was measured at the
local site by spot urine protein-creatinine ratio (uPCR) or
albumin-creatinine ratio (uACR). Values for uPCR were
converted to uACR using the method described by Sum-
ida et al. [19]. Sample preparation Differential protein levels between clusters were per-
formed with the proteome of a single cluster compared
to the remaining proteome as a “combined cluster”, i.e.,
Cluster 1 vs all remaining. The comparison was per-
formed using a linear model with multiple t-tests (R
package limma) and an adjusted-P < 0.05 (Benjamini-
Hochberg) was considered statistically significant. Canonical pathways and functional protein associa-
tion networks were visualised using String (v.11.0, URL:
https://string-db.org/) [24] and Qiagen Ingenuity Path-
way Analysis (IPA) [25]. Pathways with Z score of < − 1
or > 1 was considered significant. Plasma analysis was performed using Sequential Window
Acquisition of All Theoretical Mass Spectra (SWATH-
MS). Briefly, 10uL of plasma was depleted of highly abun-
dant proteins (albumin, IgG, transferrin, fibrinogen, IgA,
α-2-macroglobulin, α-1-antitrypsin, IgM, haptoglobin,
α1-acid glycoprotein, apolipoprotein A-I and apolipo-
protein A-II), then concentrated through centrifugal
filtration and added to digestion buffer of ammonium
bicarbonate, yielding a final volume of 80–100 uL. Pro-
teins were reduced and solubilised with dithiothreitol and
sodium deoxycholate then alkylated with iodoacetamide
and digested with trypsin. The sample was recovered
through centrifugation and peptides were lyophilised
from recovered supernatant by vacuum centrifugation
(MiVac Quattro Concentrator, SP Scientific US) and
stored at − 80 °C until use (details in Additional file 1). Samples were analysed by SWATH-MS with a micro-
flow LC–MS system comprising an Eksigent nanoLC 400
autosampler and an Eksigent nanoLC 425 pump coupled
to a Sciex 6600 Triple-TOF mass spectrometer with a
DuoSpray Ion Source. The system was controlled by Ana-
lyst software v1.7.1 and eksigent control software v4.2. This spectral library was generated on the basis of a pre-
viously published plasma protein library [21] but updated
to be compatible with a 100 variable window acquisition
method employed in the Stoller Centre. Full MS method-
ology is included in the Additional file 1. i
Clinical variables across clusters were analysed using
non-parametric tests; Chi square with Fisher’s exact test
and Kruskal–Wallis as appropriate. Those features which
are considered increased/or decreased between clusters
are reported based on descriptive numerical differences. In the regression models, finite mixture models were
used to allocate proteins with a clear bimodal distribu-
tion into undetectable/low/high groups. Other proteins
were considered binary (undetectable/detectable). SS-A 52 Ab ELISA Kit and anti-dsDNA using the Inova
Diagnostics Quanta Lite® dsDNA SC ELISA kit. SS-A 52 Ab ELISA Kit and anti-dsDNA using the Inova
Diagnostics Quanta Lite® dsDNA SC ELISA kit. visualisation, protein expression levels were standardised
to Z scores (where if expression level = x, Z score calcu-
lated as (x – mean)/standard deviation).f Sample preparation An
adaptive lasso regression model (with sample split into
training and validation datasets at a ratio of 3:1) was
used to identify proteins with a non-zero coefficient with
lambda selected via cross-validation. Analyses were per-
formed using R v.4.0.3, STATA v16.0 SE and SPSS v.26. Results i
SWATH maps were aligned with TRIC (msproteom-
icstools version 0.4.3) feature alignment algorithm. The
aligned openSWATH maps were processed with MSstats
to infer protein-level quantification based on aligned
transition-level quantitative information. With this tech-
nique, proteins in low quantities, below the threshold of
detection within the plasma, were designated as absent. Full methodology for alignment is included in the Addi-
tional file 1. Baseline patient characteristics Plasma samples were analysed from 223 patients, 198
(88.8%) were females with median (IQR) age and disease
duration of 40 (30, 51) and 10 (6, 17) years respectively. At baseline, 204 (91.5%) had at least one BILAG A and/
or two BILAG B scores. Of these, active disease (BILAG
2004 A or B score) was predominantly in mucocutaneous
(107, 51.2%), musculoskeletal (95, 45.5%) and renal (79,
37.4%) domains. Almost 40% had elevated anti-dsDNA
antibodies (87/223 [39%]) and/or low C3 or C4 comple-
ment (100/223 [44.8%]). Antibody status Sequential window acquisition of all theoretical mass
spectra (SWATH-MS) is a data independent acquisi-
tion (DIA) MS technique. It has advantages over data-
dependant acquisition (DDA) and targeted acquisition
(TA) methods as it employs non-selective analysis of pep-
tides and their fragments. These data are then compared All sera were tested for autoantibodies at the same centre
by immunoprecipitation of proteins from radiolabelled
cell lines, followed by PAGE separation and identification
of recognised autoantigens (e.g. Ro60 [SS-A], U1RNP/
Sm, La [SS-B]) by autoradiography as described else-
where [20]. Anti-Ro52 was measured using the ABNOVA Page 3 of 17 Su et al. Clinical Proteomics (2023) 20:29 Su et al. Clinical Proteomics Data analysis
Hi
hi
l Regarding treatment, 136/223 (61%) were taking regu-
lar oral corticosteroids with a median (IQR) dose of 10
(5, 14) mg/day. Almost half were taking an anti-malarial
(AM) at the time of plasma acquisition (110/223 [49.3%])
although 202/223 (90.6%) had ever taken an AM and 77
(34.5%) were taking mycophenolate mofetil. The cohort
demographics are shown in Table 1. Hierarchical clustering was used to cluster patients
based on their proteomic profile. The Average linkage
method was applied. The optimal number of clusters
was determined using the R package, nbclust [22]. The
elbow method was used to visually confirm the number
of clusters, and the final number of clusters resolved by
majority rule of 30 different validation indices (see Addi-
tional file 1). Initial data visualisation was performed on
untransformed data using t-distributed stochastic neigh-
bour embedding (t-SNE) with a perplexity of 11 (approxi-
mately 5% of the study population) [23]. For heat map Cluster analysis
A total of 894 proteins were quantified by SWATH-MS. After removing proteins which were only detectable Cluster analysis Concomitant immunosupp
ticosteroid use did not differ across clust
Table 1 Patient characteristics
Total (n = 223)
No (%)/median (IQR)
Age, years (n = 197)
40 (30, 51)
Female
198 (88.8)
Caucasian (n = 222)
132 (59.2)
Current smoker
27 (12.1)
Disease duration, years (n = 218)
10 (6, 17)
SLICC damage index (n = 206)
0 (0,1)
1997 ACR criteria at baseline (n = 203)
Number fulfilling ≥ 4 criteria
186 (83.4)
Malar rash
122 (54.7)
Photosensitivity
115 (51.6)
Discoid rash
41 (18.4)
Oral ulcers
136 (61)
Arthritis
192 (86.1)
Serositis
69 (30.9)
CNS
22 (9.9)
Renal disease
81 (36.3)
Haematologic disorder
115 (51.6)
Immunologic disorder
151 (67.7)
Positive ANA
185 (83)
Disease activity
SLEDAI score (n = 212)
8 (4.5, 14)
SLICC damage index (n = 206)
0 (0,1)
BILAG-2004 score at baseline
Constitutional
19 (9.3)
Mucocutaneous
107 (51.2)
Neuropsychiatric
23 (11.2)
Musculoskeletal
95 (45.5)
Cardiorespiratory
35 (17)
Gastrointestinal
11 (5.4)
Ophthalmic
13 (6.4)
Renal
79 (37.4)
Haematological
9 (4.4)
Baseline creatinine, umol/l (n = 162)
66 (57, 78)
BMI, kg/m2 (n = 171)
26.4 (22.9, 31.3)
Medications
Current steroid use
136 (61)
Usual daily OCS dose (mg/day)
10 (5, 14)
Current anti-malarial use
110 (49.3)
Anti-malarial use ever
202 (90.6)
Methotrexate
9 (4)
Azathioprine
11 (4.9)
Mycophenolate mofetil
77 (34.5)
Calcineurin inhibitor
3(1.3)
Serology
Ro (n = 215)
76 (35.3)
Ro52 (n = 215)
42 (18.8)
Ro60 (n = 192)
70 (31.4)
La (n = 195)
19 (8.5)
dsDNA (n = 215)
87 (39)
Table 1 (continued)
Total (n = 223)
No
U1-RNP (n = 192)
56
Low C3/C4 (n = 212)
10
IQR interquartile range, dsDNA Double stranded DNA, CNS
System, ANA Anti-nuclear antibody, SLEDAI Systemic Lupus
Disease Activity Index, SLICC Systemic Lupus International C
BILAG British Isles Lupus Assessment Group, BMI Body Mass
corticosteroids, ACR American College of Rheumatology.U1
Protein, MTX Methotrexate, AZA Azathioprine, MMF Mycop
Cyclosporin A, TAC Tacrolimus in < 25% of all samples, 588 remained. Seven more were
removed due to high relative levels (> 20) with low vari-
ance (< 1), resulting in 581 proteins for analysis.h The plurality (11/30) of clustering algorithms sug-
gested the data could be split into six clusters. Clusters
were created using hierarchical clustering and visual-
ised with t-SNE (Fig. 1). Cluster analysis The largest cluster comprised
65 patients (29.1% of the patient cohort; cluster 4, C4)
followed by 27 patients (12.1% of the cohort, cluster
1, C1), 27 patients (12.1% of the cohort, cluster 5, C5),
36 patients (16.1%, both cluster 2 and 3, C2 and C3)
and the smallest comprised 24 patients (10.7% of the
cohort; cluster 6, C6). Cluster analysis le 1 Patient characteristics
l (n = 223)
No (%)/median (IQR)
years (n = 197)
40 (30, 51)
ale
198 (88.8)
asian (n = 222)
132 (59.2)
ent smoker
27 (12.1)
ase duration, years (n = 218)
10 (6, 17)
C damage index (n = 206)
0 (0,1)
ACR criteria at baseline (n = 203)
umber fulfilling ≥ 4 criteria
186 (83.4)
alar rash
122 (54.7)
hotosensitivity
115 (51.6)
scoid rash
41 (18.4)
ral ulcers
136 (61)
thritis
192 (86.1)
rositis
69 (30.9)
NS
22 (9.9)
enal disease
81 (36.3)
aematologic disorder
115 (51.6)
mmunologic disorder
151 (67.7)
ositive ANA
185 (83)
ase activity
EDAI score (n = 212)
8 (4.5, 14)
ICC damage index (n = 206)
0 (0,1)
G-2004 score at baseline
onstitutional
19 (9.3)
ucocutaneous
107 (51.2)
europsychiatric
23 (11.2)
usculoskeletal
95 (45.5)
ardiorespiratory
35 (17)
astrointestinal
11 (5.4)
phthalmic
13 (6.4)
enal
79 (37.4)
aematological
9 (4.4)
seline creatinine, umol/l (n = 162)
66 (57, 78)
MI, kg/m2 (n = 171)
26.4 (22.9, 31.3)
cations
urrent steroid use
136 (61)
sual daily OCS dose (mg/day)
10 (5, 14)
urrent anti-malarial use
110 (49.3)
nti-malarial use ever
202 (90.6)
ethotrexate
9 (4)
zathioprine
11 (4.9)
ycophenolate mofetil
77 (34.5)
alcineurin inhibitor
3(1.3)
ogy
o (n = 215)
76 (35.3)
o52 (n = 215)
42 (18.8)
o60 (n = 192)
70 (31.4)
(n = 195)
19 (8.5)
DNA (n = 215)
87 (39)
DNA titre, IU/l
38.5 (13.9, 246.1)
Table 1 (continued)
Total (n = 223)
No (%)/median (
U1-RNP (n = 192)
56 (25.1)
Low C3/C4 (n = 212)
100 (44.8)
IQR interquartile range, dsDNA Double stranded DNA, CNS Central Nervous
System, ANA Anti-nuclear antibody, SLEDAI Systemic Lupus Erythematosus
Disease Activity Index, SLICC Systemic Lupus International Collaborating Cli
BILAG British Isles Lupus Assessment Group, BMI Body Mass Index, OCS Oral
corticosteroids, ACR American College of Rheumatology.U1-RNP U1-Ribonu
Protein, MTX Methotrexate, AZA Azathioprine, MMF Mycophenolate Mofetil
Cyclosporin A, TAC Tacrolimus in < 25% of all samples, 588 remained. Se
removed due to high relative levels (> 20
ance (< 1), resulting in 581 proteins for a
The plurality (11/30) of clustering a
gested the data could be split into six clu
were created using hierarchical clusteri
ised with t-SNE (Fig. 1). Cluster analysis The largest clu
65 patients (29.1% of the patient cohort
followed by 27 patients (12.1% of the
1, C1), 27 patients (12.1% of the cohort,
36 patients (16.1%, both cluster 2 and
and the smallest comprised 24 patients
cohort; cluster 6, C6). Antibody status and age significantly differ
between clusters
The youngest patients were in C6 (me
[19, 40.25] years) and the oldest in C3 (4
years) (p = 0.012 between clusters). C6
proportion of Caucasian patients (7/24
further description of ethnic groups
file 1. Between clusters, there was no st
ence in disease duration or SDI scores (T
There was a significant difference i
antibody positivity between clusters (p =
the highest frequency of anti-SSA/Ro (
and the lowest frequency was in C2
Similarly, anti-Ro60 was highest in C4,
and lowest in C2 (3/36 [8.3%]) (p = 0.00
whilst there was no significant differ
clusters for anti-Ro52 (K-Wallis, p = 0
comparison of C4 (19/63 [30.2%]) with
clusters combined (23/152 [15.1%)) w
significant (p = 0.014). There was no sig
ence in other autoantibodies or presenc
complement. Cluster analysis y
A total of 894 proteins were quantified by SWATH-MS. After removing proteins which were only detectable Page 4 of 17 Su et al. Clinical Proteomics (2023) 20:29 Table 1 Patient characteristics
Total (n = 223)
No (%)/median (
Age, years (n = 197)
40 (30, 51)
Female
198 (88.8)
Caucasian (n = 222)
132 (59.2)
Current smoker
27 (12.1)
Disease duration, years (n = 218)
10 (6, 17)
SLICC damage index (n = 206)
0 (0,1)
1997 ACR criteria at baseline (n = 203)
Number fulfilling ≥ 4 criteria
186 (83.4)
Malar rash
122 (54.7)
Photosensitivity
115 (51.6)
Discoid rash
41 (18.4)
Oral ulcers
136 (61)
Arthritis
192 (86.1)
Serositis
69 (30.9)
CNS
22 (9.9)
Renal disease
81 (36.3)
Haematologic disorder
115 (51.6)
Immunologic disorder
151 (67.7)
Positive ANA
185 (83)
Disease activity
SLEDAI score (n = 212)
8 (4.5, 14)
SLICC damage index (n = 206)
0 (0,1)
BILAG-2004 score at baseline
Constitutional
19 (9.3)
Mucocutaneous
107 (51.2)
Neuropsychiatric
23 (11.2)
Musculoskeletal
95 (45.5)
Cardiorespiratory
35 (17)
Gastrointestinal
11 (5.4)
Ophthalmic
13 (6.4)
Renal
79 (37.4)
Haematological
9 (4.4)
Baseline creatinine, umol/l (n = 162)
66 (57, 78)
BMI, kg/m2 (n = 171)
26.4 (22.9, 31.3)
Medications
Current steroid use
136 (61)
Usual daily OCS dose (mg/day)
10 (5, 14)
Current anti-malarial use
110 (49.3)
Anti-malarial use ever
202 (90.6)
Methotrexate
9 (4)
Azathioprine
11 (4.9)
Mycophenolate mofetil
77 (34.5)
Calcineurin inhibitor
3(1.3)
Serology
Ro (n = 215)
76 (35.3)
Ro52 (n = 215)
42 (18.8)
Ro60 (n = 192)
70 (31.4)
La (n = 195)
19 (8.5)
dsDNA (n = 215)
87 (39)
dsDNA titre, IU/l
38.5 (13.9, 246.1) in < 25% of all samples, 588 remained. Seven more w
removed due to high relative levels (> 20) with low v
ance (< 1), resulting in 581 proteins for analysis. The plurality (11/30) of clustering algorithms s
gested the data could be split into six clusters. Clus
were created using hierarchical clustering and vis
ised with t-SNE (Fig. 1). The largest cluster compr
65 patients (29.1% of the patient cohort; cluster 4,
followed by 27 patients (12.1% of the cohort, clu
1, C1), 27 patients (12.1% of the cohort, cluster 5, C
36 patients (16.1%, both cluster 2 and 3, C2 and
and the smallest comprised 24 patients (10.7% of
cohort; cluster 6, C6). Cluster analysis Antibody status and age significantly differs
between clusters
The youngest patients were in C6 (median age 3
[19, 40.25] years) and the oldest in C3 (45 [39.25, 5
years) (p = 0.012 between clusters). C6 had the l
proportion of Caucasian patients (7/24 [29.2%]);
further description of ethnic groups see Additio
file 1. Between clusters, there was no statistical dif
ence in disease duration or SDI scores (Table 2). There was a significant difference in anti-SSA
antibody positivity between clusters (p = 0.017), C4
the highest frequency of anti-SSA/Ro (29/65 [44.6
and the lowest frequency was in C2 (4/36 [11.1
Similarly, anti-Ro60 was highest in C4, (28/65 [43.1
and lowest in C2 (3/36 [8.3%]) (p = 0.008). In contr
whilst there was no significant difference betw
clusters for anti-Ro52 (K-Wallis, p = 0.059), a di
comparison of C4 (19/63 [30.2%]) with the remain
clusters combined (23/152 [15.1%)) was statistic
significant (p = 0.014). There was no significant dif
ence in other autoantibodies or presence of low C3
complement. Concomitant immunosuppressant or
ticosteroid use did not differ across clusters. Antibody status and age significantly differs
between clustersh The youngest patients were in C6 (median age 30.5
[19, 40.25] years) and the oldest in C3 (45 [39.25, 50.5]
years) (p = 0.012 between clusters). C6 had the least
proportion of Caucasian patients (7/24 [29.2%]); for
further description of ethnic groups see Additional
file 1. Between clusters, there was no statistical differ-
ence in disease duration or SDI scores (Table 2). There was a significant difference in anti-SSA/Ro
antibody positivity between clusters (p = 0.017), C4 had
the highest frequency of anti-SSA/Ro (29/65 [44.6%])
and the lowest frequency was in C2 (4/36 [11.1%]). Similarly, anti-Ro60 was highest in C4, (28/65 [43.1%])
and lowest in C2 (3/36 [8.3%]) (p = 0.008). In contrast,
whilst there was no significant difference between
clusters for anti-Ro52 (K-Wallis, p = 0.059), a direct
comparison of C4 (19/63 [30.2%]) with the remaining
clusters combined (23/152 [15.1%)) was statistically
significant (p = 0.014). There was no significant differ-
ence in other autoantibodies or presence of low C3/C4
complement. Concomitant immunosuppressant or cor-
ticosteroid use did not differ across clusters. Su et al. Clinical Proteomics (2023) 20:29 Page 5 of 17 Su et al. Clinical Proteomics A
B A
B
C1
C2
C3
C4
C5
C6
PI3K/AKT Signalling
↓
↓
Leukocyte Extravasa on
Signalling
↑
↑
↑
PAK Signalling
↑
↑
↓
Glioma Invasiveness
Signalling
↑
↑
Phospholipase C Signalling
↑
↓
RhoGDI Signalling
↓
↓
↓
↑
Neuroinflamma on Signalling
Pathway
↓
↓
↓
Rac Signalling
↑
↑
↓
Oestrogen Receptor
Signalling
↑
↓
Signalling by Rho Family
GTPases
↑
↓
Cdc42 Signalling
↑
↓
RhoA Signalling
↑
↑
↓
Ephrin Receptor Signalling
↑
↓
MSP-RON Signalling In Cancer
Cells Pathway
↓
↑
Hepa c Fibrosis Signalling
Pathway
↑
↓
↓
Tumour Microenvironment
Pathway
↓
↓
IL-8 signalling
↓
↓
SLE In T Cell Signalling
Pathway
↑
↑
C
Fig. 1 Cluster analysis of proteins in 223 patients with active SLE. A t-distributed stochastic neighbour embedding (t-SNE) plot of the 6 clusters
following hierarchal clustering. Each point represents a single patient allocated to one of n = 6 clusters based on the plasma proteome alone. B Heatmap of the 6 clusters showing only those proteins which were significantly different in at least 1 cluster. Colour shows the Z-score
with increased levels in red and decreased levels in blue. C Canonical pathways that are predicted to be activated or supressed in more than one
cluster. Antibody status and age significantly differs
between clustersh The arrows show the direction (up = activation, down = suppression) C A B B Fig. 1 Cluster analysis of proteins in 223 patients with active SLE. A t-distributed stochastic neighbour embedding (t-SNE) plot of the 6 clusters
following hierarchal clustering. Each point represents a single patient allocated to one of n = 6 clusters based on the plasma proteome alone. B Heatmap of the 6 clusters showing only those proteins which were significantly different in at least 1 cluster. Colour shows the Z-score
with increased levels in red and decreased levels in blue. C Canonical pathways that are predicted to be activated or supressed in more than one
cluster. The arrows show the direction (up = activation, down = suppression) Fig. 1 Cluster analysis of proteins in 223 patients with active SLE. A t-distributed stochastic neighbour embedding (t-SNE) plot of the 6 clusters
following hierarchal clustering. Each point represents a single patient allocated to one of n = 6 clusters based on the plasma proteome alone. B Heatmap of the 6 clusters showing only those proteins which were significantly different in at least 1 cluster. Colour shows the Z-score
with increased levels in red and decreased levels in blue. C Canonical pathways that are predicted to be activated or supressed in more than one
cluster. The arrows show the direction (up = activation, down = suppression) (60 [52,72] and C3 (60 [53, 70] and highest in C2 (75 [58,
94]) (K-Wallis between clusters p = 0.002). Differences in disease activity between clusters Marked variations in the proportion of patients with
active musculoskeletal disease (BILAG A or B score)
were found between clusters (p = 0.002); ranging from
19/27 (70.4%) in C1 to 5/24 (20.8%) in C6. Conversely,
there was no significant difference between clusters
(p = 0.177) in the number of patients with inflammatory
arthritis fulfilling the 1997 ACR criterion. In terms of ACR criteria, there was also a significant
difference in patient numbers that satisfied the renal
domain; numerically greatest in C6, (16/24 [66.7%]) and
lowest in C2, (9/36 [25%]) (p = 0.017). Oral ulcers were
numerically higher in C1 (20/27 [70.1%]) and lower in C5
(14/35 [40%]) (p = 0.036). i
Similarly, BILAG 2004 scores in renal disease was high-
est in C6 (15/24 [62.5%]) and lowest in C2 (9/36 [25%])
although the proportion of patients with renal disease
was not statistically significant between clusters (Chi-2,
p = 0.051). The comparison of C6 with the other clus-
ters combined, identified a significant difference (15/23
([65.2%]) vs. 64/188 [41.2%] respectively, p = 0.005). Although baseline creatinine was significantly different
between clusters, it was numerically equal lowest in C6 BMI Body Mass Index, dsDNA Double stranded DNA, SLEDAI Systemic Lupus Erythematosus Disease Activity Index, SLICC Systemic Lupus International Collaborating
Clinics, BILAG British Isles Lupus Assessment Group, ACR American College of Rheumatology, ANA Anti-nuclear antibody, U1-RNP U1-Ribonuclear Protein Values are n (%) or median (IQR) as appropriate. Comparisons between clusters were made using the Kruskal–Wallis test or Chi-2 test for continuous and categorical
variables respectively Body Mass Index, dsDNA Double stranded DNA, SLEDAI Systemic Lupus Erythematosus Disease Activity Index, SLICC Systemic Lupus Inter
ics, BILAG British Isles Lupus Assessment Group, ACR American College of Rheumatology, ANA Anti-nuclear antibody, U1-RNP U1-Ribonuc NA Double stranded DNA, SLEDAI Systemic Lupus Erythematosus Disease Activity Index, SLICC Systemic Lupus International Collaborating
upus Assessment Group, ACR American College of Rheumatology, ANA Anti-nuclear antibody, U1-RNP U1-Ribonuclear Protein ss Index, dsDNA Double stranded DNA, SLEDAI Systemic Lupus Erythematosus Disease Activity Index, SLICC Systemic Lupus International
G British Isles Lupus Assessment Group, ACR American College of Rheumatology, ANA Anti-nuclear antibody, U1-RNP U1-Ribonuclear Prote Values are n (%) or median (IQR) as appropriate. Comparisons between clusters were made using the Kruskal–Wallis test or Chi-2 test for continuous and categorical
variables respectively dsDNA Double stranded DNA, SLEDAI Systemic Lupus Erythematosus Disease Activity Index, SLICC Systemic Lupus International Collabora
l
L
A
t G
ACR A
i
C ll
f Rh
t l
ANA A ti
l
tib d
U1 RNP U1 Rib
l
P
t i Canonical pathways and network analysis
l
k To investigate relevant protein networks and pathways
that underpin each cluster, we identified proteins that
were significantly different (higher or lower) in each
cluster compared to all other clusters combined, using
a linear model adjusted for multiple comparisons. The
number of proteins with an adjusted p-value of < 0.05 Page 6 of 17 Su et al. Clinical Proteomics (2023) 20:29 Su et al. Clinical Proteomics Table 2 Comparison of patient characteristics between the 6 proteomic clusters Page 7 of 17 Su et al. Clinical Proteomics (2023) 20:29 Su et al. Clinical Proteomics Su et al. Clinical Proteomics (2023) 20:29 Page 8 of 17 Su et al. Clinical Proteomics Table 2 (continued)
Values are n (%) or median (IQR) as appropriate. Comparisons between clusters were made using the Kruskal–Wallis test or Chi-2 test for continuous an Su et al. Clinical Proteomics (2023) 20:29 Page 9 of 17 Page 9 of 17 was: 118 in C1, 67 in C2, 110 in C3, 24 in C4, 49 in C5
and 14 in C6. As C1 and C6 were associated with increased fre-
quency of musculoskeletal and renal disease respectively,
we performed further analyses on proteins present within
these clusters. was: 118 in C1, 67 in C2, 110 in C3, 24 in C4, 49 in C5
and 14 in C6. We performed pathways analysis using each of the
6 sets of proteins in turn. Pathways with Z score ≥ 1 or
Z ≤ − 1 were considered relevant. C1, C2, C3 and C6 each
had unique over-represented pathways which were not
observed in other clusters (Table 3). The number of these
unique pathways varied between clusters and was great-
est in C1 (17 unique pathways) and lowest in C6 (one
pathway). A total of 18 canonical pathways were common
to two or more clusters (Fig. 1C). Notably, the RhoGDI
signalling pathway was over-represented in 4 clusters but
it was decreased in C2, C4 and C5 and increased in C6. Association between plasma proteins and renal disease nodes (Fig. 2B). Furthermore, within the t-SNE plot
(Fig. 1A), the C1 cluster appeared to form 2 distinct
subclusters which we arbitrarily designated C1A as
the cluster closest to C4 (purple) and the other as C1B
(Fig. 1B). On evaluation of the heatmap of C1, a sub-
group of 49 proteins within C1 was identified as being
different between sub-clusters 1A and 1B (Fig. 3A). Of
these, 47/49 (96.0%) were numerically higher in C1B,
driving this group towards C3 and C6 where they were
also increased (the 2 numerically lower proteins were
transketolase [TKT] and coagulation factor XII). In
GO analysis, the top five pathways which were differen-
tially represented in C1A and C1B were “actin binding”,
“cytoskeletal protein binding”, “calcium ion binding”
and “Receptor for Advanced Glycation End products
(RAGE) receptor binding” and “protein binding”. Net-
work analysis (Fig. 3B) suggested that Cofilin-1 (CFL1),
was a central node in the proteins which defined C1B
compared to C1A. Seven of the 49 proteins were S100
proteins including S100A8, S100A9, S100A11 and
S100A12 (see Additional file 1). After correction for
multiple testing (t-test with Holm-Sidak correction)
2 proteins were significantly higher in C1B than C1A:
S100A4 and Transaldolase (TALDO1). We found no
differences in other disease features, medications,
or serology between clusters C1A and C1B (data not
shown). p
p
As C6 had over-representation of patients with renal
disease, we aimed to identify whether the proteins driv-
ing C6 belonged to one or more biological pathways. Of
the 14 significantly different proteins in renal disease
enriched cluster, C6, 3 were higher and 11 were lower
compared to other clusters (Fig. 4A), network analysis
did not identify a central node nor a distinct relationship
between several nodes (Fig. 4B). Most canonical path-
ways related to C6 were predicted to be downregulated
except for “Rho GDI signalling” and “SLE in T cell sig-
nalling pathway”. The only canonical pathway unique to
C6 was “Integrin signalling” (for details of the pathway
analysis related to C6 see the Additional file 1). Several
canonical pathways, including Rac/Rho/Cdc42 path-
ways, were shared between C2 and C6 but in opposing
directions. We then wanted to know whether any of these 14
proteins were associated with renal disease beyond
those patients in C6 after accounting for important
confounders. Further stratification of Cluster 1 As MSK disease was over-represented in C1, we aimed
to identify which individual proteins were increased/
decreased in C1 and whether we could identify key
biological pathways. We identified a total of 118 pro-
teins which were significantly different in C1 compared
to the other 5 clusters (Fig. 2A), and network analysis
of these 118 proteins identified a number of central Table 3 Unique canonical pathways in each cluster Table 3 Unique canonical pathways in each cluster Table 3 Unique canonical pathways in each cluster Page 10 of 17 Su et al. Clinical Proteomics (2023) 20:29 Su et al. Clinical Proteomics Fig. 2 Proteins in C1. A Volcano plot showing the individual proteins that are significantly different in C1 compared to the other clusters. The x-axis
shows log-fold change and the y-axis − Log10 adjusted P value. Proteins in red are those with a log fold change of < − 1 or > 1 and adjusted P
value < 0.05. B Network analysis of the proteins significantly different in cluster 1 compared to other clusters Fig. 2 Proteins in C1. A Volcano plot showing the individual proteins that are significantly different in C1 compared to the other clusters. The x-axis
shows log-fold change and the y-axis − Log10 adjusted P value. Proteins in red are those with a log fold change of < − 1 or > 1 and adjusted P
value < 0.05. B Network analysis of the proteins significantly different in cluster 1 compared to other clusters Association between plasma proteins and renal disease We used an adaptive lasso regression
model to select those proteins from C6 which were
associated with active renal disease (BILAG A or B)
across the whole cohort. In the model, three proteins
(Staphylococcal nuclease domain-containing protein
1 [SND1], glycogen debranching enzyme [AGL] and Page 11 of 17 Su et al. Clinical Proteomics (2023) 20:29 Su et al. Clinical Proteomics Fig. 3 Subcluster analysis of C1A and C1B. A Heatmap of proteins significantly different between C1 and the remaining clusters identifies 49
proteins that are different between C1A and C1B. B Network analysis of these 49 proteins demonstrating CFL1, LCP1 and CAP1 as central nodes Fig. 3 Subcluster analysis of C1A and C1B. A Heatmap of proteins significantly different between C1 and the remaining clusters identifies 49
proteins that are different between C1A and C1B. B Network analysis of these 49 proteins demonstrating CFL1, LCP1 and CAP1 as central nodes marginal zone B- and B1-cell-specific protein [MZB1])
were selected at lambda 57; higher levels of AGL and
lower levels of SND1 and MZB1 were associated with
active renal disease. In these models, MZB1 was con-
sidered binary (detectable/undetectable) whilst SND1
and AGL were ordinal (high/low/undetectable). Multi-
variable logistic regression models of active renal dis-
ease were constructed with age, gender, ethnicity, low
C3 and/or C4 complement and high anti-dsDNA as
covariates. The AUC for this model was 0.7346, increas-
ing to 0.7784 when the three proteins were added as
covariates (p = 0.0617). Similarly, the AUC for a model
of biopsy-proven lupus nephritis increased from 0.8115
to 0.86 (p = 0.0830) (Table 4). Adding the three proteins
to a model of proteinuria did not improve the AUC. compare values across all 6 clusters and our reporting of
over or under-represented disease features is descriptive
based on numerical values. We also identified 3 proteins,
(Staphylococcal nuclease domain-containing protein 1
(SND1), glycogen debranching enzyme (AGL) and mar-
ginal zone B- and B1-cell-specific protein (MZB1) that
were associated with the presence of active renal disease. In a previous study by Idborg et al. [26], 281 proteins
were measured by antibody suspension bead array with
the proteins selected based on existing published candi-
date biomarkers, microarray data and LC–MS data. Association between plasma proteins and renal disease Using
generalised linear models, higher levels of interferon
regulating factor 5 (IRF5), solute carrier family 22 mem-
ber 2 (SLC22A2) and S100 calcium binding protein A12
(S100A12) were identified in SLE patients compared to
healthy matched controls; in our data the S100A12 path-
way was increased in our C1B subcluster. Using unsuper-
vised clustering, Idborg identified 3 clusters of patients
characterised by rheumatoid factor (RF)-IgM, and high
or low levels of IRF5; the low IRF5 group closely resem-
bled healthy controls. As might be expected, the RF-high
group had increased frequency of anti-Ro/SSA and anti-
La/SSB antibodies and a reduced frequency of nephritis. Discussion Using an unbiased approach, we identified 6 proteomic
endotypes in a cohort of patients with active SLE (Fig. 5). These clusters were associated with some clinical or
serological features, notably inflammatory arthritis,
renal disease and anti-Ro/SSA antibodies. It should be
noted however, that the tests for statistical significance Page 12 of 17 (2023) 20:29 Su et al. Clinical Proteomics (2023) 20:2 Su et al. Clinical Proteomics A
Cluster 6 (Brown)
B
Fig. 4 Proteins in C6. A Volcano plot showing the individual proteins that are significantly different in C6 compared to the other clusters. Proteins
in red are those with a log fold change of < − 1 or > 1 and adjusted P value < 0.05. B Network analysis of the proteins significantly different in cluster
1 compared to other clusters A
Cluster 6 (Brown) B A Cluster 6 (Brown) Fig. 4 Proteins in C6. A Volcano plot showing the individual proteins that are significantly different in C6 compared to the other clusters. Proteins
in red are those with a log fold change of < − 1 or > 1 and adjusted P value < 0.05. B Network analysis of the proteins significantly different in cluster
1 compared to other clusters Table 4 Logistic regression model for active renal disease across the whole cohort
Clinical model comprises: age, gender, ethnicity, high dsDNA, low C3 and/or C4
Proteins: SND1, AGL, MZ
Outcome
Clinical model
AUC ROC
3 proteins alone
AUC ROC
Clinical model + 3
proteins
AUC ROC
p-value (clinical model vs
clinical model + proteins)
Active renal disease (BILAG A or B)
0.7346
0.6735
0.7784
0.0617
Biopsy-proven nephritis
0.8115
0.6947
0.8600
0.0830
Proteinuria (ACR > 70)
0.6691
0.6690
0.7208
0.2162
Cluster 1
Ac ve MSK disease
Previous oral ulcers
Cluster 2
Higher serum crea nine
Haematological disease (ns)
Cluster 3
White background
Cluster 4
An -Ro/SSA
Cluster 5
Older
Non-White background
Cluster 6
Younger
Non-White background
Acve renal disease (ns)
Previous renal disease
An-U1RNP (ns)
Fig. 5 Summary of the 6 proteomic clusters. Discussion Increased PAK signalling has been
observed in fibroblast-like synoviocytes (FLS) and associ-
ated with joint damage in RA patients [27]. Data visualisation suggested that C1 comprised two
distinct sub clusters (1A and 1B) suggesting that 2
molecular subtypes may exist within patients with active
MSK disease. The splitting of C1 into these sub-clusters
appeared to be due to differences in 49 proteins which
have roles in actin binding, cytoskeletal protein bind-
ing, calcium ion binding, RAGE receptor binding and
protein binding. Cofilin-1 (CFL1) was a central node in
the network analysis and has roles in actin and cytoskel-
etal protein binding. CFL1, which is stimulated by TNFα
and GM-CSF, disassembles actin filaments during cellu-
lar replication, facilitating FLS migration in RA patients
[28]. In a small study by Ooka et al., anti-CFL-1 anti-
bodies were found in 6.3% of patients with SLE but also
patients with RA, Behcet’s disease and myositis [29] sug-
gesting that CFL-1 may be an autoantigen common to
several inflammatory diseases. Proteins in the S100 fam-
ily also differed between C1A and C1B. These proteins
act as damage-associated molecular patterns (DAMPs)
and plasma levels are increased in both adult and child-
hood-onset SLE, especially those with active disease [30,
31] Whilst increased S100 proteins are associated with
lupus nephritis [32], our study suggests that they may
also be relevant to patients with lupus arthritis, support-
ing observations in other forms of inflammatory arthri-
tis [33]. S100A4 was significantly increased in C1B and
in RA, S100A4 is expressed in synovium and induces the
expression of matrix metalloproteinases [34]. Transaldo-
lase was also increased in cluster C1B. In RA, monocytes
have increased expression of Transaldolase which has
been proposed to protect RA monocytes from apoptosis,
increasing the pool of activated monocytes in inflamed
synovium [35]. g
p
p
q
Kwon et al. [41], performed MS on urinary samples of
SLE patients with and without lupus nephritis compared
to HC. They identified an increase in 143 and 67 proteins
in patients with SLE without nephritis and with nephri-
tis respectively. They did not perform clustering analysis
on their cohort; however, identified 23 common proteins
between the 2 SLE groups compared to HC, five of these
(ORM1, antithrombin‐III [SERPINC1], ceruloplasmin,
haemoglobin subunit beta [HBB] and delta [HBD]) were
significantly upregulated in patients with lupus nephri-
tis. Discussion Key clinical and serological differences in each of the 6 clusters, ns = not statistically significant
across all clusters ble 4 Logistic regression model for active renal disease across the whole cohort Cluster 1
Ac ve MSK disease
Previous oral ulcers
Cluster 2
Higher serum crea nine
Haematological disease (ns)
Cluster 3
White background
Cluster 4
An -Ro/SSA
Cluster 5
Older
Non-White background
Cluster 6
Younger
Non-White background
Acve renal disease (ns)
Previous renal disease
An-U1RNP (ns)
Fig. 5 Summary of the 6 proteomic clusters. Key clinical and serological differences in each of the 6 clusters, ns = not statistically significant
across all clusters Page 13 of 17 Su et al. Clinical Proteomics (2023) 20:29 Su et al. Clinical Proteomics (2023) 20:29 Associations between these molecular subgroups and
other clinical features of SLE are not reported. Associations between these molecular subgroups and
other clinical features of SLE are not reported. in C6, although the role of integrins in SLE remains to
be elucidated. Mutations in the Integrin Subunit Alpha
M (ITGAM) gene which encodes the CD11b chain of
the Mac-1 integrin is a risk factor for SLE [37], the exact
mechanisms by which changes in CD11b drives inflam-
mation but has been proposed to be through TLR inhibi-
tion of cytokine production [38]. In our pathway analysis,
the “integrin signalling” pathway contained the proteins
integrin αM, β1 and β3, but not the β2 integrin, which
is associated with the development of SLE [39] Although
these studies do not explore the clinical variation, one
study in 2009 by Yang et al., observed a strong association
in lupus nephritis with the mutation, and of note, also
observed a higher incidence of arthritis in the absence of
the mutation [40]. More research to identify the roles of
these integrin subunits in lupus nephritis is required. In our study there was over-representation of active
musculoskeletal disease in C1, and a reciprocal under-
representation in C6. Interestingly, there was no dif-
ference in number of patients fulfilling the 1997 ACR
arthritis criterion suggesting that the protein signature
is related to active MSK disease, or possibly non-arthritis
MSK disease (e.g. myositis). The over-represented path-
ways in C1 included intracellular proteins associated with
regulation of cell cytoskeleton. The P21 activated kinase
(PAK) pathway was predicted to be increased in C1 and
decreased in C6. Discussion Plasma levels of
PECAM-1 were not measured, but leakage of PECAM-1
from plasma into the urine in patients with proteinu-
ria may explain the lower levels that we observed in our
study. Further studies with contemporaneous measure-
ment of PECAM-1 in plasma and urine should be consid-
ered in patients with lupus nephritis. A strength of SWATH-MS is the compilation of large
spectral libraries containing all known peptide fragments
with good reproducibility [12]. Given this, SWATH-MS
has been utilised in drug discovery and biomarker iden-
tification in gastroenterology, oncology and cardiology
[54–57]. SWATH-MS has been used to identify bio-
markers in other rheumatic diseases including SS [58]
and osteoarthritis [59]. In the study of SS by Cecchettini
et al., differentially expressed inflammatory proteins in
the saliva of patients with primary SS and HC, were used
perform gene ontology analysis which identified some
biological processes which were also noted in our study
including gluconeogenesis (increased in C1) and protein
kinase A signalling (increased in C2) [58]. A strength this study design is the timing of patient
plasma collection, which allowed us to capture the pro-
teome profile of high disease activity patients. We found
no differences in drug treatment between clusters, sug-
gesting the protein signatures are the result of the disease,
rather than drug effect, although longitudinal studies are
important to validate these observations. p
p
p
MZB1 is implicated in B cell antibody production with
elevated levels reported in lymph nodes of lupus patients
[48]. MBZ1 is also expressed in plasmocytic dendritic
cells and regulates IFNα production, a key cytokine in
SLE [49]. In our study, plasma levels of MZB1 were para-
doxically lower in C6 although this may again be due to
increased excretion via the kidney. To identify whether
these biomarkers may have clinical utility, and to control
for important confounders such as ethnicity, we devel-
oped models of active renal disease using proteins identi-
fied from C6. A combination of three proteins improved
accuracy of the model to identify patients with active
nephritis. Although replication of these findings in an
independent cohort is needed, this confirms that data
reduction using cluster analysis followed by variable
selection is a valid approach for biomarker discovery in
patients with active SLE. Discussion We did not detect ORM1 in our data, but the other 4
proteins were not significantly increased in C6 compared
to the rest of the cohort, and thus were not included in
our lasso model. In a small study, serum Annexin 2 as
measured by ELISA, was associated with proliferative
lupus nephritis but not membranous disease [42]. In
our study, Annexin 2 was detected in fewer than 25% of
samples and thus excluded from further analysis. In our
data we do not have details of the subtypes of lupus renal
disease which limited any subgroup analyses of patients
with lupus nephritis.if Only three of the significantly different proteins in C6
had increased levels: filamin-C (FLNC), tyrosine-protein
kinase CSK (CSK) and glycogen debranching enzyme
(AGL). CSK is associated with autoimmunity as higher
levels in early stages of B cell maturation increase the
number auto-reactive B cells and autoantibody produc-
tion [43] and contrary to our study, was significantly
downregulated in a pilot proteomic study by Zhou et al. [44], which had a cohort of SLE mostly enriched with
cutaneous disease, the observed reduced CSK expres-
sion may reflect the differing treatment paradigms of skin
versus systemic disease but may also reflect the variation
in disease pathogenesis of SLE. Among the proteins with
reduced expression, marginal zone B- and B1-cell-spe-
cific (MZB1) and platelet endothelial cell adhesion mol-
ecule (PECAM-1) are associated with SLE. Cluster 6 had the greatest proportion of renal dis-
ease (lowest in C2); these patients were younger and
more likely to be non-Caucasian, representing the typi-
cal demographic of lupus nephritis [36]. In our data, the
integrin signalling pathway was predicted to be reduced Page 14 of 17 Page 14 of 17 Su et al. Clinical Proteomics (2023) 20:29 Su et al. Clinical Proteomics (2023) 20:29 PECAM-1 is an important regulator of B cell develop-
ment and B cell receptor activation. In murine models,
PECAM-1 deficiency leads to B cell hyper-responsiveness
and autoantibody formation [45]. It has been noted to be
elevated in both the urine and serum of patients with SLE
[46, 47]. Increased PECAM-1 in the serum of patients
with SLE may also be modulated by a greater proportion
of metabolic syndrome risk factors including increased
age, Body Mass Index (BMI) and waist circumference
which may increase PECAM-1 levels. Discussion An important limitation is that only active SLE patients
were included in this study, and therefore we cannot
determine how the plasma proteome would compare to
patients with inactive SLE or HC. As this is also a cross
sectional study, it would also be important to perform
a longitudinal study with HC to further assess the clini-
cal associations of identified proteins. A study in 2022 by
Zhang et al. explored the metabolic profile of 21 HC, 52
SLE patients and 43 LN patients using Ultra high-perfor-
mance liquid/gas chromatography-tandem mass spec-
trometry (UPL/GC–MS/MS) [60]. This study discovered
28 differential metabolites, five of which were discrimi-
natory for LN from SLE and significantly associated with
urea, creatinine, Cystatin C and C1q, not observed in
healthy controls and reiterating the importance of a con-
trol group for validation. The Rac/Rho/Cdc42 pathways, which are associated
with cell motility, were differentially modulated in C2
and C6, and the role of these proteins in plasma war-
rants further investigation. The Rho/Rho kinase pathway
is implicated in the pathogenesis of lupus as Rho kinase
inhibitors ameliorate SLE disease activity in murine
models [50, 51]. The RhoA-Rho kinase pathway is also
implicated in B cell activation and survival [52]. If dysreg-
ulation of this pathway, reduces B cells activation or sur-
vival, this could explain the lower levels of autoantibodies
in C2. Between the clusters, there was also a significant
difference in number of patients with anti-SSA/Ro anti-
bodies; highest in C4 and lowest in C2. This observa-
tion was clearer for Ro60 isotype than Ro52, although
this may reflect insufficient power to detect differences
between groups. Interestingly, there was no increase in
clinical features often associated with anti-SSA/Ro anti-
bodies including mucocutaneous, MSK, neurological or
pulmonary disease in C4 compared to other clusters (nor
reduced frequency of these features in C2) [53]. Furthermore, as most patients recruited into BILAG-
BR, are patients with refractory disease requiring biologi-
cal therapy, patients with early or naïve disease are not
represented in this study, noting that the activity scores
used to stratify patients are for purposes of disease moni-
toring and making treatment decisions, but may not
reflect disease biology. The lack of significant differences
in SDI and disease duration between clusters at least
implies that the patients are relatively homogeneous in
terms active and severe disease. Availability of data and materials The datasets used during the current study may be available from the cor‑
responding author on reasonable request providing anonymisation can be
maintained. Funding The MASTERPLANS Consortium was funded by a grant from the Medical
Research Council (MR/M01665X/1). BILAG BR has been funded by unrestricted
educational donations from Roche, GSK and LUPUS UK. Professor Bruce is a
National Institute for Health Research (NIHR) Emeritus Senior Investigator and
is funded by the NIHR Manchester Biomedical Research Centre. Equipment
used in the Stoller Biomarker Discovery Centre is funded by a donation
received from the Stoller Charitable Trust and a research grant awarded by the
Medical Research Council (MR/M008959/1). MRC/EPSRC Molecular Pathology
Node provided additional financial support by a grant from the Medical
Research Council and Engineering & Physical Sciences Research Council (MR/
N00583X/1). Supplementary Information The online version contains supple
org/10.1186/s12014-023-09420-1. The online version contains supplementary material available at https://doi.
org/10.1186/s12014-023-09420-1. Additional file 1: Figure S1. ‘Elbow’ Methods of determining optimal
cluster number with the RStudio package ‘nbclust’. Table S1. Detailed
ethnicity data for the study population. Table S2. Details of ethnicity
between clusters. Table S3. List of Proteins in Cluster 1 with Significant
FC and P value. Table S4. Cluster 1 Proteins associated with subcluster
1A and 1B. Figure S2. Dissimilarity matrices for each of the 6 clusters. Table S5. Proteins and canonical pathways relating to the clusters 1A and
1B. Figure S3. Gene Ontology of all proteins in cluster 1 utilising CLUEgo,
a Cytoscape plugin for biological interpretation (4). Table S6. Pathway
analysis of the proteins which are increased or decreased in C6 compared
to the rest of the cohort. Acknowledgements The authors would also like to acknowledge the work of Patrick Doherty (MAS‑
TERPLANS data architect), Gillian Armitt (MASTERPLANS project manager) and
the MASTERPLANS Patient Partners. Abbreviations
ACR
American College of Rheumatology
AGL
Glycogen debranching enzyme
AM
Anti-malarial
BEL
Belimumab
BILAG-BR
British Isles Lupus Assessment Group-Biologics Regist
BMI
Body Mass Index
C1
Cluster 1
C2
Cluster 2
C3
Cluster 3
C4
Cluster 4
C5
Cluster 5
C6
Cluster 6
CFL-1
Cofilin1
CSK
Tyrosine-protein kinase
DAMPs
Damage-associated molecular patterns
DDA
Data-dependant acquisition
FLNC
Filamin-C
FLS
Fibroblast-like synoviocytes
HC
Healthy Control
IRF5
Interferon regulating factor 5 (IRF5)
ITGAM
Integrin subunit alpha M
LC-MS
Liquid chromatography mass spectrometry
MALDI-TOF MS
Matrix assisted laser desorption ionization-time of
flight mass spectrometry
MMF
Mycophenolate Mofetil
MS
Mass-spectrometry
MZB1
Marginal zone B- and B1-cell-specific protein
NICE
National Institute for Health and Care Excellence
PAK
P21-activated kinases
PECAM-1
Platelet endothelial cell adhesion molecule
PLC
Phospholipase C
RA
Rheumatoid arthritis
RAGE
Receptor for advanced glycation end products
RF
Rheumatoid factor
RTX
Rituximab
S100A12
S100 calcium binding protein A12
SS
Sjogren’s Syndrome
SLC22A2
Solute carrier family 22 member 2
SLE
Systemic lupus erythematosus Conclusion In conclusion, SWATH-MS is a valid method for iden-
tifying proteomic differences in patients with SLE and
can identify proteins which may be useful biomarkers
for features of active disease, notably MSK and renal
involvement. The pathways and proteins identified may
serve as potential biomarkers and/or therapeutic targets
and investigation of their role in SLE pathogenesis is
warranted. Author contributions KYCS: Conceptualization, formal analysis, methodology, project administration,
software, validation, visualisation, writing—original draft, writing—review and
edit. JAR: Conceptualization, formal analysis, methodology, project administra‑
tion, validation, visualization, supervision, software, writing—review and edit. RR: Investigation, Data Curation, Software. RDS: Investigation, Data Curation,
Software. JK: Investigation, Data Curation, Software. IB-J: Investigation, Data
Curation, Software. DL: Investigation, Data Curation, Software. ADW: Investiga‑
tion, Data Curation, Software, writing—review and edit. NG: Investigation,
writing—review and edit. NMH: Investigation, Data Curation, Software, writ‑
ing—review and edit. INB: Investigation, Data Curation, Software, writing—
review and edit, Funding acquisition (As per: https://credit.niso.org/). Discussion As each of the 6 clusters were relatively small, this study
may lack power to identify differences in some clinical
or serological features. Similarly, as the most frequent
active organ domains were mucocutaneous, MSK and
renal, we may lack power to identify differences in other
domains such as neuropsychiatric disease. Importantly, Su et al. Clinical Proteomics (2023) 20:29 Page 15 of 17 Su et al. Clinical Proteomics (2023) 20:29 Staphylococcal nuclease domain-containing protein 1
Sequential window acquisition of all theoretical mass
spectra mass spectrometry
Targeted acquisition
T-distributed stochastic neighbour embedding
Albumin-creatinine ratio
Urine protein-creatinine ratio SWATH-MS is not suited to detecting low level pro-
teins such as cytokines and chemokines and future stud-
ies including multiplex cytokine/chemokine data should
be integrated into proteomic analysis. Interestingly, in
our data there was a lower-than-expected proportion
of patients taking AM, which may reflect the refractory
stage of disease in many patients within the BILAG-BR
or incomplete data capture; the proportion of ever used
AM was more than 90%. Importantly, we saw no differ-
ence in AM use between clusters considering ‘current’, or
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educational consulting. John A Reynolds: None to disclose. Rachel Reed:
None to disclose. Rachael Da Silva: None to disclose. Janet Kelsall: None to
disclose. Ivona Baricevic-Jones: None to disclose. David Lee: None to disclose. Anthony D Whetton: None to disclose. Nophar Geifman: None to disclose. Neil
McHugh: None to disclose. Ian N Bruce has received grant/research support
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? Choose BMC and benefit from: 47. References Ramirez-Bello J, Sun C, Valencia-Pacheco G, Singh B, Barbosa-Cobos RE,
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Efficient Transfer Entropy Analysis of Non-Stationary Neural Time Series
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PloS one
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cc-by
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Abstract Information theory allows us to investigate information processing in neural systems in terms of information transfer,
storage and modification. Especially the measure of information transfer, transfer entropy, has seen a dramatic surge of
interest in neuroscience. Estimating transfer entropy from two processes requires the observation of multiple realizations of
these processes to estimate associated probability density functions. To obtain these necessary observations, available
estimators typically assume stationarity of processes to allow pooling of observations over time. This assumption however,
is a major obstacle to the application of these estimators in neuroscience as observed processes are often non-stationary. As
a solution, Gomez-Herrero and colleagues theoretically showed that the stationarity assumption may be avoided by
estimating transfer entropy from an ensemble of realizations. Such an ensemble of realizations is often readily available in
neuroscience experiments in the form of experimental trials. Thus, in this work we combine the ensemble method with a
recently proposed transfer entropy estimator to make transfer entropy estimation applicable to non-stationary time series. We present an efficient implementation of the approach that is suitable for the increased computational demand of the
ensemble method’s practical application. In particular, we use a massively parallel implementation for a graphics processing
unit to handle the computationally most heavy aspects of the ensemble method for transfer entropy estimation. We test the
performance and robustness of our implementation on data from numerical simulations of stochastic processes. We also
demonstrate the applicability of the ensemble method to magnetoencephalographic data. While we mainly evaluate the
proposed method for neuroscience data, we expect it to be applicable in a variety of fields that are concerned with the
analysis of information transfer in complex biological, social, and artificial systems. Martı´nez-Zarzuela M, Vicente R, Dı´az-Pernas FJ, Wibral M (2014) Efficient Transfer Entropy Analysis of Non-Stationary Neural Time
02833. doi:10.1371/journal.pone.0102833 Editor: Daniele Marinazzo, Universiteit Gent, Belgium Received December 20, 2013; Accepted June 24, 2014; Published July 28, 2014 Copyright: 2014 Wollstadt et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: MW and RV received financial support from LOEWE Grant ‘‘Neuronale Koordination Forschungsschwerpunkt Frankfurt (NeFF)’’. MMZ received financial
support from the University of Valladolid. Abstract 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. * Email: p.wollstadt@stud.uni-frankfurt.de * Email: p.wollstadt@stud.uni-frankfurt.de . These authors contributed equally to this work. Patricia Wollstadt1*., Mario Martı´nez-Zarzuela4., Raul Vicente2,3, Francisco J. Dı´az-Pernas4,
Michael Wibral1 1 MEG Unit, Brain Imaging Center, Goethe University, Frankfurt, Germany, 2 Frankfurt Institute for Advanced Studies (FIAS), Goethe University, Frankfurt, Germany,
3 Max-Planck Institute for Brain Research, Frankfurt, Germany, 4 Department of Signal Theory and Communications and Telematics Engineering, University of Valladolid,
Valladolid, Spain July 2014 | Volume 9 | Issue 7 | e102833 Patricia Wollstadt1*., Mario Martı´nez-Zarzuela4., Raul Vicente2,3, Francisco J. Dı´az-Pernas4,
Mi h
l Wib
l1 lstadt1*., Mario Martı´nez-Zarzuela4., Raul Vicente2,3, Francisco J. Dı´az-Pernas4, Introduction For example, the analysis of magne-
toencephalographic data presented here would require a runtime
of 8200 h for 15 subjects and a single experimental condition. It is
easy to see that any practical application of the methods hinges on
a substantial speed-up of the computation. causal interactions and information transfer, because causal
interactions will subserve all three components of information
processing (transfer, storage, modification). However, it is infor-
mation transfer, rather than causal interactions, we might be
interested in when trying to understand a computational process in
the brain [48]. On the practical side, efforts to apply measures of information
transfer in neuroscience have been hampered by two obstacles: (1)
the need to analyze the information processing in a multivariate
manner, to arrive at unambiguous conclusions that are not
clouded by spurious traces of information transfer, e.g. due to
effects of cascades and common drivers; (2) the fact that available
estimators of information transfer typically require the processes
under investigation to be stationary. Fortunately, the algorithms involved in ensemble-based TE
estimation, lend themselves easily to data-parallel processing, since
most of the algorithm’s fundamental parts can be computed
simultaneously. Thus, our problem matches the massively parallel
architecture of Graphics Processing Unit (GPU) devices well. GPUs were originally devised only for computer graphics, but are
routinely used to speed up computations in many areas today
[58,59]. Also in neuroscience, where applied algorithms continue
to grow faster in complexity than the CPU performance, the use of
GPUs with data-parallel
methods is becoming increasingly
important [60] and GPUs have successfully been used to speedup
time series analysis in neuroscientific experiments [61–66]. The first obstacle can in principle be overcome by conditioning
TE on all other processes in a system, using a fully multivariate
approach that had already been formulated by Schreiber [14]. However, the naive application of this approach normally fails
because the samples available for estimation are typically too few. Therefore, recently four approaches to build an approximate
representation of the information transfer network have been
suggested: Lizier and Rubinov [50], Faes and colleagues [44], and
Stramaglia and colleagues [51] presented algorithms for iterative
inclusion of processes into an approximate multivariate descrip-
tion. In the approach suggested by Stramaglia and colleagues,
conditional mutual information terms are additionally computed
at each level as a self-truncating series expansion, following a
suggestion by Bettencourt and colleagues [52]. Introduction moment in time subserves any of the three component functions to
a varying degree (see [5] for an example of time-varying storage). In neural systems it is indeed crucial to understand where and
when information storage, transfer and modification take place, to
constrain possible algorithms run by the system. While there is still
a struggle to properly define information modification [6,7] and its
proper measure [8–12], well established measures for (local active)
information storage [13], information transfer [14], and its
localization in time and space [15,16] exist, and are applied in
neuroscience (for information storage see [5,17,18], for informa-
tion transfer see below). We typically think of the brain as some kind of information
processing system, albeit mostly without having a strict definition
of information processing in mind. However, more formal
accounts of information processing exist, and may be applied to
brain research. In efforts dating back to Alan Turing [1] it was
shown that any act of information processing can be broken down
into the three components of information storage, information
transfer, and information modification [1–4]. These components
can be easily identified in theoretical or technical information
processing systems, such as ordinary computers, based on the
specialized machinery for and the spatial separation of these
component functions. In these examples, a separation of the
components of information processing via a specialized mathe-
matical formalism seems almost superfluous. However, in biolog-
ical systems in general, and in the brain in particular, we deal with
a form of distributed information processing based on a large
number of interacting agents (neurons), and each agent at each Especially the measure for information transfer, transfer entropy
(TE), has seen a dramatic surge of interest in neuroscience [19–
41], physiology [42–44], and other fields [6,15,31,45,46]. Never-
theless, conceptual and practical problems still exist. On the
conceptual side, information transfer has been for a while confused
with causal interactions, and only some recent studies [47–49]
made clear that there can be no one-to-one mapping between July 2014 | Volume 9 | Issue 7 | e102833 1 July 2014 | Volume 9 | Issue 7 | e102833 PLOS ONE | www.plosone.org Non-Stationary Transfer Entropy Therefore, the use of the ensemble method has remained a
theoretical possibility so far, especially in combination with the
nearest neighbor-based estimation techniques by Kraskov and
colleagues [57] that provide the most precise, yet computationally
most heavy TE estimates. Background Our study focuses on making the application of ensemble-based
estimation of TE from non-stationary data practical using a GPU-
based algorithm. For the convenience of the reader, we will also
present the necessary background on stationarity, TE estimation
using the Kraskov-Sto¨gbauer-Grassberger (KSG) estimator [19],
and the ensemble method of Gomez-Herrero et al. [55] in
condensed form in a short background section below. Readers well
familiar with these topics can safely skip ahead to the Implemen-
tation section below. Fortunately, in neuroscience we can often obtain many
realizations
of
the
processes
in
question
by
repeating
an
experiment. In fact, this is the typical procedure in neuroscience
- we repeat trials under conditions that are kept as constant as
possible (i.e we create a cyclostationary process). The possibility to
use such an ensemble of data to estimate the time resolved TE has
already been demonstrated theoretically by Gomez-Herrero and
colleagues
[55]. Practically,
however,
the
statistical
testing
necessary for this ensemble-based method leads to an increase in
computational cost by several orders of magnitude, as some
shortcuts in statistical validation that can be taken for stationary
data cannot be used for the ensemble approach (see [56]): For
stationary data, TE is calculated per trial and one set of trial-based
surrogate data may be used for statistical testing. The ensemble
method does not allow for trial-based TE estimation as TE is
estimated across trials. Instead, the ensemble method requires the
generation of a sufficiently large number of surrogate data sets, for
all of which TE has to be estimated, thus multiplying the
computational demand by the number of surrogate data sets. Introduction In contrast to these
approaches that explicitly compute conditional TE terms, we
recently suggested an approximation based on a reconstruction of
information transfer delays [53] and a graphical pruning algorithm
[54]. While the first three approaches will eventually be closer to
the ground truth, the graphical method may be better applicable
to very limited amounts of data. In sum, the first problem of
multivariate analysis can be considered solved for practical
purposes, given enough data are available. Thus, in order to overcome the limitations set by the
computational demands of TE analysis from an ensemble of data,
we developed a GPU implementation of the algorithm, where the
neighbor searches underlying the binless TE estimation [57] are
executed
in parallel
on the GPU. After parallelizing this
computationally most heavy aspect of TE estimation we were
able to use the ensemble method for TE estimation proposed by
[55], to estimate time-resolved TE from non-stationary neural
time-series in acceptable time. Using the new GPU-based TE
estimation tool on a high-end consumer graphics card reduced
computation time by a factor of 50 compared to the CPU
optimized TE search used previously [67]. In practical terms, this
speedup shortens the duration of an ensemble-based analysis for
typical neural data sets enough to make the application of the
ensemble method feasible for the first time. The second obstacle of dealing with non-stationary processes is
also not a fundamental one, as the definition of TE relies on the
availability of multiple realizations of (two or more) random
processes, that can be obtained by running an ensemble of many
identical copies of the processes in question, or by running one
process multiple times. Only when obtaining data from such
copies or repetitions is impossible, we have to turn to a stationarity
assumption in order to evaluate the necessary probability density
functions (PDF) based on a single realization. pYt(Yt~bj) Stationarity and non-stationarity in experimental time
series We
may obtain a reliable estimation of pXHzt(:) from this ensemble by
evaluating p:(:) over all observations xHzt,VH[R. For the sake of
readability, we will refer to these observations from the ensemble
as xt(r), where t refers to a time point t, relative to the beginning
of the process at time H, and r~1, . . . ,R refers to the index of the
repetition. If a process is repeated periodically, i.e. the repetitions
are spaced by a fixed interval T, we call such a process
cyclostationary [68]: Here, u is the assumed delay of the information transfer
between processes X and Y [53]; yt(r) denotes the future
observation of Y in repetition r~1, . . . ,R; ydY
t{1(r) denotes the
past state of Y in repetition r and xdX
t{u(r) denotes the past state of
X in repetition r. Note, that the functional TESPO used here is a
modified form of the original TE formulation introduced by
Schreiber [14]. Schreiber defined TE as a conditional mutual
information TE X?Y,t
ð
Þ~I(Yt; Xdx
t{1DYdy
t{1), whereas the func-
tional in eq. 3 implements the conditional mutual information
TESPO X?Y,t,u
ð
Þ~I(Yt; Xdx
t{uDYdy
t{1) [53]. The latter functional,
TESPO, contains the definition of Schreiber as a special case for
u~1. Note that the two functionals are identical if TESPO is used
with the physically correct delay d (i.e. u~d) and a proper
embedding for the source, and the Schreiber measures is used with
an over-embedding such that the source state at (t{d) is still fully
covered by the source embedding. I
ddi i
h
i i
l f
l i
f TE
i
[53] h ð2Þ AT : Vt pXt(aj)~pXnTzt(aj) Vn, t[N, tvT, Vaj[AXt:
ð2Þ In neuroscience, ensemble evaluation for the estimation of
information theoretic functionals becomes relevant as physical
copies of a process are typically not available and stationarity of a
process can not necessarily be assumed. Gomez-Herrero and
colleagues recently showed how ensemble averaging may be used
to nevertheless estimate information theoretic functionals from
cyclostationary processes [55]. In neuroscience for example, a
cyclostationary process, and thus an ensemble of data, is obtained
by repeating an experimental manipulation, e.g. the presentation
of a stimulus; these repetitions are often called experimental trials. Stationarity and non-stationarity in experimental time
series PDFs in neuroscience are typically not known a priori, so in
order to estimate information theoretic functionals, these PDFs
have to be reconstructed from a sufficient amount of observed
realizations of the process. How these realizations are obtained
from data depends on whether the process in question is stationary
or non-stationary. Stationarity of a process means that PDFs of the
random variables that form the random process do not change
over time, such that pXt(Xt~aj)~pXt’(Xt’~aj), Vt,t’[N. Any PDF
pXt(:) may then be estimated from one observation of process X by
means of collecting realizations xt’ over time t’[f1, . . . ,Ng. For processes that do not fulfill the stationarity-assumption,
temporal pooling is not applicable as PDFs vary over time t and
some random variables Xt, Xs (at least two) are associated with
different PDFs pXt(:), pXs(:) (Figure 1). To still gain the necessary
multiple observations of a random variable Xt we may resort to
either run multiple physical copies of the process X or – in cases
where physical copies are unavailable – we may repeat a process in
time. If we choose the number of repetitions large enough, i.e. there is a sufficiently large set R of time points H, at which the
process is repeated, we can assume that TESPO X?Y,t,u
ð
Þ~I(Yt; X
dX
t{uDY
dY
t{1),
ð3Þ ð3Þ where I(:; :D:) is the conditional mutual information, and Yt, Y
dY
t{1,
and X
dX
t{u are the current value and the dY-dimensional past state
variables of the target process Y, and the dX-dimensional past state
variable at time t{u of the source process X, respectively (see next
paragraph for an explanation of states). Rewriting this, taking into account repetitions r of the random
processes explicitly we obtain: AR(N ^ R= : pXHzt(aj)~pXH’zt(aj)
Vt[N : tvmin DH{H’D
ð
Þ, VH,H’[R, Vaj[AXt,
ð1Þ TESPO X?Y,t,u
ð
Þ~
X
yt(r),y
dY
t{1(r),x
dX
t{u(r)
[A
Yt,YdY
t{1,XdX
t{u
p yt(r),y
dY
t{1(r),x
dX
t{u(r)
log
p yt(r)Dy
dY
t{1(r),x
dX
t{u(r)
p yt(r)Dy
dY
t{1(r)
ð4Þ
ð4Þ ð1Þ ð4Þ
ð4Þ i.e. PDFs pXHzt(:) at time point t relative to the onset of the
repetition at H are equal over all R~DRD repetitions. We call the
repeated observations of a process an ensemble of time series. Transfer entropy estimation from an ensemble of time
series Transfer entropy estimation from an ensemble of time
series Ensemble-based TE functional. When independent repe-
titions of an experimental condition are available, it is possible to
use ensemble evaluation to estimate various PDFs from an
ensemble of repetitions of the time series [55]. By eliminating the
need for pooling data over time, and instead pooling over
repetitions, ensemble methods can be used to estimate information
theoretic functionals for non-stationary time series. Here, we
follow the approach of [55] and present an ensemble TE
functional
that
extends
the
TE
functional
presented
in
[19,20,53] and also takes into account an extension of the original
formulation of TE, presented in [53], guaranteeing self prediction
optimality
(indicated
by
the
subscript
SPO). In
the
next
subsection, we will then present a practical and data-efficient
estimator of this functional. The functional reads Notation To describe practical TE estimation from time series recorded
in a system of interest X (e.g. a brain area), we first have to
formalize these recordings mathematically: We define an observed
time series x~(x1,x2, . . . ,xt, . . . ,xN) as a realization of a random
process X~(X1,X2, . . . ,Xt, . . . ,XN). A random process here is
simply a collection of individual random variables sorted by an
integer index t[f1, . . . ,Ng, representing time. TE or other
information theoretic functionals are then calculated from the
random variables’ joint PDFs pXsYt(Xs~ai,Yt~bj) and condi-
tional PDFs pXsDYt(Xs~aiDYt~bj) (with s,t[f1, . . . ,Ng), where
AXs~fa1,a2, . . . ,ai, . . . ,aIg and BYt~fb1,b2, . . . ,bj, . . . ,bJg are
all possible outcomes of the random variables Xs and Yt, and
where pXsDYt(Xs~aiDYt~bj)~ pXsYt(Xs~ai,Yt~bj)
pYt(Yt~bj)
. July 2014 | Volume 9 | Issue 7 | e102833 July 2014 | Volume 9 | Issue 7 | e102833 PLOS ONE | www.plosone.org 2 Non-Stationary Transfer Entropy next demonstrate a computationally efficient approach to the
estimation of TE using the ensemble method proposed in [55]. We call information theoretic quantities functionals as they are
defined as functions that map from the space of PDFs to the real
numbers. If we have to estimate the underlying probabilities from
experimental data first, the mapping from the data to the
information theoretic quantity (a real number) is called an
estimator. July 2014 | Volume 9 | Issue 7 | e102833 Stationarity and non-stationarity in experimental time
series In the remainder of this article, we will use the term repetition, and
interpret trials from a neuroscience experiment as a special case of
repetitions of a random process. Building on such repetitions, we In addition to the original formulation of TESPO in [53], here
we explicitly state that the necessary realizations of the random
variables in question are obtained through ensemble evaluation
over repetitions r – assuming the underlying processes to be
repeatable or cyclostationary. Furthermore, we note explicitly that July 2014 | Volume 9 | Issue 7 | e102833 PLOS ONE | www.plosone.org 3 Non-Stationary Transfer Entropy Figure 1. Pooling of data over an ensemble of time series for transfer entropy (TE) estimation. (A) Schematic account of TE. Two sca
time series Xr and Yr recorded from the rth repetition of processes X and Y, coupled with a delay d (indicated by green arrow). Colored bo
indicate delay embedded states xdX
t{u(r), ydY
t{1(r) for both time series with dimension dX~dY~3 samples (colored dots). The star on the Y time se
indicates the scalar observation yt that is obtained at the target time of information transfer t. The red arrow indicates self-information-transfer fr
the past of the target process to the random variable Yt at the target time. u is chosen such that u~d and influences of the state xdX
t{u(r) arr
exactly at the information target variable Yt. Information in the past state of X is useful to predict the future value of Y and we obtain nonzero TE. To estimate probability density functions for xdX
t{u(r), ydY
t{1(r) and yt(r) at a certain point in time t, we collect their realizations from observ
repetitions r~1, . . . ,R. (C) Realizations for a single repetition are concatenated into one embedding vector and (D) combined into one ensem
state space. Note, that data are pooled over the ensemble of data instead of time. Nearest neighbor counts within the ensemble state space can th
be used to derive TE using the Kraskov-estimator proposed in [57]. doi:10.1371/journal.pone.0102833.g001 Figure 1. Pooling of data over an ensemble of time series for transfer entropy (TE) estimation. (A) Schematic account of TE. Two scalar
time series Xr and Yr recorded from the rth repetition of processes X and Y, coupled with a delay d (indicated by green arrow). Stationarity and non-stationarity in experimental time
series Colored boxes
indicate delay embedded states xdX
t{u(r), ydY
t{1(r) for both time series with dimension dX~dY~3 samples (colored dots). The star on the Y time series
indicates the scalar observation yt that is obtained at the target time of information transfer t. The red arrow indicates self-information-transfer from
the past of the target process to the random variable Yt at the target time. u is chosen such that u~d and influences of the state xdX
t{u(r) arrive
exactly at the information target variable Yt. Information in the past state of X is useful to predict the future value of Y and we obtain nonzero TE. (B)
To estimate probability density functions for xdX
t{u(r), ydY
t{1(r) and yt(r) at a certain point in time t, we collect their realizations from observed
repetitions r~1, . . . ,R. (C) Realizations for a single repetition are concatenated into one embedding vector and (D) combined into one ensemble
state space. Note, that data are pooled over the ensemble of data instead of time. Nearest neighbor counts within the ensemble state space can then
be used to derive TE using the Kraskov-estimator proposed in [57]. doi:10.1371/journal.pone.0102833.g001 this ensemble-based functional introduces the possibility of time
resolved TE estimates. information I(Yt; Xdx
t{u) by the additional conditioning on the past
of the target time series, YdY
t{1. This additional conditioning serves
two important functions. First, as mentioned already by Schreiber
in the original paper [14], and later detailed by Lizier [4] and
Wibral and colleagues [39,53], it removes the information about
the future of the target time-series Yt that is already contained in
its own past, YdY
t{1. Second, this additional conditioning allows for
a discovery of information transfer from the source XdX
t{u to the
target that can only be seen when taking into account information
from the past of the target YdY
t{1 [69]. In the second case, the past
information from the target serves to ‘decode’ this information
transfer, and acts like a key in cryptography. As a consequence of
this importance of the past of the target process it is very important We recently showed that the estimator presented in [53] can
also be used to recover an unknown information transfer delay d
between two processes X and Y, as TESPO X?Y,t,u
ð
Þ is maximal
when the assumed delay u is equal to the true information transfer
delay d [53]. July 2014 | Volume 9 | Issue 7 | e102833 Stationarity and non-stationarity in experimental time
series This holds for the extended estimator presented here,
thus d~ arg max
u
TESPO X?Y,t,u
ð
Þ
ð
Þ:
ð5Þ ð5Þ State
space
reconstruction
and
practical
estimator. Transfer entropy differs from the lagged mutual PLOS ONE | www.plosone.org July 2014 | Volume 9 | Issue 7 | e102833 4 Non-Stationary Transfer Entropy to take all the necessary information in this past into account when
evaluating the TE as in equation 4. TESPO X?Y,t,u
ð
Þ~H Y
dY
t{1,X
dX
t{u
{H Yt,Y
dY
t{1,X
dX
t{u
zH Yt,Y
dY
t{1
{H Y
dY
t{1
,
ð8Þ To this end we need to form a collection of past random
variables ð8Þ Y
dY
t{1~(Yt{1,Yt{1{t, . . . ,Yt{1{(dY {1)t),
ð6Þ ð6Þ The Shannon differential entropies in equation 8 can be
estimated in a data efficient way using nearest neighbor techniques
[72,73]. Nearest neighbor estimators yield a non-parametric
estimate of entropies, assuming only a smoothness of the
underlying PDF. It is however problematic to simply apply a
nearest neighbor estimator (for example the Kozachenko-Leo-
nenko estimator [72]) to each term appearing in eq. 8. This is
because the dimensionality of each space associated with the terms
differs largely over terms. Thus, a fixed number of neighbors for
the search would lead to very different spatial scales (range of
distances) for each term. Since the error bias of each term is
dependent on these scales, the errors would not cancel each other
but accumulate. We therefore use a modified KSG estimator
which handles this problem by only fixing the number of
neighbors k in the highest dimensional space (k-nearest neighbor
search, kNNS) and by projecting the resulting distances to the
lower dimensional spaces as the range to look for and count
neighbors there (range search, RS) (see [57], type 1 estimator, and
[56,74]). In the ensemble variant of TE estimation we proceed by
searching for nearest neighbors across points from all repetitions
instead of searching the same repetition as the point of reference of
the search – thus we form an ensemble search space by combining
points over repetitions. Finally, the ensemble estimator of TE
reads such that their realizations, such that their realizations, y
dY
t{1(r)~(yt{1(r),yt{1{t, . . . ,yt{1{(dY {1)t),
ð7Þ ð7Þ are maximally informative about the future of the target process,
Yt. Stationarity and non-stationarity in experimental time
series This task is complicated by the fact the we often deal with
multidimensional systems, of which we only observe a scalar
variable (here modeled as our random processes X,Y). To see this,
think for example of a pendulum (which is a two dimensional
system) of which we record only the current position Yt. If the
pendulum is at its lowest point, it could be standing still, going left,
or going right. To properly describe which state the pendulum is
in, we need to know at least the realization of one more random
variable Yt{1 back in time. Collections of such past random
variables whose realizations uniquely describe the state of a
process are called state variables. Such a sufficient collection of past variables, called a delay
embedding vector, can always be reconstructed from scalar
observations for low dimensional deterministic systems, such as
the above pendulum, as shown by Takens [70]. Unfortunately,
most real world systems are high-dimensional stochastic dynamic
systems (best described by non-linear Langevin equations) rather
than low-dimensional deterministic ones. For these systems it is not
obvious that a delay embedding similar to Takens’ approach
would yield the desired results. In fact, many systems can be shown
to require an infinite number of past random variables when only
a scalar observable of the high-dimensional stochastic process is
accessible. Nevertheless, as shown by Ragwitz and Kantz [71], the
behavior of scalar observables of most of these systems can be
approximated very well by a finite collection of such past variables
for all practical purposes; in other words, these systems can be
approximated well by a finite order, one-dimensional Markov-
process. TESPO X?Y,t,u
ð
Þ~y k
ð ÞzSy n
ydY
t{1(r)z1
{y n
yt(r) ydY
t{1(r)z1
{y n
ydY
t{1(r) xdX
t{u(r)z1
Tr ,
ð9Þ ð9Þ where y denotes the digamma function and the angle brackets
(v:wr) indicate an averaging over points in different repetitions r
at time instant t. The distances to the k-th nearest neighbor in the
highest dimensional space (spanned by Yt,Y
dY
t{1,X
dX
t{u) define the
radius of the spheres for the counting of the number of points (n:)
in these spheres around each state vector (:) involved. For practical TE estimation using equation 4, we therefore
proceed by first reconstructing the state variables of such
approximated Markov processes for the two systems X, Y from
their scalar time series. Stationarity and non-stationarity in experimental time
series Then, we use the statistics of nearest
ensemble neighbors with a modified KSG estimator for TE
evaluation [57]. In cases where the number of repetitions is not sufficient to
provide the necessary amount of data to reliably estimate Shannon
entropies through an ensemble average, one may combine
ensemble evaluation with collecting realizations over time. In
these cases, we count neighbors in a time window t’[½t{,tz with
t{ƒt’ƒtz, where Dt~tz{t{ controls the temporal resolution
of the TE estimation: Thus, we select a delay embedding vector of the form
YdY
t{1~(Yt{1,Yt{1{t, . . . ,Yt{1{(dY {1)t) from equation 6 as our
collection of past random variables – with realizations in repetition r
given by ydY
t{1(r)~(yt{1(r),yt{1{t, . . . ,yt{1{(dY {1)t). Here, dY is
called the embedding dimension and t the embedding delay. These
embedding parameters dY and t, are chosen such that they
optimize a local predictor [71], as this avoids an overestimation of
TE [53]; other approaches related to minimizing non-linear
prediction errors are also possible [44]. In particular, dY:t is chosen
such that YdY
t
is conditionally independent of any YdY
e
with
evt{d:t given YdY
t{1. The same is done for the process X at time
t{u. TESPO X?Y,t’,u
ð
Þ~y k
ð ÞzSy n
ydY
t’{1(r)z1
{y n
yt’(r) ydY
t’{1(r)z1
{y n
ydY
t’{1(r) xdX
t’{u(r)z1
Tr,t’ :
ð10Þ ð10Þ Next, we decompose TESPO into a sum of four individual
Shannon entropies: July 2014 | Volume 9 | Issue 7 | e102833 PLOS ONE | www.plosone.org 5 Non-Stationary Transfer Entropy Figure 2. Transfer entropy estimation using the ensemble
method in TRENTOOL 3.0. (A) Data preparation and optimization of
embedding parameters in function
TEprepare.m ; (B) transfer
e n t r o p y
( T E )
e s t i m a t i o n
f r o m
p r e p a r e d
d a t a
i n
TEsurrogatestats_ensemble.m (yellow boxes indicate variables
being passed between sub-functions). TE is estimated via iterating over
all channel combinations provided in the data. Stationarity and non-stationarity in experimental time
series For each channel
combination: (1) Data is embedded individually per repetition and
combined over repetitions into one ensemble state space (chunk), (2) S
surrogate data sets are created by shuffling the repetitions of the target
time series, (3) each surrogate data set is embedded per repetition and
combined into one chunk (forming S chunks in total), (4) Sz1 chunks
of original and surrogate data are passed to the GPU where nearest
neighbor searches are conducted in parallel, (5) calculation of TE values
from returned neighbor counts for original data and S surrogate data
sets using the KSG-estimator [57], (6) statistical testing of original TE
value against distribution of surrogate TE values; (C) output of
TEsurrogatestats_ensemble.m, an array with dimension [no. channels|5], where rows hold results for all channel combinations: (1)
p-value of TE for this channel combination, (2) significance at the
designated alpha level (1 - significant, 0 - not significant), (3)
significance after correction for multiple comparisons, (4) absolute
difference between the TE value for original data and the median of
surrogate TE values, (5) presence of volume conduction (this is always
set to 0 when using the ensemble method as instantaneous mixing is
by default controlled for by conditioning on the current state of the
source time series xt(r) [119]). doi:10 1371/journal pone 0102833 g002 ( )
doi:10.1371/journal.pone.0102833.g002 Implementation The estimation of TE from finite time series consists of the
estimation of joint and marginal entropies as shown in equations 9
and 10, calculated from nearest neighbor statistics, i.e. distances
and the count of neighbors within these distances. In practice we
obtain these neighbor counts by applying kNNS and RS to
reconstructed state spaces. In particular, we use a kNNS in the
highest dimensional space to determine the k-th nearest neighbor
of a data point and the associated distance. This distance is then
used as the range for the RS in the marginal spaces, that return the
point counts n. Both searches have a high computational cost. This cost increases even further in a practical setting, where we
need to calculate TE for a sufficient number of surrogate data sets
for statistical testing (see [19] and below for details). To enable TE
estimation and statistical testing despite its computational cost, we
implemented ad-hoc kNNS and RS algorithms in NVIDIAH
CUDATM C/C++ code [75]. This allows to run thousands of
searches in parallel on a modern GPU. To allow for a better understanding of the parallelization used,
we will now briefly describe the main work flow of TE analysis in
the open source MathWorksH MATLABH toolbox TRENTOOL
[56], which implements the approach to TE estimation described
in the Background section. The work flow includes the steps of
data preprocessing prior to the use of the GPU algorithm for
neighbor searches as well as the statistical testing of resulting TE
values. In a subsequent section we will describe the core
implementation of the algorithm in more detail and present its
integration into TRENTOOL. Main analysis work flow in TRENTOOL Main analysis work flow in TRENTOOL
Practical TE estimation in TRENTOOL. The practical
GPU-based TE estimation in TRENTOOL 3.0 is divided into the
two steps of data preparation and TE estimation (see Figure 2 and
the TRENTOOL 3.0 manual: http: www.trentool.de). As a first
step, data is prepared by optimizing embedding parameters for
state space reconstruction (Figure 2, panel A). As a second step,
TE is estimated by following the approach for ensemble-based TE
estimation lined out in the preceding section (Figure 2, panel B). PLOS ONE | www.plosone.org July 2014 | Volume 9 | Issue 7 | e102833 6 Non-Stationary Transfer Entropy Non-Stationary Transfer Entropy Figure 3. Creation of surrogate data sets. (A) Original time series with information transfer (solid arrow) from a source state xdx
(t{u)(r) to a
corresponding target time point yt(r), given the time point’s history ydy
(t{1)(r). Solid arrows indicate the direction of transfer entropy (TE) analysis,
while information transfer is present. (B) Shuffled target time series, repetitions are permutes, such that yt(w(r)) and ydy
(t{1)(w(r)), where w denotes a
random permutation. Dashed arrows indicate the direction of TE analysis, while no more information flow is present. doi:10.1371/journal.pone.0102833.g003 Figure 3. Creation of surrogate data sets. (A) Original time series with information transfer (solid arrow) from a source state xdx
(t{u)(r) to a
corresponding target time point yt(r), given the time point’s history ydy
(t{1)(r). Solid arrows indicate the direction of transfer entropy (TE) analysis,
while information transfer is present. (B) Shuffled target time series, repetitions are permutes, such that yt(w(r)) and ydy
(t{1)(w(r)), where w denotes a
random permutation. Dashed arrows indicate the direction of TE analysis, while no more information flow is present. doi:10.1371/journal.pone.0102833.g003 TRENTOOL estimates TESPO X?Y,t,u
ð
Þ (eq. 4) for a given pair
of processes X and Y and given values for u and t. For each pair,
we call X the source and Y the target process. proportion of surrogate TE values TESPO X?Y’,t,u
ð
Þ equal or
larger than TESPO X?Y,t,u
ð
Þ. This p-value is then compared to a
critical alpha level (see for example [56,76]). Reconstruction of information transfer delays. TESPO
X?Y,t,u
ð
Þ may be used to reconstruct the interaction transfer
delay dXY between X and Y (eq. 5, [53]). Implementation of the GPU algorithm Parallelized nearest neighbor searches. The KSG esti-
mator used for estimating TESPO X?Y,t,u
ð
Þ in eq. 9 and 10 uses
neighbor
(distance-)statistics
obtained
from
kNNS
and
RS
algorithms to estimate Shannon differential entropies. Thus, the
choice of computationally efficient kNNS and RS algorithms is
crucial to any practical implementation of the TESPO estimator. kNNS algorithms typically return a list of the k nearest neighbors
for each reference point, while RS algorithms typically return a list
of all neighbors within a given range for each reference point. kNNS and RS algorithms have been studied extensively because of
their broad potential for application in nearest neighbor searches
and related problems. Several approaches have been proposed to
reduce their high computational cost: partitioning of input data
into k-d Trees, Quadtrees or equivalent data structures [77] or
approximation algorithms (ANN: Approximate Nearest Neigh-
bors) [78,79]. Furthermore, some authors have explored how to
parallelize the kNNS algorithm on a GPU using different
implementations: exhaustive brute force searches [80,81], tree-
based searches [82,83] and ANN searches [83,84]. The proposed GPU algorithm is accessed in step (4). As we will
further explain below (see paragraph on Input data), the GPU
implementation uses the fact that all of the necessary computations
on surrogate data sets and the original data are independent and
can thus be performed in parallel. The proposed GPU algorithm is accessed in step (4). As we will
further explain below (see paragraph on Input data), the GPU
implementation uses the fact that all of the necessary computations
on surrogate data sets and the original data are independent and
can thus be performed in parallel. TE calculation and statistical testing against surrogate
data. Estimated TE values need to be tested for their statistical
significance [56] (step (6) of the main TRENTOOL work flow). For this statistical test under a null hypothesis of no information
transfer between a source X and target time series Y, we estimate
TESPO X?Y,t,u
ð
Þ and compare it to a distribution of TE values
calculated from surrogate data sets. Surrogate data sets are formed
by
shuffling
repetitions
in
Y
to
obtain
Y’,
such
that
ydY
t (r)?ydY
t (w(r)) and yt(r)?yt(w(r)), where w denotes a random
permutation of the repetitions r (Figure 3). From this surrogate
data set, we calculate surrogate TE values TESPO X?Y’,t,u
ð
Þ. Main analysis work flow in TRENTOOL dXY may be reconstructed
by scanning possible values for u: TESPO X?Y,t,u
ð
Þ is estimated
for all values in u; The value that maximizes the TESPO X?Y,t,u
ð
Þ
is kept as the reconstructed information transfer delay. We used the
reconstruction of information transfer delays as an additional
parameter when testing the proposed implementation for correct-
ness and robustness. After data preparation TESPO X?Y,t,u
ð
Þ (eq. 9 and 10) is
estimated in six steps: (1) using optimized embedding parameters,
original data is embedded per repetition and repetitions are
concatenated forming the ensemble search space of the original
data, (2) S sets of surrogate data are created from the original data
by shuffling the repetitions of the target process Y, (3) each
surrogate dataset is embedded per repetition and concatenated
forming S additional ensemble search spaces for surrogate data, (4)
all Sz1 search spaces of embedded original and surrogate data
are passed to a wrapper function that calls the GPU functions to
perform individual neighbor searches for each search space in
parallel (in the following, we will refer to each of the Sz1
ensembles as one data chunk), (5) TE values are calculated for
original and surrogate data chunks from the neighbor counts using
the KSG- estimator [57], (6) TE values for original data are tested
statistically against the distribution of surrogate TE values. Implementation of the GPU algorithm July 2014 | Volume 9 | Issue 7 | e102833 Implementation of the GPU algorithm By
repeating this process a sufficient number of times S, we obtain a
distribution of values TESPO X?Y’,t,u
ð
Þ. To asses the statistical
significance of TESPO X?Y,t,u
ð
Þ, we calculate a p-value as the TE calculation and statistical testing against surrogate
data. Estimated TE values need to be tested for their statistical
significance [56] (step (6) of the main TRENTOOL work flow). For this statistical test under a null hypothesis of no information
transfer between a source X and target time series Y, we estimate
TESPO X?Y,t,u
ð
Þ and compare it to a distribution of TE values
calculated from surrogate data sets. Surrogate data sets are formed
by
shuffling
repetitions
in
Y
to
obtain
Y’,
such
that
ydY
t (r)?ydY
t (w(r)) and yt(r)?yt(w(r)), where w denotes a random
permutation of the repetitions r (Figure 3). From this surrogate
data set, we calculate surrogate TE values TESPO X?Y’,t,u
ð
Þ. By
repeating this process a sufficient number of times S, we obtain a
distribution of values TESPO X?Y’,t,u
ð
Þ. To asses the statistical
significance of TESPO X?Y,t,u
ð
Þ, we calculate a p-value as the Although performance of existing implementations of kNNS for
GPU was promising, they were not applicable to TE estimation. The most critical reason was that existing implementations did not
allow for the concurrent treatment of several problem instances by
the GPU and maximum performance was only achieved for very July 2014 | Volume 9 | Issue 7 | e102833 July 2014 | Volume 9 | Issue 7 | e102833 7 PLOS ONE | www.plosone.org Non-Stationary Transfer Entropy Figure 4. GPU implementation of the parallelized nearest neighbor search in TRENTOOL 3.0 Chunks of data are prepared on the CPU
(embedding and concatenation) and passed to the GPU. Data points are managed in the global memory as Structures of Arrays (SoA). To make
maximum use of the memory bandwidth, data is padded to ensure coalesced reading and writing from and to the streaming multiprocessor (SM)
units. Each SM handles one chunk in one thread block (dashed box). One block conducts brute force neighbor searches for all data points in the
chunk and collects results in its shared memory (red and blue arrows and shaded areas). Results are eventually returned to the CPU. doi:10.1371/journal.pone.0102833.g004 Figure 4. Implementation of the GPU algorithm Our implementation is able to handle several data chunks
simultaneously to make maximum use of the GPU resources. Thus, several chunks may be combined, using an additional index
vector to encode the sizes of individual chunks. These chunks are
then passed at once to the GPU algorithm to be searched in
parallel. Input data. As input, the proposed RS and kNNS algorithms
expect a set of data points representing the search space and a
second set of data points that serve as reference points in the
searches. One such problem instance is considered one data
chunk. Our implementation is able to handle several data chunks
simultaneously to make maximum use of the GPU resources. Thus, several chunks may be combined, using an additional index
vector to encode the sizes of individual chunks. These chunks are
then passed at once to the GPU algorithm to be searched in
parallel. Our implementation is written in CUDA (Compute Unified
Device Architecture) [75] (a port to OpenCLTM [85] is work in
progress). CUDA is a parallel computing framework created by
NVIDIA that includes extensions to high level languages such as
C/C++, giving access to the native instruction set and memory of
the parallel computational elements in CUDA enabled GPUs. Accelerating an algorithm using CUDA includes translating it into
data-parallel sequences of operations and then carefully mapping
these operations to the underlying resources to get maximum
performance [58,59]. To understand the implementation suggest-
ed here, we will give a brief explanation of these resources, i.e. the
GPU’s hardware architecture, before explaining the implementa-
tion in more detail (additionally, see [58,59,75]). Our implementation is written in CUDA (Compute Unified
Device Architecture) [75] (a port to OpenCLTM [85] is work in
progress). CUDA is a parallel computing framework created by
NVIDIA that includes extensions to high level languages such as
C/C++, giving access to the native instruction set and memory of
the parallel computational elements in CUDA enabled GPUs. Accelerating an algorithm using CUDA includes translating it into
data-parallel sequences of operations and then carefully mapping
these operations to the underlying resources to get maximum
performance [58,59]. To understand the implementation suggest-
ed here, we will give a brief explanation of these resources, i.e. the
GPU’s hardware architecture, before explaining the implementa-
tion in more detail (additionally, see [58,59,75]). Implementation of the GPU algorithm GPU implementation of the parallelized nearest neighbor search in TRENTOOL 3.0 Chunks of data are prepared on the CPU
(embedding and concatenation) and passed to the GPU. Data points are managed in the global memory as Structures of Arrays (SoA). To make
maximum use of the memory bandwidth, data is padded to ensure coalesced reading and writing from and to the streaming multiprocessor (SM)
units. Each SM handles one chunk in one thread block (dashed box). One block conducts brute force neighbor searches for all data points in the
chunk and collects results in its shared memory (red and blue arrows and shaded areas). Results are eventually returned to the CPU. doi:10.1371/journal.pone.0102833.g004 large kNNS problem instances. Unfortunately, the problem
instances typically expected in our application are numerous (i.e. Sz1 problem instances per pair of time series), but rather small
compared to the main memory on a typical GPU device in use
today. Thus, an implementation that handled only one instance at
a time would not have made optimal use of the underlying
hardware. Therefore, we designed an implementation that is able
to handle several problem instances at once to perform neighbor
searches for chunks of embedded original and surrogate data in
parallel. Moreover, we aimed at a flexible GPU implementation of
kNNS and RS that maximized the use of the GPU’s hardware
resources for variable configurations of data – thus making the
implementation independent of the design of the neuroscientific
experiment. Threads are grouped in blocks, which are in turn organized in a
grid. The grid is the entry point to the GPU resources. It handles
one kernel call at a time and executes it on multiple data in
parallel. Within the grid, each block of threads is executed by one
SM. The SM executes the threads of a block by issuing them in
groups of 32 threads, called warps. Threads within one warp are
executed concurrently, while as many warps as possible are
scheduled per SM to be resident at a time, such that the utilization
of all the cores is maximized. Input data. As input, the proposed RS and kNNS algorithms
expect a set of data points representing the search space and a
second set of data points that serve as reference points in the
searches. One such problem instance is considered one data
chunk. July 2014 | Volume 9 | Issue 7 | e102833 Implementation of the GPU algorithm Each individual search is handled by one
CUDA thread. Parallelization of these searches on the GPU
happens in two ways: (1) the GPU algorithm is able to handle
several chunks, (2) each chunk can be searched in parallel, such
that individual searches within one chunk are handled simulta-
neously. An individual search is conducted by a CUDA thread by
brute-force measuring the infinity norm distance of the given
reference point to any other point within the same chunk. Simultaneously, other threads measure these distances for other
points in the same chunk or handle a different chunk altogether. Searching several chunks in parallel is an essential feature of the
proposed
solution, that maximizes the utilization of GPU
resources. From the GPU execution point of view, simultaneous
searches are realized by handling a variable number of kNNS (or
RS) problem instances through one grid launch. The number of
searches that can be executed in parallel is thus only limited by the
device’s global memory that holds the input data and the number
of threads that can be started simultaneously (both limitations are
taken into account). Furthermore, the solution is implemented
such that optimal performance is guaranteed. Figure 5. Practical performance measures of the ensemble
method for GPU compared to CPU. Combined execution times in s
for serial and parallel implementations of k-nearest neighbor and range
search as a function of input size (number of data chunks). Execution
times were measured for the serial implementation running on a CPU
(black) and for our parallel implementation using one of three GPU
devices (blue, red, green) of varying computing power. Computation
using a GPU was considerably faster than using a CPU (by factors 22, 33
and 50 respectively). doi 10 1371/jo rnal pone 0102833 g005 p
p
g
Low-level
implementation
details. There
are
several
strategies that are essential for optimal performance when
implementing algorithms for GPU devices. Most important are
the reduction of memory latencies and the optimal use of
hardware resources by ensuring high occupancy (the ratio of
number of active warps per SM to the maximum number of
possible active warps [58]). To maximize occupancy, we designed
our algorithm’s kernels such that always more than one block of
threads (ideally many) are loaded per SM [58]. We can do this
since many searches are executed concurrently in every kernel
launch. Implementation of the GPU algorithm By maximizing occupancy, we both ensure hardware
utilization and improve performance by hiding data memory
latency from the GPU’s global memory to the SMs’ registers [75]. Moreover, in order to reduce memory latencies we take care of
input data memory alignment and guarantee that memory
readings issued by the threads of a warp are coalesced into as
few memory transfers as possible. Additionally, with the aim of
minimizing sparse data accesses to memory, data points are
organized as Structures of Arrays (SoA). Finally, we use the shared
memory inside the SMs (a self-programmed intermediate cache
between global memory and SMs) to keep track of nearest
neighbors associated information during searches. The amount of
shared memory and registers is limited in a SM. The maximum
possible occupancy depends on the number of registers and shared
memory needed by a block, which in turn depends on the number
of threads in the block. For our implementation, we used a suitable
block size of 512 threads. p
y
doi:10.1371/journal.pone.0102833.g005 Evaluation To evaluate the proposed algorithm we investigated four
properties: first, whether the speedup is sufficient to allow the
application of the method to real-world neural datasets; second,
the correctness of results on simulated data, where the ground
truth is known; third, the robustness of the algorithm for limited
sample sizes; fourth, whether plausible results are achieved on a
neural example dataset. Implementation of the GPU algorithm In the estimation of TESPO X?Y,t,u
ð
Þ, according to the work
flow described in paragraph Practical TE estimation in TREN-
TOOL, we used the proposed implementation to parallelize
neighbor searches over surrogate data sets for a given pair of time
series x and y and given values for u and t. Thus, in one call to the
GPU algorithms Sz1 data chunks were passed as input, where
chunks represented the search space for the original pair of time
series and S search spaces for corresponding surrogate data sets. Points within the search spaces may have either been collected
through temporal or ensemble pooling of embedded data points or
a combination of both (eq. 9 or 10). GPU
resources. GPU
resources comprise of massively
parallel processors with up to thousands of cores (processing
units). These cores are divided among Stream Multiprocessors
(SMs) in order to guarantee automatic scalability of the algorithms
to different versions of the hardware. Each SM contains 32 to 192
cores that execute operations described in the CUDA kernel code. Operations executed by one core are called a CUDA thread. Core algorithm. In the core GPU-based search algorithm,
the kNNS implementation is mapped to CUDA threads as
depicted in Figure 4 (the RS implementation behaves similarly). Each chunk consists of a set of data points that represents the Core algorithm. In the core GPU-based search algorithm,
the kNNS implementation is mapped to CUDA threads as
depicted in Figure 4 (the RS implementation behaves similarly). Each chunk consists of a set of data points that represents the July 2014 | Volume 9 | Issue 7 | e102833 July 2014 | Volume 9 | Issue 7 | e102833 PLOS ONE | www.plosone.org 8 Non-Stationary Transfer Entropy Figure 5. Practical performance measures of the ensemble
method for GPU compared to CPU. Combined execution times in s
for serial and parallel implementations of k-nearest neighbor and range
search as a function of input size (number of data chunks). Execution
times were measured for the serial implementation running on a CPU
(black) and for our parallel implementation using one of three GPU
devices (blue, red, green) of varying computing power. Computation
using a GPU was considerably faster than using a CPU (by factors 22, 33
and 50 respectively). doi:10.1371/journal.pone.0102833.g005 search space and are at the same time used as reference points for
individual searches. Ethics statement The neural example dataset was taken from an experiment
described in [86]. All subjects gave written informed consent
before the experiment. The study was approved by the local ethics
committee (Johann Wolfgang Goethe University, Frankfurt,
Germany). July 2014 | Volume 9 | Issue 7 | e102833 Non-Stationary Transfer Entropy Non-Stationary Transfer Entropy To put these numbers into perspective, we note that in a
neuroscience experiment the number of chunks to be processed is
the product of (typical numbers): channel pairs for TE (100) *
number of surrogate data sets (1000) * experimental conditions (4)
* number of subjects (15). This results in a total computational
load on the order of 6 106 chunks to be processed. Given an
execution time of 24.1 s/50 on the NVIDIA GTX Titan for a
typical test dataset, these computations will take 2:9 106 s or 4.8
weeks on a single GPU, which is feasible compared to the initial
duration of 240 weeks on a single CPU. Even when considering a
trivial parallelization of the computations over multiple CPU cores
and CPUs, the GPU based solution is by far more cost and energy
efficient than any possible CPU-based solution. If in addition a
scanning of various possible information transfer delays is
important, then parallelization over multiple GPUs seems to be
the only viable option. because of the duplicated chunk data). Note that for both, CPU
and GPU implementations, data handling prior to nearest
neighbor searches is identical. We were thus able to confine the
testing of performance differences to the respective kNNS and RS
algorithms only, as all data handling prior to nearest neighbor
searches was conducted using the same, highly optimized
TRENTOOL functionalities. because of the duplicated chunk data). Note that for both, CPU
and GPU implementations, data handling prior to nearest
neighbor searches is identical. We were thus able to confine the
testing of performance differences to the respective kNNS and RS
algorithms only, as all data handling prior to nearest neighbor
searches was conducted using the same, highly optimized
TRENTOOL functionalities. Analogous to TE estimation implemented in TRENTOOL, we
conducted one kNNS (with k~4, TRENTOOL default, see also
[87]) in the highest dimensional space and used the returned
distances for a RS in one lower dimensional space. Both functions
were called for increasing numbers of chunks to obtain the
execution time as a function of input size. One chunk of data from
the highest dimensional space had dimensions [30094|17] and
size 1.952 MB (single precision); one chunk of data from the lower
dimensional space had dimensions [30094|8] and size 0.918 MB
(single precision). Evaluation on Lorenz systems To test the ability of the presented implementation to
successfully reconstruct information transfer between systems with
a non-stationary coupling, we simulated various coupling scenarios
between stochastic and deterministic systems. We introduced non-
stationary into the coupling of two processes by varying the
coupling strength over the course of a repetition (all other
parameters
were
held
constant). Simulations
for individual
scenarios are described in detail below. For the estimation of TE
we used MathWork’s MATLAB, and the TRENTOOL toolbox
extended by the implementation of the ensemble method proposed
above (version 3.0, see also [56] and http: www.trentool.de). For a
detailed testing of the used estimator TESPO (eq. 4) refer to [53]. To obtain reliable results for the serial implementation we ran
both kNNS and RS 200 times on the data, receiving an average
execution time of 1.26 s for kNNS and an average execution time
of 24.1 s for RS. We extrapolated these execution times to higher
numbers of chunks and compared them to measured execution
times of the parallel searches on three NVIDIA GPU devices. On
average, execution times on the GPU compared to the CPU were
faster by a factor of 22 on the NVIDIA Tesla C2075, by a factor of
33 for the NVIDIA GTX 580 and by a factor of 50 for the
NVIDIA GTX Titan (Figure 5). Coupled Lorenz systems. Simulated data was taken from
two unidirectionally coupled Lorenz systems labeled X and Y. Systems interacted in direction X?Y according to equations: _Ui(t)~s(Vi(t){Ui(t)),
_Vi(t)~Ui(t)(ri{Wi(t)){Vi(t)z
X
i,j~X,Y
cijV2
j (t{dij),
_W i(t)~Ui(t)Vi(t){bWi(t),
ð11Þ ð11Þ Figure 6. Transfer entropy reconstruction from non-stationary Lorenz systems. We used two dynamically coupled Lorenz systems (A) to
simulate non-stationarity in data generating processes. A coupling cXY~0:3 was present during a time interval from 1000 to 2000 ms only (cXY~0
otherwise). The information transfer delay was set to dXY~45ms. Transfer entropy (TE) values were reconstructed using the ensemble method
combined with the scanning approach proposed in [53] to reconstruct information transfer delays. Assumed delays u were scanned from 35 to 55 ms
(1 ms resolution). In (B) the maximum TE values for original data over this interval are shown in blue. Red bars indicate the corresponding mean over
surrogate TE values (error bars indicate 1 SD). Significant TE was found for the second time window only; here, the delay was reconstructed as
u~49ms. doi:10.1371/journal.pone.0102833.g006 Figure 6. Transfer entropy reconstruction from non-stationary Lorenz systems. Non-Stationary Transfer Entropy Performance testing of the serial implementation
was carried out on an Intel Xeon CPU (E5540, clocked at
2.53 GHz), where we measured execution times of the TSTOOL
kNNS (functions ‘nn_prepare.m’ and ‘nn_search.m’) and the
TSTOOL RS (function ‘range_search.m’). Testing of the parallel
implementation was carried out three times on GPU devices of
varying processing power (NVIDIA Tesla C2075, GeForce GTX
580 and GeForce GTX Titan). On the GPUs, we measured
execution times for the proposed kNNS (‘fnearneigh_gpu.mex’)
and RS (‘range_search_all_gpu.mex’) implementation. When the
GPU’s global memory capacity was exceeded by higher input
sizes, data was split and computed over several runs (i.e. calls to
the GPU). All performance testing was done by measuring
execution times using the MATLAB functions tic and toc. Evaluation of computational speedup To test for an increase in performance due to the parallelization
of neighbor searches, we compared practical execution times of
the proposed GPU implementation to execution times of the serial
kNNS and RS algorithms implemented in the MATLAB toolbox
TSTOOL (http: www.dpi.physik.uni-goettingen.de/tstool/). This
toolbox wraps a FORTRAN implementation of kNNS and RS,
and has proven the fastest CPU toolbox for our purpose. All
testing was done in MATLAB 2008b (MATLAB 7.7, The
MathWorks Inc., Natick, MA, 2008). As input, we used increasing
numbers of chunks of simulated data from two coupled Lorenz
systems, further described below. Repetitions of simulated time
series were embedded and combined to form ensemble state
spaces, i.e. chunks of data (c.f. paragraph Input Data). To obtain
increasing input sizes, we duplicated these chunks the desired
number of times. While the CPU implementation needed to
iteratively perform searches on individual chunks, the GPU
implementation searched chunks in parallel (note that chunks
are treated independently here, so that there is no speedup Implementation interface. The GPU functionality is ac-
cessed through MATLAB scripts for kNNS (‘fnearneigh_gpu.-
mex’) and RS (‘range_search_all_gpu.mex’), which encapsulate all
the associated complexity. Both scripts are called from TREN-
TOOL using a wrapper function. In its current implementation in
TRENTOOL (see paragraph Practical TE estimation in TREN-
TOOL), the wrapper function takes all Sz1 chunks as input and
launches a kernel that searches all chunks in parallel through the
mex-files for kNNS and RS. The wrapper makes sure that the
input size does not exceed the GPU device’s available global
memory and the maximum number of threads that can be started
simultaneously. If necessary, the wrapper function splits the input
into several kernel calls; it also manages the output, i.e. the
neighbor counts for each chunk, which are passed on for TE
calculation. July 2014 | Volume 9 | Issue 7 | e102833 PLOS ONE | www.plosone.org 9 Evaluation on Lorenz systems We simulated three cou-
pling scenarios, where the coupling varied in strength over the
course of a repetition (duration 3000 ms): (1) unidirectional
coupling X?Y with a coupling onset around 1000 ms; (2)
unidirectional coupling with a two-step increase in coupling X?Y
at around 1000 ms and around 2000 ms; (3) bidirectional coupling
X?Y with onset around 1000 ms and Y?X with onset around
2000 ms. See table 1 for specific parameter values used in each
scenario. For each scenario, 500 surrogate data sets were computed to
allow for statistical testing of the reconstructed information
transfer. Surrogate data were created by permutation of data
points in blocks of the target time series (Figure 3), leaving each
repetition intact. The value k for the nearest neighbor search was
set to 4 for all analyses (TRENTOOL default, see also [87]). We realized a varying coupling strength cXY(t) (and cYX(t) for
scenario (3)) by modulating coupling parameters bYX, bXY with a
hyperbolic tangent function. No coupling was realized by setting
b:~0. For scenarios (1) and (3) we used the coupling y
(
[
])
Results. We analyzed data from three time windows from
200 to 450 ms, 1600 to 1850 ms and 2750 to 3000 ms using the
estimator proposed in eq. 10 with Dt~250ms, assuming local
stationarity (Figure 6, panel A). For each time window, we
scanned assumed delays in the interval u~½35,55. Figure 6,
panel B, shows the maximum TE value from original data (blue)
over all assumed u and the corresponding mean surrogate TE
value (red). Significant differences between original TE and
surrogate TE were found in the second time window only
(indicated by an asterisk). No significant information transfer was
found during the non-coupling intervals. The information transfer
delay reconstructed for the second analysis window was 49 ms
(true information transfer delay dXY~45ms). Thus, the proposed
implementation was able to reliably detect a coupling between
both systems and reconstructed the corresponding information
transfer delay with an error of less than 10%. cYX~bYX 0:5 1z tanh 0:05(t{2000)
½
ð
Þ
ð14Þ ð14Þ cXY~bXY 0:5 1z tanh 0:05(t{1000)
½
ð
Þ,
ð15Þ ð15Þ where 0.05 was the slope and 2000 and 1000 are the inflection
points of the hyperbolic tangent respectively. Note that we
additionally scaled the tanh function such that function value
ranged from 0 to 1. Evaluation on Lorenz systems For coupling scenario (2), the two-step increase
in cXY was expressed as: cXY~bXY 0:5 0:5 1z tanh 0:05(t{1000)
½
ð
Þ
½
z0:5 1z tanh 0:05(t{2000)
½
ð
Þ
ð16Þ ð16Þ Evaluation on Lorenz systems We used two dynamically coupled Lorenz systems (A) to
simulate non-stationarity in data generating processes. A coupling cXY~0:3 was present during a time interval from 1000 to 2000 ms only (cXY~0
otherwise). The information transfer delay was set to dXY~45ms. Transfer entropy (TE) values were reconstructed using the ensemble method
combined with the scanning approach proposed in [53] to reconstruct information transfer delays. Assumed delays u were scanned from 35 to 55 ms
(1 ms resolution). In (B) the maximum TE values for original data over this interval are shown in blue. Red bars indicate the corresponding mean over
surrogate TE values (error bars indicate 1 SD). Significant TE was found for the second time window only; here, the delay was reconstructed as
u~49ms. doi:10.1371/journal.pone.0102833.g006 July 2014 | Volume 9 | Issue 7 | e102833 PLOS ONE | www.plosone.org 10 Non-Stationary Transfer Entropy Table 1. Parameter settings for simulated autoregressive processes. Testcase
aX
aY
bYX
bXY
dYX
dXY
Unidirectional
0.75
0.35
0
20.35
0
10
Two-step
unidirectional
0.75
0.35
0
20.35
0
10
Bidirectional
0.475
0.35
20.4
20.35
20
10
doi:10.1371/journal.pone.0102833.t001 where i,j~X,Y, dij is the coupling delay and cij is the coupling
strength; s, r and b are the Prandtl number, the Rayleigh number,
and a geometrical scale. Note, that cYX~cXX~cYY~0 for the
test cases (no self feedback, no coupling from Y to X). Numerical
solutions to these differential equations were computed using the
dde23 solver in MATLAB and results were resampled such that
the delays amounted to the values given below. For analysis
purposes we analyzed the V-coordinates of the systems. y(t)~aYy(t{1)zcXY(t)x(t{dXY)zgY(t),
ð13Þ ð13Þ where aX, aY are the AR parameters, cYX(t), cXY(t) denote
coupling strength, dYX, dXY are the coupling delays and gX, gY
denote uncorrelated, unit-variance, zero-mean Gaussian white
noise terms. We introduced non-stationarity in the coupling between both
systems by varying the coupling strength c over time. In particular,
a coupling cXY~0:3 was set for a limited time interval only,
whereas before and after the coupling interval cXY was set to 0. A
constant information transfer delay dXY~45ms was simulated for
the whole coupling interval. We simulated 150 repetitions with
3000 data points each, with a coupling interval from approxi-
mately 1000 to 2000 data points (see Figure 6, panel A). Simulated coupling scenarios. x(t)~aXx(t{1)zcYX(t)y(t{dYX)zgX(t),
ð12Þ Evaluation on autoregressive processes repetitionwise) permutation of data
points in the target time series. The value k for the nearest
neighbor search was set to 4 for all analyses (TRENTOOL
default, see also [87]). showed a small error in the reconstructed information transfer
delay. This may be due to too little data to detect the weaker
coupling at this epoch of the simulated coupling (see below). Results – Scenario (2), unidirectional coupling with two-
step increase. For scenario (2), we again used the scanning
approach for TE reconstruction, using an interval of assumed
delays
u~½1,20,
where
the
true
delay
was
simulated
at
dXY~10ms. No TE was detected prior to the coupling onset
around 1 s. TE was detected for analysis windows 4, 5, and 6 (1.1
to 1.4, 1.4 to 1.7, 1.7 to 2.0 s) with reconstructed information
transfer delays of 10, 4, and 7 ms respectively. Further, significant
TE was found for analysis windows 7 and 8 (after the second
increase in coupling strength around 2 s). Here, the correct
coupling of 10 ms was reconstructed. One false positive result was
obtained in window 6 (1.7 to 2.0 s), where significant TE was
found in the direction Y?X. Results – Scenario (1), unidirectional coupling. For
scenario (1) of two unidirectionally coupled AR(1)-processes with
a delay dXY~10ms, we used a scanning approach [53] to
reconstruct TE and the corresponding information transfer delay. We scanned assumed delays in the interval u~½1,20 and used
four analysis windows of length 300 ms each, ranging from 0.2 to
1.4 s. For the first two analysis windows, no significant information
transfer was found (0.2 to 0.5 and 0.5 to 0.8 s). For the third and
fourth analysis window we detected significant TE, where we
found a maximum significant TE value at 7 ms for the third
analysis window (0.8 to 1.1 s) and a maximum at 9 ms for the
fourth window (1.1 to 1.4 s). Thus, the proposed implementation
was able to detect information transfer between both processes if
present (later than 1.1 s). During the transition in coupling
strength between 0.8 and 1.1 s TE was detected, but the method Note, that the method’s ability to recover information transfer
from data depends on the strength of the coupling relative to the
amount of data that is available for TE estimation. Evaluation on autoregressive processes To asses the performance of the proposed implementation on
non-abrupt changes in coupling, we simulated various coupling
scenarios for two autoregressive processes X, Y of order 1 (AR(1)-
processes) with variable couplings over time. In each scenario,
couplings were modulated using hyperbolic functions to realize a
smooth transition between uncoupled and coupled regimes. The
AR(1)-processes were simulated according to the equations We chose the arguments of the hyperbolic function such that
the function’s slope led to a smooth increase in the coupling over
an epoch of approximately 200 ms around the inflection points at
1 and 2 s respectively (Figure 7, panels A–D). For each scenario,
we simulated 50 trials of length 3000 ms with a sampling rate of
1000 Hz. We then estimated time resolved TE for analysis
windows of length Dt~300ms. Again, we mixed temporal and
ensemble pooling according to eq. 10. For the scenario with
unidirectional coupling (1) we used four analysis windows to cover
the change in coupling (from 0.2 to 0.5 s, 0.5 to 0.8 s, 0.8 to 1.1 s,
and 1.1 to 1.4 s, see Figure 7, panel E), for the two-step increase ð12Þ x(t)~aXx(t{1)zcYX(t)y(t{dYX)zgX(t),
ð12Þ PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 11 July 2014 | Volume 9 | Issue 7 | e102833 Non-Stationary Transfer Entropy PLOS ONE | www.plosone.org
12
July 2014 | Volume 9 | Issue 7 | e102833 PLOS ONE | www.plosone.org
12
July 2014 | Volume 9 | Issue 7 | e102833 July 2014 | Volume 9 | Issue 7 | e102833 12 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org Non-Stationary Transfer Entropy Figure 7. Transfer entropy reconstruction from coupled autoregressive processes. We simulated two dynamically coupled autoregressive
processes (A) with coupling delays dXY~10ms and dYX~20ms, and coupling scenarios: (B) unidirectional coupling X?Y (blue line) with onset
around 1 s, coupling Y?X set to 0 (red line); (C) unidirectional coupling X?Y (blue line) with onset around 1 s and an increase in coupling
strength at around 2 s, coupling Y?X set to 0 (red line); (D) bidirectional coupling X?Y (blue line) with onset around 1 s and Y?X (red line) with
onset around 2 s. (E-G) Time-resolved transfer entropy (TE) for both directions of interaction, blue and red lines indicate raw TE values for X?Y and
Y?X respectively. Dashed lines denote significance thresholds at 0.01% (corrected for multiple comparisons over signal combinations). Evaluation on autoregressive processes Shaded
areas (red and blue) indicate the maximum absolute TE values for significant information transfer (indicated by asterisks in red and blue). (E) TE values
for unidirectional coupling; (F) unidirectional coupling with a two-step increase in coupling strength; (G) bidirectional coupling. doi:10.1371/journal.pone.0102833.g007 Figure 7. Transfer entropy reconstruction from coupled autoregressive processes. We simulated two dynamically coupled autoregressive
processes (A) with coupling delays dXY~10ms and dYX~20ms, and coupling scenarios: (B) unidirectional coupling X?Y (blue line) with onset
around 1 s, coupling Y?X set to 0 (red line); (C) unidirectional coupling X?Y (blue line) with onset around 1 s and an increase in coupling
strength at around 2 s, coupling Y?X set to 0 (red line); (D) bidirectional coupling X?Y (blue line) with onset around 1 s and Y?X (red line) with
onset around 2 s. (E-G) Time-resolved transfer entropy (TE) for both directions of interaction, blue and red lines indicate raw TE values for X?Y and
Y?X respectively. Dashed lines denote significance thresholds at 0.01% (corrected for multiple comparisons over signal combinations). Shaded
areas (red and blue) indicate the maximum absolute TE values for significant information transfer (indicated by asterisks in red and blue). (E) TE values
for unidirectional coupling; (F) unidirectional coupling with a two-step increase in coupling strength; (G) bidirectional coupling. doi:10.1371/journal.pone.0102833.g007 (2) and bidirectional (3) scenarios, we used eight analysis windows
each (from 0.2 to 0.5 s, 0.5 to 0.8 s, 0.8 to 1.1 s, 1.1 to 1.4 s, 1.4 to
1.7 s, 1.7 to 2.0 s, 2.0 to 2.3 s, and 2.3 to 2.6 s, see Figure 7,
panels F and G). As for the Lorenz systems, 500 surrogate data sets
were used for the statistical testing in each analysis. Surrogate data
were created by blockwise (i.e. repetitionwise) permutation of data
points in the target time series. The value k for the nearest
neighbor search was set to 4 for all analyses (TRENTOOL
default, see also [87]). (2) and bidirectional (3) scenarios, we used eight analysis windows
each (from 0.2 to 0.5 s, 0.5 to 0.8 s, 0.8 to 1.1 s, 1.1 to 1.4 s, 1.4 to
1.7 s, 1.7 to 2.0 s, 2.0 to 2.3 s, and 2.3 to 2.6 s, see Figure 7,
panels F and G). As for the Lorenz systems, 500 surrogate data sets
were used for the statistical testing in each analysis. Surrogate data
were created by blockwise (i.e. Evaluation on autoregressive processes This is
observable in the reconstructed TE in the third analysis window
for scenario (1) and (2): in scenario (2) no TE is detected, whereas
in scenario (1) weak information transfer is already reconstructed
for the third window. Note, that in scenario (2) the simulated
coupling between 1 and 2 s is much weaker than the coupling in Figure 8. Robustness of transfer entropy estimation with respect to limited amounts of data. Estimated transfer entropy (TE) values
TEX?Y for estimations using varying numbers of data points (color coded) as a function of u. Data was sampled from two Lorenz systems X and Y
with coupling X?Y. The simulated information transfer delay dXY~45ms is indicated by a vertical dotted line. Sampled data was embedded and
varying numbers of embedded data points (500, 2000, 5000, 10000, 30000) were used for TE estimation. For each estimation, the maximum TEX?Y
values for all values of u are indicated by solid dots. Dashed lines indicate significance thresholds (pv0:05). doi:10.1371/journal.pone.0102833.g008 Figure 8. Robustness of transfer entropy estimation with respect to limited amounts of data. Estimated transfer entropy (TE) values
TEX?Y for estimations using varying numbers of data points (color coded) as a function of u. Data was sampled from two Lorenz systems X and Y
with coupling X?Y. The simulated information transfer delay dXY~45ms is indicated by a vertical dotted line. Sampled data was embedded and
varying numbers of embedded data points (500, 2000, 5000, 10000, 30000) were used for TE estimation. For each estimation, the maximum TEX?Y
values for all values of u are indicated by solid dots. Dashed lines indicate significance thresholds (pv0:05). doi:10.1371/journal.pone.0102833.g008 July 2014 | Volume 9 | Issue 7 | e102833 PLOS ONE | www.plosone.org 13 13 Non-Stationary Transfer Entropy igure 9. Transfer entropy reconstruction from electrophysiological data. Time resolved reconstruction of transfer entropy (TE)
magnetoencephalographic (MEG) source data, recorded during a face recognition task. (A) Face stimulus [88]. (B) Cortical sources after beamform
f MEG data (L, left; R, right: L orbitofrontal cortex (OFC); R middle frontal gyrus (MiFG); L inferior frontal gyrus (IFG left); R inferior frontal gyrus Figure 9. Transfer entropy reconstruction from electrophysiological data. Time resolved reconstruction of transfer entropy (TE) from
magnetoencephalographic (MEG) source data, recorded during a face recognition task. (A) Face stimulus [88]. 14
July 2014 | Volume 9 | Issue 7 | e102833 Evaluation of the robustness of ensemble-based
TE-estimation To investigate differences in source activation in the face and
non-face condition, we used a frequency domain beamformer [90]
at frequencies of interest that had been identified at the sensor
level (80 Hz with a spectral smoothing of 20 Hz). We computed
the frequency domain beamformer filters for combined data
epochs (‘‘common filters’’) consisting of activation (multiple
windows, duration, 200 ms; onsets at every 50 ms from 0 to
450 ms) and baseline data (2350 to 2150 ms) for each analysis
interval. To compensate for the short duration of the data
windows, we used a regularization of l~5% [91]. A sufficiently accurate reconstruction was reached for 10000
and 30000 data points (Figure 8). For 5000 data points estimation
was off by approximately 7% (the reconstructed information
transfer delay was 48 ms), less data entering the estimation led to a
further decline in accuracy of the recovered information transfer
delay (here, reconstructed delays were 50 ms and 54 ms for 2000
and 500 data points respectively). Evaluation on autoregressive processes This
resulted in smaller and non-significant absolute TE values and in
reconstructed information transfer delays that were less precise. the unidirectional scenario (1) (Figure 7, panels C and B). This
resulted in smaller and non-significant absolute TE values and in
reconstructed information transfer delays that were less precise. magnetoencephalographic (MEG) recordings from a perceptual
closure experiment described in [86]. Subjects. MEG data were obtained from 15 healthy subjects
(11 females; mean + SD age, 25.4 + 5.6 years), recruited from
the local community. Results
–
Scenario
(3),
bidirectional
coupling. For
scenario (3), we used the scanning approach for TE reconstruction,
using an interval of assumed delays u~½1,30, where the true delay
was simulated at dXY~10ms and dYX~20ms. No TE in either
direction was detected prior to the first coupling onset around 1 s. TE for the first direction X?Y was detected after coupling onset
around 1 s for analysis windows 4, 5, 6, 7, and 8. Reconstructed
information transfer delays were 8 and 2 ms for analysis windows
4 and 5. For each of the following analysis windows 6 to 8 the
correct delay of 10 ms was reconstructed. Task. Subjects were presented with a randomized sequence
of degraded black and white picture of human faces [88] (Figure 9,
panel A) and scrambled stimuli, where black and white patches
were randomly rearranged to minimize the likelihood of detecting
a face. Subjects had to indicate the detection of a face or no-face
by a button press. Each stimulus was presented for 200 ms, with a
random inter-repetition interval (IRI) of 3500 to 4500 ms (9, panel
E). For further analysis we used repetitions with correctly
identified face conditions only. TE for the second direction Y?X was detected after coupling
onset around 2 s for analysis windows 7 and 8, where also the
correct coupling of 20 ms was reconstructed. Thus, the proposed
implementation was able to reconstruct information transfer in
bidirectionally coupled systems. MEG and MRI data acquisition. MEG data were recorded
using a 275-channel whole-head system (Omega 2005, VSM
MedTech Ltd., BC, Canada) at a rate of 600 Hz in a synthetic
third order axial gradiometer configuration. The data were filtered
with 4th order Butterworth filters with 0.5 Hz high-pass and
150 Hz low-pass. Behavioral responses were recorded using a fiber
optic response pad (Lumitouch, Photon Control Inc., Burnaby,
BC, Canada). Evaluation of the robustness of ensemble-based
TE-estimation We tested the robustness of the ensemble method for cases
where the amount of data available for TE estimation was severely
limited. We created two coupled Lorenz systems X, Y from which
we sampled a maximum number of 300 repetitions of 300 ms each
at 1000 Hz, using a coupling delay of dXY~45ms (see equation
11). We
embedded
the
resulting
data
with
their
optimal
embedding parameters for different values of the assumed delay
u (30 to 60 ms, step size of 1 ms, also see equation 4). From the
embedded data, we used subsets of data points with varying size M
(M~f500,2000,5000,10000,30000g) to estimate TE according to
equation 10 (we always used the first M consecutive data points for
TE estimation). For each u and number of data points M, we
created surrogate data to test the estimated TE value for statistical
significance. Furthermore, we reconstructed the corresponding
information transfer delay for each M by finding the maximum
TE value over all values for u. A reconstructed TE value was
considered a robust estimation of the simulated coupling if the
reconstructed delay value was able to recover the simulated
information transfer delay of 45ms with an error of +5%, i.e. 45+1:125ms. Structural magnetic resonance images (MRI) were obtained
with a 3 T Siemens Allegra, using 3D magnetization-prepared
rapid-acquisition gradient echo sequence. Anatomical images were
used
to
create
individual
head
models
for
MEG
source
reconstruction. Data analysis. MEG data were analyzed using the open
source MATLAB toolboxes FieldTrip (version 2008-12-08; [89]),
SPM2 (http://www.fil.ion.ucl.ac.uk/spm/), and TRENTOOL
[56]. We will briefly describe the applied analysis here, for a more
in depth treatment refer to [86]. Data analysis. MEG data were analyzed using the open
source MATLAB toolboxes FieldTrip (version 2008-12-08; [89]),
SPM2 (http://www.fil.ion.ucl.ac.uk/spm/), and TRENTOOL
[56]. We will briefly describe the applied analysis here, for a more
in depth treatment refer to [86]. For data preprocessing, data epochs (repetitions) were defined
from the continuously recorded MEG signals from 21000 to
1000 ms with respect to the onset of the visual stimulus. Only data
repetitions with correct responses were considered for analysis. Data epochs contaminated by eye blinks, muscle activity, or jump
artifacts in the sensors were discarded. Data epochs were baseline
corrected by subtracting the mean amplitude during an epoch
ranging from 2500 to 2100 ms before stimulus onset. Evaluation on autoregressive processes (B) Cortical sources after beamforming
of MEG data (L, left; R, right: L orbitofrontal cortex (OFC); R middle frontal gyrus (MiFG); L inferior frontal gyrus (IFG left); R inferior frontal gyrus (IFG PLOS ONE | www.plosone.org
14
July 2014 | Volume 9 | Issue 7 | e102833 PLOS ONE | www.plosone.org 14 Non-Stationary Transfer Entropy right); L anterior inferotemporal cortex (aTL left); L cingulate gyrus (cing); R premotor cortex (premotor); R superior temporal gyrus (STG); R anterior
inferotemporal cortex (aTL right); L fusiform gyrus (FFA); L angular/supramarginal gyrus (SMG); R superior parietal lobule/precuneus (SPL); L caudal
ITG/LOC (cITG); R primary visual cortex (V1)). (C) Reconstructed TE in three single subjects (red box) in three time windows (02150 ms, 1502300 ms,
3002450 ms). Each link (red arrows) corresponds to significant TE on single subject level (corrected for multiple comparisons). (D) Thresholded TE
links over 15 subjects (blue box) in three time windows (02150 ms, 1502300 ms, 3002450 ms). Each link (black arrows) corresponds to significant
TE in eight and more individual subjects (pvv0:0001, after correction for multiple comparisons). Blue arrows indicate differences between time
windows, i.e. links that occur for the first time in the respective window. (E) Experimental design: stimulus was presented for 200 ms (gray shading),
during the inter stimulus interval (ISI, 1800 ms) a fixation cross was displayed. doi:10.1371/journal.pone.0102833.g009 right); L anterior inferotemporal cortex (aTL left); L cingulate gyrus (cing); R premotor cortex (premotor); R superior temporal gyrus (STG); R anterior
inferotemporal cortex (aTL right); L fusiform gyrus (FFA); L angular/supramarginal gyrus (SMG); R superior parietal lobule/precuneus (SPL); L caudal
ITG/LOC (cITG); R primary visual cortex (V1)). (C) Reconstructed TE in three single subjects (red box) in three time windows (02150 ms, 1502300 ms,
3002450 ms). Each link (red arrows) corresponds to significant TE on single subject level (corrected for multiple comparisons). (D) Thresholded TE
links over 15 subjects (blue box) in three time windows (02150 ms, 1502300 ms, 3002450 ms). Each link (black arrows) corresponds to significant
TE in eight and more individual subjects (pvv0:0001, after correction for multiple comparisons). Blue arrows indicate differences between time
windows, i.e. links that occur for the first time in the respective window. (E) Experimental design: stimulus was presented for 200 ms (gray shading),
during the inter stimulus interval (ISI, 1800 ms) a fixation cross was displayed. doi:10.1371/journal.pone.0102833.g009 the unidirectional scenario (1) (Figure 7, panels C and B). Evaluation on neural time series from
magnetoencephalography To find significant source activations in the face versus non-face
condition, we first conducted a within-subject t-test for activation
versus baseline effects. Next, the t-values of this test statistic were
subjected to a second-level randomization test at the group level to
obtain effects of differences between face and no-face conditions; a To demonstrate the proposed method’s suitability for time-
resolved reconstruction of information transfer and the corre-
sponding
delays
from
biological
time
series,
we
analyzed PLOS ONE | www.plosone.org July 2014 | Volume 9 | Issue 7 | e102833 July 2014 | Volume 9 | Issue 7 | e102833 15 Non-Stationary Transfer Entropy Table 2. Reconstructed information transfer delays for magnetoencephalographic data. Source
Target
0–150 ms
150–300 ms
300–450 ms
SPL
IFG left
5.00
-
5.50
SPL
cITG
-
-
5.00
cITG
IFG left
5.00
5.00
5.00
cITG
FFA
5.00
5.00
5.00
cITG
SMG
-
5.00
-
STG
aTL right
5.00
5.00
5.00
STG
Premotor
-
-
5.83
STG
FFA
-
5.50
-
aTL right
STG
5.00
5.00
5.00
aTL right
Premotor
5.00
5.60
-
SMG
SPL
5.00
-
-
SMG
V1
5.00
-
-
SMG
IFG left
-
5.20
-
SMG
FFA
-
5.22
5.20
OFC
IFG left
5.18
5.00
5.00
OFC
FFA
-
5.00
5.20
MiFG
IFG right
5.00
5.00
5.00
MiFG
Premotor
5.00
5.00
5.00
IFG right
MiFG
5.00
5.00
5.00
IFG right
Premotor
5.00
5.00
5.00
IFG left
SPL
5.00
5.00
-
IFG left
cITG
5.00
-
5.00
IFG left
SMG
5.00
5.40
5.00
IFG left
OFC
5.00
5.00
5.00
IFG left
FFA
5.00
5.00
5.00
FFA
cITG
5.00
5.00
5.22
FFA
OFC
5.00
-
-
FFA
IFG left
5.00
-
-
FFA
SMG
-
5.00
-
V1
cITG
5.25
-
5.25
V1
SMG
-
-
5.00
Premotor
STG
5.00
-
-
Premotor
MiFG
5.00
5.00
5.00
Premotor
IFG right
5.00
5.00
5.00
Cing
STG
5.25
-
-
Cing
FFA
-
-
5.67
Mean over reconstructed interaction delays for significant information transfers in three analysis windows. Information transfer delays were investigated in steps of
2 ms, from 5–17 ms. Fractional numbers arise from averaging over subjects. doi:10.1371/journal.pone.0102833.t002 Mean over reconstructed interaction delays for significant information transfers in three analysis windows. Information transfer delays were investigated in steps of
2 ms, from 5–17 ms. Fractional numbers arise from averaging over subjects. doi:10.1371/journal.pone.0102833.t002 Mean over reconstructed interaction delays for significant information transfers in three analysis windows. Evaluation on neural time series from
magnetoencephalography Information transfer delays were investigated in steps of
2 ms, from 5–17 ms. Fractional numbers arise from averaging over subjects. doi:10.1371/journal.pone.0102833.t002 furthermore reconstructed information transfer delays for signif-
icant information transfer by scanning over a range of assumed
delays from 5 to 17 ms (resolution 2 ms), following the approach
in [53]. We corrected the resulting information transfer pattern for
cascade effects as well as common drive effects using a graph-based
post-hoc correction proposed in [54]. p-value v0.01 was considered significant. We identified 14
sources with differential spectral power between both conditions
in the frequency band of interest in occipital, parietal, temporal,
and frontal cortices (see Figure 9, panel B, and [86] for exact
anatomical locations). We then reconstructed source time courses
for TE analysis, this time using a broadband beamformer with a
bandwidth of 10 to 150 Hz. Results. Time-resolved GPU-based TE analysis revealed
significant information transfer at the group-level (pvv0:001
corrected for multiple comparison; binomial test under the null
hypothesis of the number of occurrences k of a link being
B(kDp0,n)-distributed, where p0~0:05 and n~15), that changed
over time (Figure 9, panel D and table 2 for reconstructed Results. Time-resolved GPU-based TE analysis revealed
significant information transfer at the group-level (pvv0:001
corrected for multiple comparison; binomial test under the null
hypothesis of the number of occurrences k of a link being
B(kDp0,n)-distributed, where p0~0:05 and n~15), that changed
over time (Figure 9, panel D and table 2 for reconstructed We estimated TE between beamformer source time courses
using our ensemble method with a mixed pooling of embedded
time points over repetitions r and time windows t’ (eq. 10). We
analyzed three non-overlapping time windows Dt of 150 ms each
(0–150 ms, 150–300 ms, 300–450 ms, Figure 9, panel C). We July 2014 | Volume 9 | Issue 7 | e102833 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 16 Non-Stationary Transfer Entropy information transfer delays). Our preliminary findings of informa-
tion transfer are in line with hypothesis formulated in [92], [93]
and [86], and the time-dependent changes show the our method’s
sensitivity to the dynamics of information processing during
experimental stimulation, in line with the simulation results above. Available data and choice of window size. As available
data is often limited in neuroscience and other real-world
applications, the user has to make sure that enough data enters
the analysis, such that a reliable estimation of TE is possible. Efficient transfer entropy estimation from an ensemble of
time series Note, that the embedding window should not be confused with
the analysis window. The analysis window strictly describes the
data points, for which neighbor statistics enter TE estimation –
where neighbor counts may be averaged over an epoch Dt or may
come from a single point in time t only. The embedding window
however, describes the data points that enter the embedding of a
single point in time. Thus, the temporal resolution of TE analysis
may still be in single time steps t (i.e. only one time point entering
the analysis), even though the embedding window spans several
points in time that contain the history for this single point. Efficient transfer entropy estimation from an ensemble of
time series We presented an efficient implementation of the ensemble
method for TE estimation proposed by [55]. As laid out in the
introduction, estimating TE from an ensemble of data allows to
analyze information transfer between time series that are non-
stationary and enables the estimation of TE in a time-resolved
fashion. This is especially relevant to neuroscientific experiments,
where rapidly changing (and thus non-stationary) neural activity is
believed to reflect neural information processing. However, up
until now the ensemble method has remained out of reach for
application in neuroscience because of its computational cost. Only with using parallelization on a GPU, as presented here, the
ensemble method becomes a viable tool for the analysis of neural
data. Thus, our approach makes it possible for the first time to
efficiently analyze information transfer between neural time series
on short time scales. This allows us to handle the non-stationarity
of underlying processes and makes a time- resolved estimation of
TE possible. To facilitate the use of the ensemble method it has
been implemented as part of the open source toolbox TREN-
TOOL (version 3.0). Thus, the choice of an appropriate analysis window is crucial to
guarantee reliable TE estimation, while still resolving the temporal
dynamics under investigation. A further data limiting factor is the
need for an appropriate embedding of the scalar time series. To
embed the time series at a given point t, enough history for this
sample (embedding dimension times the embedding delay in
sample points) has to be recorded. We call this epoch the
embedding window. The need for an appropriate embedding thus
constitutes another constraint for the data necessary for TE
estimation. Thus, the choice of an optimal embedding dimension
(e.g. through the use of Ragwitz’ criterion [71]) is crucial as the use
of larger than optimal embedding dimensions wastes available
data and may lead to a weaker detection rate in noisy data [56]. Even though we will focus on neural data when discussing
applications of the ensemble method for TE estimation below, this
approach is well suited for applications in other fields. For
example, TE as defined in [14] has been applied in physiology
[42–44], climatology [94,95], financial time series analysis [45,96],
and in the theory of cellular automata [48]. Large datasets from
these and other fields may now be easily analyzed with the
presented approach and its implementation in TRENTOOL. Evaluation on neural time series from
magnetoencephalography In the
proposed implementation of the ensemble method for TE
estimation the amount of data entering the estimation directly
depends on the size of the chosen analysis window and the amount
of available repetitions of the process being analyzed. Further-
more, the choice of the embedding parameters lead to varying
numbers of embedded data that can be obtained from scalar time
series. When estimating TE from neural data, we therefore
recommend to control the amount of data in one analysis window
that is available after embedding and to design experiments
accordingly. For example, the presented MEG data set was
sampled at 600 Hz, with 137 repetitions of the stimulus on
average, which - after embedding - led to 8800 data points per
analysis window of 150 ms. In comparison, for simulated data TE
was reconstructed correctly for 10000 data points and more. Thus,
in our example MEG data set, shorter analysis windows would not
have been advisable because of an insufficient amount of data per
analysis window for reliable TE estimation. If shorter analysis
windows are necessary, they will have to be counterbalanced by a
higher number of experimental repetitions. Notes on the practical application of the ensemble
method for TE estimation Applicability to simulated and real world experimental
data. To validate the proposed implementation of the ensemble
method, we applied it to simulated data as well as MEG
recordings. For simulated data, information transfer could reliably
be reconstructed despite the non-stationarity in the underlying
generating processes. For MEG data the obtained speed-up was
large enough to analyze these data in practical time. Information
transfer reconstructed in a time-resolved fashion from the MEG
source data was in line with findings by [86,92,93], as discussed
below. July 2014 | Volume 9 | Issue 7 | e102833 Repeatability of neuronal processes Within the category of event-related activity,
contributions can be further distinguished into phase-locked and
non phase-locked contributions (the latter is commonly called
induced activity). Phase-locked activity has a fixed polarity and
latency with respect to the stimulus and – on averaging over
repetitions – contributes to an event-related potential or field
(ERP/F). Phase-locked activity is further distinguished into two
types of contributions, that are discussed as mechanisms in the
ERP/F-generation (e.g. [97–99]): (1) additive evoked contributions,
i.e. neural activity that is in addition to ongoing activity and
represents the stereotypical response of a neural population to the
presented stimulus in each repetition [100–102]; (2) phase- reset
contributions, i.e. the phase of ongoing activity is reset by the
stimulus, such that phase-aligned signals no longer cancel each
other out on averaging over repetitions [103–106]. In contrast to
these two subtypes of phase-locked activity, induced activity is
event-related activity that is not phase-locked to the stimulus, such
that latency and polarity vary randomly over repetitions and
induced activity averages out over repetitions. LTE(X?Y,t,d)~log p(ytDyt{1,xt{d)
p(ytDyt{1)
ð17Þ ð17Þ LTE relates to TE in the same way Shannon information relates
to Shannon entropy – by means of taking an expected value under
the common PDF p(yt,yt{1,xt{d) of the collection of random
variables fXtg,fYtg that form the processes X, Y, which exchange
information. Stationarity here guarantees that all the random
variables X1,X2, . . . (Y1,Y2, . . .) have a common PDF (as the PDF
is not allowed to change over time): ð18Þ TE(X?Y,t,d)~vLTE(X?Y,t,d)wp(yt,yt{1,xt{d)
ð18Þ In contrast, the approach presented here does not assume that
the random processes X, Y are stationary, but that either
replications if the process can be obtained, or that the process is
cyclostationary. Under these constraints a local PDF can be
obtained. The events drawn from this PDF may then be analyzed
in terms of their average information transfer, i.e. using TE as
presented here, or by inspecting them individually, computing
LTE for each event. In this sense, the approach suggested here is
aimed at extracting the proper local PDFs, while local information
dynamics comes into play once these proper PDFs have been
obtained. We are certain that both approaches can be fruitfully
combined in future studies. We therefore have to consider four types of contributions to
neural recordings: (1) additive evoked contributions, (2) phase-reset
contributions, (3) induced contributions and (4) spontaneous
ongoing
contributions,
the
last
being
stimulus-independent. Repeatability of neuronal processes When applying the ensemble method to estimate TE from
neural recordings, we treat experimental repetitions as multiple
realizations of the neural processes under investigation. In doing
so, we assume stationarity of these processes over repetitions. We
claim that in most cases this assumption of stationarity is justified
for processes concerned with the processing of experimental
stimuli and that the assumption also holds for stimulus-indepen-
dent processes that contribute to neural recordings. We will first
present the different contributions to neural recordings and
subsequently discuss their individual statistical properties, i.e. their
stationarity over repetitions. Note, that the term stationarity refers
to the stability of the probability distribution underlying the
observed realizations of contributions over repetitions and does
not require individual realizations to be identical; i.e. stationarity
does not preclude a variability in observed realizations, but rather Note, that even though our proposed implementation of the
ensemble method reduces analysis times by a significant amount,
the estimation of TE from neural time series is still time consuming
relative to other measures of connectivity. For the example MEG
data set presented in this paper, TE estimation for one subject and
one analysis window took 93 hours on average (when scanning
over seven values for the assumed information transfer delay u and
reconstructing TE for all possible combinations of 14 sources). Thus, for 15 subjects with three analysis windows each, the whole
analysis would take approximately six months when carried out in
a serial fashion on one computer equipped with a modern GPU
(e.g. NVIDIA GTX Titan). This time may however be reduced by
parallelizing the analysis over subjects and analysis windows on
multiple GPUs, as it was done for this study. July 2014 | Volume 9 | Issue 7 | e102833 PLOS ONE | www.plosone.org 17 July 2014 | Volume 9 | Issue 7 | e102833 Non-Stationary Transfer Entropy Non-Stationary Transfer Entropy implies some variance in observed realizations, that is reflective of
the variance in the underlying probability distribution. The local information dynamics approach to information
transfer computes information transfer for stationary random
processes from their joint and marginal PDFs for each process
step, thereby fully localizing information transfer in time. The
quantity proposed for this purpose is the LTE [15]: Contributions to neural recordings may either be stimulus-
related (event-related activity) or stimulus-independent (spontane-
ous ongoing activity). Relation of the ensemble method to other measures of
connectivity for non-stationary data Linear Granger causality (GC) is – as has been shown recently
by [26] – equivalent to TE for variables with a jointly Gaussian
distribution. Thus, for data that exhibit such a distribution,
information transfer may be analyzed more easily within the GC
framework. Similar to the ensemble method for TE estimation,
extensions to GC estimation have been proposed that deal with
non-stationary data by fitting time-variant parameters. For
example, Mo¨ller and colleagues presented an approach that fitted
multivariate autoregressive models (MVAR) with time-dependent
parameters to an ensemble of EEG signals [108]. Similar
measures, that fit time-dependent parameters in autoregressive
models to data ensembles, were used by [109] and [110]. A
different approach to dealing with non-stationarity was taken by
Leistritz and colleagues [111]. These authors proposed to use self-
exciting threshold autoregressive (SETAR) models to model neural
time series within a GC framework. SETAR models extend
traditional AR models by introducing state-dependent model
parameters and allow for the modeling of transient components in
the signal. To sum up the statistical properties of different contributions to
neural data and their relevance for using an ensemble approach to
TE estimation, we conclude that all contributions to neural
recordings can be considered stationary over repetitions by
default. Non-stationarity over repetitions will only be a problem
in paradigms that introduce (slow) drifts or trends in the recorded
signal, for example by facilitating learning during the experiment. Testing for drifts may be done by comparing mean and variance
in a split-half analysis. Repeatability of neuronal processes Stationarity can be assumed for all these contributions if no
learning effects occur during the experiment. Learning effects may
lead to slow drifts, i.e. changing mean and variances, in the
recorded signal. Such learning effects may easily be tested for by
comparing the first and second half of recorded repetitions with
respect to equal variances and means. If variances and means are
equal, learning effects can most likely be excluded. Empirically,
the stationarity assumption, specifically of phase-locked contribu-
tions,
can
also
be
verified
using
a
modified
independent
component analysis recently proposed in [107]. July 2014 | Volume 9 | Issue 7 | e102833 Application to MEG source activity in a perceptual
closure task Application of the ensemble-based TE estimation to MEG
source activities revealed a time varying pattern of information
transfers, as expected in the nonstationary setting of the visual task. While a full discussion of the revealed information transfer pattern
is beyond the scope of this study, we point out individual
connections transferring information that underline the validity of
our results. Notable connections in the first time window transfer
information from the early visual cortices (V1) to the orbitofrontal
cortex (OFC) – in line with earlier findings by Bar an colleagues
[92], that suggest a role of the OFC in early visual scene
segmentation and gist perception. Another brain area receiving
information from early visual cortex is the caudal inferior temporal
gyrus (cITG)[115], an area responsible for the processing of shape-
from-shading information, which is thought to be essential for
perception of Mooney stimuli as they were used here. Both of these
areas, OFC and cITG at later stages of processing exchange
information with the fusiform face area, which is essential for the
processing of faces [116–118], and thereby expected to receive
information from other areas in this task. Indeed, FFA seems to be
an essential hub in the task-related network investigated in this
study and receives increasing amounts of incoming information
transfer as the task progresses in time. This is in line with the fact
that the most pronounced task-related differences in FFA activity
were found at latencies w 200 ms previously [86]. Conclusion and further directions We presented an implementation of the ensemble method for
TE presented in [55], that uses a GPU to handle computationally
most demanding aspects of the analysis. We chose an implemen-
tation that is flexible enough to scale well with different
experimental designs as well as with future hardware develop-
ments. Our implementation was able to successfully reconstruct
information transfer in simulated and neural data in a time-
resolved fashion. Nearest neighbor searches using a GPU
exhibited substantially reduced execution times. The implemen-
tation has been made available as part of the open source
MATLAB toolbox TRENTOOL [56] for the use with CUDA-
enabled GPU devices. Relation of the ensemble method to local information
dynamics We will now discuss the relation of the ensemble approach
suggested here to the local transfer entropy (LTE) approach of
Lizier [4,15]. This may be useful as both approaches at first glance
seem to have a similar goal, i.e. assessing information transfer
more locally in time. As we will show, the approaches differ in
what quantities they localize. From this difference it also follows
that they can (and should be) combined when necessary. The presented methods for the estimation of time-variant linear
GC may yield a computationally less expensive approach to the
estimation of information transfer from an ensemble of data. However, linear GC is equivalent to TE regarding the full
recovery of information transfer for data with a jointly Gaussian
distribution only. For non-Gaussian data, linear GC may fail to
capture higher order interactions. As neural data are most likely
non-Gaussian, the application of TE may have an advantage for In detail, the ensemble approach used here tries to exploit
cyclostationarity or repeatability of random processes to obtain
multiple PDFs from the different (quasi-) stationary parts of the
repeated process cycle, or a PDF for each step in time from
replications of a process, respectively. In contrast, local informa-
tion dynamics localizes information transfer in time (and space)
given the PDF of a stationary process. PLOS ONE | www.plosone.org July 2014 | Volume 9 | Issue 7 | e102833 July 2014 | Volume 9 | Issue 7 | e102833 18 Non-Stationary Transfer Entropy [113] use ensemble pooling of delay-embedded time series in
combination with nearest neighbor statistics as a general approach
to the estimation of arbitrary non-linear dependency measures. However, the practical application of ensemble pooling and
nearest neighbor statistics together with the necessary generation
of a sufficient amount of surrogate data sets (typically w 1000 in
neuroscience applications where correction for multiple compar-
isons is necessary) was always hindered by its high computational
cost. Only with the presentation of a GPU algorithm for nearest
neighbor searches, we provide an implementation of the ensemble
method that allows its practical application. Note that even though
we use ensemble pooling and GPU search algorithms to
specifically estimate TE, the presented implementation may easily
be adapted to other dependency measures that are calculated from
(conditional) mutual informations estimated from nearest neighbor
statistics. the analysis of information transfer in this type of data. Relation of the ensemble method to local information
dynamics The non-
Gaussian nature of neural data can for example be seen, when
comparing brain electrical source signals from physical inverse
methods to time courses of corresponding ICA components [112]. Here, ICA components and extracted brain signals closely match. Given that ICA components are as non-Gaussian as possible (by
definition of the ICA), we can infer that brain signals are very
likely non-Gaussian. We also note that a nonstationary measure of coupling between
dynamic systems building on repetitions of time series and next-
neighbor statistics was suggested by Andrzejak and colleagues
[113]. The key difference of their approach to the ensemble
method suggested here is that the previous states of the target time
series are not taken into account explicitly in their method. Hence,
their measure is not (and was not intended to be) a measure of
information transfer (see [53] for details why a measure of
information transfer needs to include the past state of target time
series, and [48] for the difference between measures of (causal)
coupling and information transfer). In addition, their methods
explicitly tries to determine the direction of coupling between to
systems. This implies that there should be a dominant direction of
coupling in order to obtain meaningful results. Transfer entropy,
in contrast, easily separates and quantifies both directions of
information transfer related to bidirectional coupling, under some
mild conditions related to entropy production in each of the two
coupled systems [53]. Relation of the ensemble method to the direct method
for the calculation of mutual information of Strong and
colleagues The ensemble method proposed here shares the use of
replications (or trials) with the so called ‘direct method’ of Strong
and colleagues [114]. The authors introduced this method to
calculate mutual information between a controlled stimulus set
and neural responses. Similarities also exist in the sense that the
surrogate data method for statistical evaluation used in our
ensemble method builds on trial-to-trial variability, as does
Strong’s
method
(by
looking
at
intrinsic
variability
versus
variability driven by stimulus changes). However, the two methods differ conceptually on two accounts:
First, the quantity estimated is different – symmetric mutual
information in Strong’s method compared to inherently asym-
metric conditional mutual information in the case of TE. Second,
the method of Strong and colleagues requires a direct intervention
in the source of information (i.e. the stimuli) to work, whereas TE
in general is independent of such interventions. This has far
reaching consequences for the interpretation of the two measures:
The intervention inherent in Strong’s method places it somewhat
closer to causal measures such as Ay and Polani’s causal
information flow [47], whereas intervention-free TE has a clear
interpretation
as
the
information
transfered
in
relation
to
distributed computation [48]. As a consequence, TE maybe easily
applied to quantify neural information transfer from one neuron
or brain area to another even under constant stimulus conditions. In contrast, using Strong’s method inside a neural system in this
way would require precisely setting of the activity of the source
neuron or brain area, something that may often be difficult to do. Our data also clearly show a great variability in information
transfer pattern across subjects, which we relate to the limited
amount of data per subject, rather than to true variation. Moreover, future investigations will have to show whether more
fine grained temporal segmentation of the neural information
processing in this task is possible and whether it will provide
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effect. Eur Phys J B 73: 605–615. 49. Author Contributions Conceived and designed the experiments: MW RV MMZ PW FDP. Performed the experiments: PW MMZ MW. Analyzed the data: PW
MMZ MW. Contributed reagents/materials/analysis tools: MMZ PW
MW RV FDP. Wrote the paper: PW MMZ RV MW. Conceived and
designed the parallel algorithm: MW MMZ. Implemented the algorithm:
MMZ PW MW FDP. Designed the software used in analysis: MW RV
MMZ PW FDP. Application of the proposed implementation to other
dependency measures The use of ensemble pooling of observations for the estimation
of time-resolved dependency measures has been proposed in a
variety of frameworks. For example, Andrzejak and colleagues PLOS ONE | www.plosone.org July 2014 | Volume 9 | Issue 7 | e102833 19 Non-Stationary Transfer Entropy Non-Stationary Transfer Entropy We conclude that the ensemble method in its presented
implementation is a suitable tool for the analysis of non-stationary
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Ligneous Periodontitis in a Patient with Type 1 Plasminogen Deficiency: A Case Report and Review of the Literature
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Case Report
Ligneous Periodontitis in a Patient with Type 1 Plasminogen
Deficiency: A Case Report and Review of the Literature Arun Sadasivan
,1 Roshni Ramesh
,2 and Deepu George Mathew
3
1Department of Periodontics, Sree Mookambika Institute of Dental Sciences, Kulashekaram, Tamil Nadu, India
2Department of Periodontics, Government Dental College, Thrissur, Kerala, India
3Department of Oral and Maxillofacial Pathology, Annoor Dental College, Muvattupuzha, Kerala, India Arun Sadasivan
,1 Roshni Ramesh
,2 and Deepu George Mathew
3
1Department of Periodontics, Sree Mookambika Institute of Dental Sciences, Kulashekaram, Tamil Nadu, India
2Department of Periodontics, Government Dental College, Thrissur, Kerala, India
3Department of Oral and Maxillofacial Pathology, Annoor Dental College, Muvattupuzha, Kerala, India Correspondence should be addressed to Arun Sadasivan; sadasivan_arun@hotmail.com Received 23 October 2019; Accepted 3 March 2020; Published 26 March 2020 Academic Editor: Mine Dündar Copyright © 2020 Arun Sadasivan et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background. Ligneous periodontitis or destructive membranous periodontal disease is a rare condition involving gingival tissues,
which is due to plasminogen deficiency and fibrin deposition. Plasminogen deficiency is an ultrarare autosomal recessive
disease. The disease is characterized by gingival enlargement and periodontal tissue destruction that leads to rapid tooth loss
despite treatment attempts. A defect in fibrinolysis and abnormal wound healing are the main pathogenesis of this condition. It
is caused by mutations in PLG, the gene coding for plasminogen, which results in decreased levels and functional activity. Case
Presentation. In this case report, clinical and histopathological findings of a 26-year-old male patient who presented with
generalized membranous gingival enlargement are presented. He was the third child of consanguineous parents and had
multicystic congenital hydrocephalus at birth. Besides the gingival enlargement, he also presented ligneous conjunctivitis since
childhood. The intraoral examination revealed generalized periodontal breakdown. Radiographs showed alveolar bone loss
present in every quadrant. All blood investigations were normal except for plasminogen deficiency. A biopsy sample was excised
from affected gingiva and a series of histopathological evaluation was performed. Based on clinical and histopathological
evidence, a diagnosis of destructive membranous periodontal disease or ligneous periodontitis was made. A clinical exome assay
for the PLG gene was also done. It was confirmed as Type 1 plasminogen deficiency. Conclusion. Ligneous periodontitis has
been rarely reported in India. The reasons could be because of the rarity of the disease or missed diagnosis. Case Report
Ligneous Periodontitis in a Patient with Type 1 Plasminogen
Deficiency: A Case Report and Review of the Literature The need to take a
proper history and perform a proper clinical examination and histopathologic evaluation has to be stressed when diagnosing
and treating gingival enlargements. If a genetic condition is suspected, genetic screening is also needed. All these will help the
clinician in correctly diagnosing the disease and formulating a proper treatment plan for managing the condition. Hindawi
Case Reports in Dentistry
Volume 2020, Article ID 5680535, 8 pages
https://doi.org/10.1155/2020/5680535 Hindawi
Case Reports in Dentistry
Volume 2020, Article ID 5680535, 8 pages
https://doi.org/10.1155/2020/5680535 1. Introduction Haemostasis depends on many factors like vasoconstric-
tion, formation of blood coagulation factors, and fibrinoly-
sins,
especially
plasmin. Plasmin
is
derived
from
plasminogen (PLG), which is its proenzyme; it is synthesized
in the liver and circulates in the plasma. PLG plays an impor-
tant role in intravascular and extravascular fibrinolysis and
wound healing. It is converted to plasmin by cleavage of the
Arg561-Val562 peptide bond by either a tissue-type PLG
activator (tPA) or a urokinase-type PLG activator (uPA). Activation of PLG by tPA is the major pathway that leads
to efficient lysis of fibrin clots in the blood stream, whereas
activation of PLG by uPA seems to be mainly responsible
for mediating PLG activation in association with cell surfaces Gingival enlargements are a common feature seen in patients
which present aesthetic, functional, psychological, and peri-
odontal problems. The etiology in most of the cases has an
inflammatory, hormonal, drug-induced, idiopathic, or can-
cerous origin. There are, however, certain rare conditions in
which the etiology is obscure. One such condition is destruc-
tive membranous periodontal disease or ligneous periodonti-
tis (LP). This condition has been attributed to a plasminogen
deficiency. Other possible causes such as autoimmune or
hypersensitivity reactions, trauma, and viral or bacterial
infections have been reported [1, 2]. 2 Case Reports in Dentistry Figure 1: Intraoral examination of a 26-year-old male patient with
PLG deficiency (Type 1) showing membranous gingival enlargement. especially wound healing and tissue remodelling [3]. Plas-
min also acts as a broad spectrum proteolytic factor either
directly by degrading extracellular matrix proteins, e.g.,
laminin, fibronectin, and proteoglycans, or indirectly by
activating latent metalloproteinases. Therefore, it plays a
crucial role in tissue homeostasis, e.g., remodelling, angio-
genesis, and wound healing. Moreover, plasmin has also
been found to play an important role in host defence
against infections [4, 5]. g
Plasminogen deficiency is a rare (1.6 in 1 million individ-
uals) autosomal recessive disease caused by homozygote or
compound-heterozygote mutations of the plasminogen gene
PLG. The PLG gene maps to chromosome 6q26-q27. It spans
about 52.5 kilobases (kb) of DNA and consists of 19 exons
and 18 introns. The PLG cDNA of 2.7 kb encodes a protein
consisting of 791 amino acid residues [6]. 1. Introduction The periodontal evaluation revealed signs of
generalized severe periodontitis, ulceration, and white-
yellow fibrinous pseudomembranes (Figure 1). Probing
showed increased pocket depths ranging from 8 to 10mm
in many teeth, bleeding on probing in over 30% of sites,
and generalized tooth mobility. The panoramic radiograph
showed generalized alveolar bone loss of more than 50%,
especially in the posterior teeth (Figure 2). Recurrent con-
junctivitis was seen as a white membrane in the mucosa of
the upper and lower eye lids (Figure 3). The patient’s growth 2.1. Clinical and Radiographic Examination. The intraoral
examination revealed solid, nodular, and fragile erythema-
tous and hyperplastic gingival enlargements involving the
marginal and attached gingiva in both maxillary and man-
dibular arches. The periodontal evaluation revealed signs of
generalized severe periodontitis, ulceration, and white-
yellow fibrinous pseudomembranes (Figure 1). Probing
showed increased pocket depths ranging from 8 to 10mm
in many teeth, bleeding on probing in over 30% of sites,
and generalized tooth mobility. The panoramic radiograph
showed generalized alveolar bone loss of more than 50%,
especially in the posterior teeth (Figure 2). Recurrent con-
junctivitis was seen as a white membrane in the mucosa of
the upper and lower eye lids (Figure 3). The patient’s growth 1. Introduction There are two types
of plasminogen deficiency: (1) hypoplasminogenemia (Type
I PLG deficiency), in which there is a marked decrease in
the levels of PLG antigen ≤1:9 mg/dL (normal range 6 to
25 mg/dL) and functional activity of up to 33% (normal
range 80 to 120%), and (2) dysplasminogenemia (Type II
PLG deficiency), in which the level of immunoreactive PLG
is within normal range but the specific activity of PLG is
reduced [7–10]. Figure 1: Intraoral examination of a 26-year-old male patient with
PLG deficiency (Type 1) showing membranous gingival enlargement. Figure 2: Panoramic radiograph showing extensive generalized
periodontal breakdown. Figure 2: Panoramic radiograph showing extensive generalized
periodontal breakdown. Figure 3: Ligneous conjunctivitis. Gingival enlargement in hypoplasminogenemia has been
described as “amyloidaceous ulcerated gingival hyperplasia”
or destructive membranous periodontal disease (ligneous
periodontitis). The term ligneous periodontitis (LP) was first
coined by Gunhan et al. to describe a destructive membra-
nous periodontal disease [11]. LP generally starts in child-
hood with conjunctiva as well as gingival mucosa as its
most affected sites [1, 12]. Ligneous periodontitis is charac-
terized by gingival enlargement and severe attachment loss,
which is associated with plasminogen deficiency, the
accumulation of amyloid-like material in the lamina propria,
and deposition of fibrin [5, 11, 13]. Most cases of LP have
been reported in association with ligneous conjunctivitis
(LC), which suggests that both clinical manifestations may
be related. LC is a rare form of chronic conjunctivitis that
usually affects children, girls more often than boys (3 : 1),
and can occur at any age. It is characterized by fibrin-rich
pseudomembranes mainly on tarsal conjunctivae [1, 8]. Type
II PLG deficiency patients, however, have never reported
developing pseudomembranous lesions. Figure 3: Ligneous conjunctivitis. with multicystic hydrocephalus which was drained by ventri-
culoperitoneal shunt. Since an early age, he also presented
with profuse granulomatous growth in conjunctiva in both
eyes. Corneal involvement and chronic obstruction led to
the progressive blindness of the left eye by the age of 15. His-
topathological evaluations led to a diagnosis of ligneous
conjunctivitis. In this case report, the clinical, histopathological, and
genetic evaluation of a 26-year-old male patient who pre-
sented with generalized membranous gingival enlargement
is presented. 2.1. Clinical and Radiographic Examination. The intraoral
examination revealed solid, nodular, and fragile erythema-
tous and hyperplastic gingival enlargements involving the
marginal and attached gingiva in both maxillary and man-
dibular arches. 2. Case Presentation A 26-year-old male patient reported to our clinic with com-
plaints of generalized swelling of gums and pain and bleeding
from gums. Gingival enlargement was present since child-
hood and was slowly increasing in size. The enlargements
had been previously localized in sites and had been surgically
excised at 8 years of age, but recurrence occurred. He had
periodic oral prophylaxis done. He was the third child of sec-
ond degree consanguineous parents. At birth, he presented 3 Case Reports in Dentistry 3 (a)
(b)
(c)
Figure 4: (a) H&E stain; original magnification is ×4, wh
magnification in (b) is ×10 and magnification in (c) is ×4
Photomicrograph showing parakeratinised stratified squamo
surface epithelium with branching and anastomosing rete ridg
and
connective
tissue
showing
subepithelial
deposits
homogenous eosinophilic material that resembled amyloid. Th
deeper parts show a diffuse mixed inflammatory cell infiltra (a) The observed variation lies in the kringle domain of the
plasminogen protein [18] and has previously been reported
as pathogenic as per ClinVar and SwissVar [19, 20]. The
p.Arg235His variant has not been reported in the 1000
Genomes, ExAC, and the internal database of the MedGen-
ome laboratories. The in silico predictions of the variant are
probably damaging by PolyPhen-2 (HumDiv) and damaging
by SIFT and MutationTaster 2. The reference codon is con-
served across species. For the OMIM phenotype, Type 1 plas-
minogen
deficiency
(OMIM#217090)
is
caused
by
homozygous or compound heterozygous mutations in the
PLG gene (OMIM∗173350). 2.5. Treatment Done. After the diagnosis of LP, the manage-
(a)
(b)
(c)
Figure 4: (a) H&E stain; original magnification is ×4, while
magnification in (b) is ×10 and magnification in (c) is ×40
Photomicrograph showing parakeratinised stratified squamous
surface epithelium with branching and anastomosing rete ridges
and
connective
tissue
showing
subepithelial
deposits
o
homogenous eosinophilic material that resembled amyloid. The
deeper parts show a diffuse mixed inflammatory cell infiltrate
comprising predominantly of neutrophils. and development were within normal limits, and no other
family member reported similar findings. 2.2. Laboratory Investigations. Haematological assessment
and biochemical tests were done all of which were within
normal parameters except for plasma plasminogen activity
which showed a level of <5% (biological reference=low, refer-
ence interval: 75‐140%), a lactate dehydrogenase (LDH) level
of 331 IU/L (reference range = 135‐214), SGOT/AST levels of
60 IU/L (reference range = 15‐37), and SGPT/ALT levels of
66 IU/L (reference range = 30‐65). 2. Case Presentation Plasminogen deficiency
was diagnosed as due to hypoplasminogenemia which is
named Type I PLG plasminogen deficiency. Haematological
evaluation and biochemical tests such as liver function tests,
serum proteins, azotemia, glycemia, hormonal values, and
kidney function tests were within normal limits. (a) (b) 2.3. Histopathological Examination. Under the effect of local
anaesthesia, an incisional biopsy of the gingival enlargement
was done. The haematoxylin-and-eosin-stained soft tissue
section showed a parakeratinised stratified squamous surface
epithelium with branching and anastomosing rete ridges in
association with a fibrovascular connective tissue. The con-
nective tissue showed subepithelial deposits of homogenous
eosinophilic material that resembled amyloid. These areas
were relatively acellular. The deeper parts of the connective
tissue showed a diffuse mixed inflammatory cell infiltrate
comprised predominantly of neutrophils, lymphocytes, and
plasma cells. Sections also showed capillary vessels and
extravasated red blood cells. Gingival biopsies all stained for
fibrin but stained (Congo Red) negatively for amyloid, lipid,
keratin, immunoglobulins, and glycogen. Masson’s tri-
chrome was negative and PAS variably positive (Figure 4). 2.3. Histopathological Examination. Under the effect of local
anaesthesia, an incisional biopsy of the gingival enlargement
was done. The haematoxylin-and-eosin-stained soft tissue
section showed a parakeratinised stratified squamous surface
epithelium with branching and anastomosing rete ridges in
association with a fibrovascular connective tissue. The con-
nective tissue showed subepithelial deposits of homogenous
eosinophilic material that resembled amyloid. These areas
were relatively acellular. The deeper parts of the connective
tissue showed a diffuse mixed inflammatory cell infiltrate
comprised predominantly of neutrophils, lymphocytes, and
plasma cells. Sections also showed capillary vessels and
extravasated red blood cells. Gingival biopsies all stained for
fibrin but stained (Congo Red) negatively for amyloid, lipid,
keratin, immunoglobulins, and glycogen. Masson’s tri-
chrome was negative and PAS variably positive (Figure 4). (b) (c) (c) (c) Figure 4: (a) H&E stain; original magnification is ×4, while
magnification in (b) is ×10 and magnification in (c) is ×40. Photomicrograph showing parakeratinised stratified squamous
surface epithelium with branching and anastomosing rete ridges
and
connective
tissue
showing
subepithelial
deposits
of
homogenous eosinophilic material that resembled amyloid. The
deeper parts show a diffuse mixed inflammatory cell infiltrate
comprising predominantly of neutrophils. Figure 4: (a) H&E stain; original magnification is ×4, while
magnification in (b) is ×10 and magnification in (c) is ×40. Photomicrograph showing parakeratinised stratified squamous
surface epithelium with branching and anastomosing rete ridges
and
connective
tissue
showing
subepithelial
deposits
of
homogenous eosinophilic material that resembled amyloid. 2. Case Presentation The
deeper parts show a diffuse mixed inflammatory cell infiltrate
comprising predominantly of neutrophils. 2.4. Genetic Testing and Variant Interpretation. After the
clinical and histopathological diagnosis of ligneous peri-
odontitis associated with Type 1 plasminogen deficiency
was made, DNA tests were done to the patient at Med-
Genome laboratories, Bangalore, India. The test methodol-
ogy performed was targeted gene sequencing. Selective
capture and sequencing of the protein coding regions of
the genome/genes was performed. Mutations identified in
the exonic regions are generally actionable when compared
to variations that occur in noncoding regions. Targeted
sequencing represents a cost-effective approach to detect
variants present in multiple/large genes in an individual. DNA extracted from blood was used to perform targeted
gene capture using a custom capture kit. The libraries
were sequenced to mean > 80‐100x coverage on an Illu-
mina sequencing platform. The sequences obtained are
aligned to a human reference genome (GRCh37/hg19)
using the BWA program [14, 15]. It was analyzed using
Picard and GATK version 3.6 to identify variants relevant
to the clinical condition [16, 17] (Figure 5). The observed variation lies in the kringle domain of the
plasminogen protein [18] and has previously been reported
as pathogenic as per ClinVar and SwissVar [19, 20]. The
p.Arg235His variant has not been reported in the 1000
Genomes, ExAC, and the internal database of the MedGen-
ome laboratories. The in silico predictions of the variant are
probably damaging by PolyPhen-2 (HumDiv) and damaging
by SIFT and MutationTaster 2. The reference codon is con-
served across species. For the OMIM phenotype, Type 1 plas-
minogen
deficiency
(OMIM#217090)
is
caused
by
homozygous or compound heterozygous mutations in the
PLG gene (OMIM∗173350). 2.5. Treatment Done. After the diagnosis of LP, the manage-
ment of the dental condition was done. Supra- and subgingi-
val debridement was performed under local anaesthesia. Oral
hygiene instructions were given to the patient who was
advised to rinse twice daily with 0.2% chlorhexidine
digluconate for a period of two weeks. A marked reduction
in pocket depth and bleeding on probing was noticed after Variant interpretation was a homozygous missense
variation in exon 7 of the PLG gene (chr6:161137712G>A;
depth: 50x) that results in the amino acid substitution of
histidine
for
arginine
at
codon
235
(p.Arg23His;
ENST00000308192.9). Inheritance was autosomal recessive. 2. Case Presentation Figure 5: Sequence chromatogram and alignment to the reference sequence showing the variation in exon 7of the PLG gene
(chr6:161137712G>A; c.704G>A; p.Arg235His) detected in homozygous condition in the patient. 4
Case Reports in Dentistry Case Reports in Dentistry 4 Figure 5: Sequence chromatogram and alignment to the reference sequence showing the variation in exon 7of the PLG gene
(chr6:161137712G>A; c.704G>A; p.Arg235His) detected in homozygous condition in the patient. eight weeks, but the enlargements still persisted. Surgical
excision of the lesion was done by internal bevel gingivec-
tomy on the upper right quadrant and by a diode laser in
the upper left quadrant. However, recurrence of the lesion
was seen after 3 months (Figure 6). The patient was also
referred to an ophthalmology clinic for the management of
ligneous conjunctivitis. (juvenile colloid milium: 1%). Congenital occlusive hydro-
cephalus was seen in 12% of the patients. A few (5%) patients
exhibited Dandy-Walker malformation, a congenital hypo-
plasia, and upward rotation of the cerebellar vermis and cys-
tic dilation of the fourth ventricle [7, 22–25]. In the present
case, the patient presented with congenital hydrocephalus,
ligneous conjunctivitis, and enlargements in both jaws since
childhood. Based on the clinical and radiological findings, a
provisional diagnosis of LP was considered. 3. Discussion Other manifestations included the
involvement of the upper and lower respiratory tract (20%),
the middle ear (15%), the female genital tract (9%), the gas-
trointestinal tract (2.7%), the kidneys (4%), and the skin 5 Case Reports in Dentistry Case Reports in Dentistry 5 (a)
(b)
(c)
(d)
Figure 6: (a) Surgical excision by internal bevel gingivectomy on the right maxillary region. (b) 3-Month follow-up shows recurrence of th
esion. (c) Excision by diode laser on left maxillary region. (d) 3-Month follow-up shows recurrence of the lesion. 5
Case Reports in Dentistry ( ) (b) (b) (b) (a) (d) (c) (c) (d) Figure 6: (a) Surgical excision by internal bevel gingivectomy on the right maxillary region. (b) 3-Month follow-up shows recurrence of the
lesion. (c) Excision by diode laser on left maxillary region. (d) 3-Month follow-up shows recurrence of the lesion. nective tissue showed subepithelial deposits of homogenous
eosinophilic material that resembled amyloid. These areas
were relatively acellular. Gingival biopsies all stained for
fibrin but stained (Congo Red) negatively for amyloid, lipid,
keratin, immunoglobulins, and glycogen. Masson’s tri-
chrome was negative and PAS variably positive. These find-
ings confirmed the diagnosis of LP. additional significant role in the pathogenesis of this dis-
ease [5, 10, 31]. The average age of patients reported in
the literature with ligneous periodontitis is 12-18 years,
while isolated cases have been reported in older patients;
these differences may reflect variability in PLG activity
due to different plasminogen gene mutations [32]. Histopathological examination was done to confirm the
diagnosis. Severe acute inflammation and irregular acantho-
sis of the epithelium with extensive fibrin leakage and the
accumulation of an amyloid-like material in the lamina pro-
pria is the characteristic feature of this disease at histopatho-
logical examination. This condensed fibrin accumulation
lacks reticulin fibres [11]. A variety of other hyaline deposits,
ranging from classical amyloid to mixtures of immunoglobu-
lins, fibrin, fibrinogen, and albumin, might be found in the
gingiva. Indeed, amyloid is itself a nonspecific term for a
spectrum of entities, including immunoglobulin light chains,
serum amyloid fibrillar proteins, β2-microglobulin, keratin
transthyretin, and others [33]. In the present case too, the
section showed parakeratinised stratified squamous surface
epithelium with branching and anastomosing rete ridges in
association with a fibrovascular connective tissue. 3. Discussion p
g
The exact pathophysiology of the clinical presentation
seen in LP is still not clear. In vitro data and animal stud-
ies indicated that alterations in tissue repair and host
defense mechanisms are responsible for the onset and
the progression of periodontal destruction [4, 26]. Plas-
minogen plays an important role in intravascular and
extravascular fibrinolysis, wound healing, cell migration,
tissue remodelling, angiogenesis, and embryogenesis [27]. Local extracellular fibrinolysis by plasmin is required for
the initial removal of the fibrin-rich matrix as well as for
the remodelling of the granulation tissue and completion
of wound healing [10, 28, 29]. Impairment of the pathway
due to hypoplasminogenemia leads to fibrin accumulation
and an increased inflammatory reaction. Consequently, the
process of wound healing stops at the stage of granulation
tissue formation and cellular proteolysis, which may then
further support the invasion of pathogens. This process
is notably pronounced in mucous membranes such as
the periodontal tissues [29, 30]. The fact that only 32%
of patients who suffer from PLG Type 1 deficiency develop
ligneous periodontitis strongly supports the notion that
external triggers, i.e., trauma or infection, may play an In this report, a case of LP has been presented along with its
corresponding clinical, radiographic, histopathologic, and
genetic findings in addition to its management by excision. Only a limited number of cases with plasminogen deficiency
and oral lesions have been reported in the literature [5]. Con-
genital plasminogen deficiency is a rare autosomal recessive
disease which is clinically characterized by chronic mucosal
pseudomembranous, nodular enlargements in both the max-
illa and mandible depending on the subepithelial fibrin depo-
sition and inflammation [11, 21]. It is now well documented
that hypoplasminogenemia is a multisystemic disease. Clini-
cal manifestations in a collection of 74 patients with severe
(i.e., homozygous or compound-heterozygous) hypoplasmi-
nogenemia showed a median age of first clinical manifesta-
tion of 9.54 months (range 3 days to 61 years). The female-
to-male ratio was 1.43 : 1. The majority of the affected
patients suffered from ligneous conjunctivitis (81%) and lig-
neous gingivitis (30%). 3. Discussion The authors reported about a combination of gingivectomy,
an administration of 20 mg doxycycline daily, and the use
of a 0.12% chlorhexidine digluconate mouth rinse. One week
after surgery, the patient started with 5 mg warfarin daily for
an indefinite time [42]. Silva et al. reported a complete regres-
sion of oral mucous lesions after systemic and topical cortico-
steroids [43]. A recent clinical trial by Shapiro et al. was the
first to evaluate the clinical utility of Glu-plasminogen in
children and adults with congenital plasminogen deficiency. Human Glu-plasminogen replacement therapy was adminis-
tered intravenously at 6.6 mg/kg every 2-4 days to 14 patients
for a period of 12 weeks. Upon administration, results show
the achievement of physiological levels of plasminogen activ-
ity that temporally coincided with clinical efficacy and
improved disease management [44]. In the present case, the
26-year-old male patient reported with gingival enlargements
present since childhood. Excision of the lesion was done with
a surgical blade on one side and with a diode laser on the
other side. However, recurrences of the lesions were seen
ithi
3
th P353A, P491R, A505V, R513H, S575P, W597C, A675T,
P744S, C765G, and R776H; nonsense mutations include
C133X, K378X, Q380X, E460X, R471X, Q540X, and W597X;
frameshift mutations include W417fsX432, E455fsX493,
V563fsX580, and L650fsX652; and splice site mutations
include IVS11-2A/G, IVS11-7T/G, IVS11+1G/A, and IVS17
+1delG as well as amino acid deletion mutation K212del and
amino acid insertion mutation T319_N320insN [7, 22–25]. Congenital heterozygous hypoplasminogenemia is a rare
condition and was first described in 1982 in a patient with
thromboembolic disease [34]. It was earlier thought that het-
erozygous hypoplasminogenemia might be a risk factor for
venous thrombosis; however, a study by Drew et al. has sug-
gested that isolated heterozygous or homozygous/com-
pound-heterozygous hypoplasminogenemia by itself is not
a risk factor for deep venous thrombosis [35]. In 1997, Schus-
ter et al. demonstrated distinct homozygous and compound-
heterozygous mutations in the PLG gene and confirmed the
autosomal-recessive inheritance of this disorder [23]. Studies
have shown the incidence rate of hypoplasminogenemia
worldwide to be less than 1%. A large epidemiologic study
performed in blood donors in the United Kingdom demon-
strated an observed rate of heterozygous (asymptomatic)
plasminogen deficiency of 25 out of 9611 patients or 0.26%
[36]. 3. Discussion The con- Inherited
homozygous
or
compound-heterozygous
hypoplasminogenemia is a very rare disorder (with an
incidence estimated at below 1 : 10) which is associated with
deficient extravascular PLG-dependent fibrinogen clearance
leading to impaired wound healing mainly of mucous mem-
branes. In affected patients, the wound healing capacity
seems to be arrested at the stage of granulation tissue forma-
tion [8]. Studies suggest that the K19E mutation is the most
common
molecular
genetic
defect
in
patients
with
hypoplasminogenemia worldwide [25]. A variety of other
genetic abnormalities have been identified in the PLG gene in
subjects with heterozygous, homozygous, and compound-
heterozygous hypoplasminogenemia: missense mutations
include T9N, L128P, R134K, G142R, G176D, T181P, R216H,
D219N, R234H, P285A, P285T, R306H, N307I, T352I, 6 Case Reports in Dentistry Case Reports in Dentistry to decrease the inflammation and thus the progression of
the disease have been attempted; these procedures include
scaling and root planing, chlorhexidine rinsing, administra-
tion of antibiotics, and periodontal surgery [9, 25, 30]. Neer-
ing et al. have reported that patients with PLG-deficiency
Type I may benefit from nonsurgical periodontal therapy
including full mouth disinfection in combination with
adjunctive antibiotic therapy and a strict supportive peri-
odontal therapy regime every three months [32]. Most of
the reports have been described as failures due to rapid gingi-
val regrowth and progressive bone loss [5]. Scully et al. sug-
gested that gingival lesions can be controlled by topical
heparin or intravenous purified plasminogen [41]. Gunhan
et al. stated that systemic fibrinolytic and antithrombotic
agents may prove more beneficial than local treatments,
because the ligneous lesions tend to involve several mucosal
areas [11]. Traditional periodontal treatment options for
managing gingival enlargements include either gingivectomy
or flap surgeries. The main objective is to excise the enlarge-
ments and reestablish healthy gingival margins and contours. The healing pattern in flap surgeries is by primary intention,
whereas in gingivectomy, it involves healing by secondary
intention. These healing mechanisms involve the formation
of a fibrin clot, which creates a framework for normal peri-
odontal tissues to grow back. However, in patients with plas-
minogen deficiency, the inadequacy of plasmin results in the
overgrowth of fibrin. This is the main reason for the failure of
conventional periodontal treatment modalities in these
patients. A case report indicated that the treatment with war-
farin exerts protection against relapsing gingival hyperplasia
over an observation period of 3 years in a 54-year-old patient. 3. Discussion The incidence rate of (heterozygous) hypoplasminogen-
emia has been roughly estimated in different geographic
regions at 0.35% (only in white subjects) in Minnesota,
USA [37], at 0.13% in Southern Germany [38], and at
0.42% in Japan [39]. p
In our case, the GATK Best Practices framework was
followed for the identification of variants in the sample. Gene
annotation of the variants was performed using the VEP pro-
gram against the Ensembl release 87 human gene model [40]. Clinically relevant mutations were annotated using published
variants in the literature and a set of disease databases—Clin-
Var, OMIM, GWAS, HGMD, and SwissVar. Common
variants were filtered based on allele frequency in 1000
Genomes phase 3, ExAC, EVS, dbSNP147, 1000 Japanese
Genome, and our internal Indian population database. Nonsynonymous variant effects were calculated using mul-
tiple algorithms such as PolyPhen-2, SIFT, MutationTaster
2, MutationAssessor, and LRT. Only nonsynonymous and
splice site variants found in the clinical exome panel con-
sisting of 8332 genes were used for clinical interpretation. Silent variations that do not result in any change in amino
acid in the coding region were not reported. A homozy-
gous missense variation in exon 7 of the PLG gene
(chr6:161137712G>A; depth: 50x) that results in the
amino acid substitution of histidine for arginine at codon
235
(p.Arg23His;
ENST00000308192.9)
was
detected. Inheritance was autosomal recessive. The observed varia-
tion lies in the kringle domain of the plasminogen protein
and has previously been reported as pathogenic as per
ClinVar and SwissVar [18–20]. Consent [13] T. Baykul and Y. Bozkurt, “Destructive membranous peri-
odontal disease (ligneous periodontitis): a case report and 3
years follow-up,” British Dental Journal, vol. 197, no. 8,
pp. 467-468, 2004. The authors certify that they have obtained the appropriate
patient consent form. In the form, the patient has given con-
sent for his clinical information to be reported in the journal. The patient understands that his name and initials will not be
published, and due efforts will be made to conceal his iden-
tity; however, anonymity cannot be guaranteed. [14] H. Li and R. Durbin, “Fast and accurate long-read alignment
with Burrows-Wheeler transform,” Bioinformatics, vol. 26,
no. 5, pp. 589–595, 2010. [15] L. R. Meyer, A. S. Zweig, A. S. Hinrichs et al., “The UCSC
genome browser database: extensions and updates 2013,”
Nucleic Acids Research, vol. 41, no. D1, pp. D64–D69, 2012. There are no conflicts of interest. [16] A. McKenna, M. Hanna, E. Banks et al., “The Genome Analy-
sis Toolkit: a MapReduce frame work for analyzing next-
generation DNA sequencing data,” Genome Research, vol. 20,
no. 9, pp. 1297–1303, 2010. Abbreviations LP:
Ligneous periodontitis
PLG:
Plasminogen
LC:
Ligneous conjunctivitis
SGOT:
Serum glutamic oxaloacetic transaminase
AST:
Aspartate amino transferase
SGPT:
Serum glutamic pyruvic transaminase
ALT:
Alanine transaminase
PAS:
Periodic acid-Schiff
BWA program: Burrows-Wheeler aligner program
SIFT:
Scale invariant feature transform
OMIM:
Online Mendelian Inheritance in Man
GATK:
Genome Analysis Toolkit
VEP:
Variant effect predictor
GWAS:
Genome-wide association study
HGMD:
Human Gene Mutation Database. [8] V. Schuster and S. Seregard, “Ligneous conjunctivitis,” Survey
of Ophthalmology, vol. 48, no. 4, pp. 369–388, 2003. [9] I. Kurtulus, A. Gokbuget, A. Efeoglu et al., “Hypoplasmino-
genemia with ligneous periodontitis: a failed local therapeutic
approach,”
Journal
of
Periodontology,
vol. 78,
no. 6,
pp. 1164–1175, 2007. [10] V. Schuster, B. Hügle, and K. Tefs, “Plasminogen deficiency,”
Journal of Thrombosis and Haemostasis, vol. 5, no. 12,
pp. 2315–2322, 2007. [11] Ö. Günhan, M. Günhan, E. Berker, C. A. Gürgan, and
H. Yildirim, “Destructive membranous periodontal disease
(ligneous periodontitis),” Journal of Periodontology, vol. 70,
no. 8, pp. 919–925, 1999. [12] Ö. Günhan, B. Celasun, F. Perrini et al., “Generalized gingival
enlargement due to accumulation of amyloid like material,”
Journal of Oral Pathology & Medicine, vol. 23, no. 9,
pp. 423–428, 1994. 4. Conclusion This case report discusses the diagnosis of ligneous periodon-
titis associated with Type 1 plasminogen deficiency in a 26-
year-old male patient. The clinical presentation was that of
a generalized gingival enlargement. Through this case report,
we would like to stress on the importance of a thorough The most characteristic features of this destructive
periodontal disease are widespread membranous, nodular
gingival enlargements in both maxilla and mandible leading
to rapid tooth loss despite several treatment attempts [11]. Several therapeutic approaches to reduce the bacterial load 7 Case Reports in Dentistry history taking, intraoral clinical evaluation, radiographic
evaluation, and haematological examination, as a varied etio-
pathogenesis is reported for gingival enlargements. A histo-
pathological examination of the biopsy specimen helped in
confirming the diagnosis. Genetic testing helped in precisely
identifying the gene, location, variant, zygosity, and inheri-
tance pattern. Excision of the enlargements by means of sur-
gical and laser techniques were not successful and recurrence
occurred. The lack of efficacious therapy has increased the
suffering of affected patients under the burden of this very
rare blood disease. [4] R. Sulniute, T. Lindh, M. Wilczynska, J. Li, and T. Ny, “Plas-
min is essential in preventing periodontitis in mice,” The
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Documentation of body mass index and control of associated risk factors in a large primary care network
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Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available
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nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA Permanent link http://nrs.harvard.edu/urn-3:HUL.InstRepos:4725503 Citation Rose, Stephanie A., Alexander Turchin, Richard W. Grant, and James B. Meigs. 2009. Documentation of body mass index and control of associated risk factors in a large primary care
network. BMC Health Services Research 9: 236. Rose, Stephanie A., Alexander Turchin, Richard W. Grant, and James B. Meigs. 2009. Documentation of body mass index and control of associated risk factors in a large primary care
network. BMC Health Services Research 9: 236. Published Version
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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. Open Ac
Research article
Documentation of body mass index and control of associated risk
factors in a large primary care network
Stephanie A Rose*1, Alexander Turchin2, Richard W Grant3 and
James B Meigs4 Open Access Open Ac
Research article
Documentation of body mass index and control of associated risk
factors in a large primary care network
Stephanie A Rose*1 Ale ander Turchin2 Richard W Grant3 and Address: 1Division of General Internal Medicine, University of Kentucky, 740 South Limestone Street, Kentucky Clinic K507, Lexington, KY 40536,
USA, 2Clinical Informatics Research and Development, Harvard University, and Division of Endocrinology, Brigham and Women's Hospital, 221
Longwood Avenue, Boston, MA, 02115, USA, 3General Medicine Division, Massachusetts General Hospital, 50 Staniford Street, Boston, MA,
02114, USA and 4General Medicine Division, Massachusetts General Hospital, 50 Staniford Street, Boston, MA, 02114, USA Email: Stephanie A Rose* - sro226@uky.edu; Alexander Turchin - aturchin@partners.org; Richard W Grant - rgrant@partners.org;
James B Meigs - jmeigs@partners.org * Corresponding author Received: 17 May 2009
Accepted: 16 December 2009 BMI Measurement
f The intent of the HEDIS initiative is to measure and
increase documentation of BMI. We obtained BMI from
height and weight data recorded in the EHR, where they
are used to automatically calculate and display BMI. For
this analysis we calculated BMI from the most recent
weight in the 18-month period and the most recent height
prior to 12/31/06 from structured coded entries in the
EHR. For completeness, we also searched the text of narra-
tive notes in the EHR using a validated Natural Language
Processing abstraction tool that computationally abstracts
weight, height, and BP values from the free text of clini-
cian notes [19,20]. The sensitivity and specificity of the
approach to abstraction (compared with a trained chart
abstractor) are 87.9% and 99% for detection of height,
91.8% and 92.1% for detection of weight, and 91% and
96% for detection of BP [19,20]. Because of high rates of
missing heights, we confirmed with clinic site medical
directors that we had searched in all the appropriate
places in the clincial record for height and weight infor-
mation. Given the magnitude and clinical impact of the current
obesity epidemic and its risk factors, and the potential
implementation of the HEDIS BMI Assessment measure,
we aimed to examine current care in BMI and risk factor
assessment for patients in a large network of primary care
practices that use a common electronic health record
(EHR). We investigated the prevalence of BMI measure-
ment and documentation and control of the CVD and dia-
betes risk factors blood pressure (BP), low density
lipoprotein (LDL), and fasting and casual glucose. We
examined these risk factors according to the BMI catego-
ries normal weight, overweight, and obese. We hypothe-
sized that documentation of BMI would vary widely in
clinical practice, and that patients who were obese would
be more likely to have better documentation, but poorer
control, of risk factors than patients of normal weight. We categorized BMI using Centers for Disease Control
(CDC) definitions, with underweight equal to BMI <18.5
kg/m2, normal weight equal to a BMI of 18.5-<25 kg/m2,
overweight BMI equal to 25-<30 kg/m2, and obese BMI
≤30 kg/m2 [21]. We included underweight patients when
measuring documentation of BMI, but excluded them
otherwise. Among those with a documented BMI, we
examined documentation and control of three risk fac-
tors: blood pressure (BP), LDL cholesterol, and fasting or
casual glucose levels, or both. http://www.biomedcentral.com/1472-6963/9/236 http://www.biomedcentral.com/1472-6963/9/236 http://www.biomedcentral.com/1472-6963/9/236 BMC Health Services Research 2009, 9:236 Background nity health centers in and around Boston, Massachusetts. These twelve practices use a common EHR that contains
all clinical and utilization data for each patient. The data
from the EHR data are searchable in the Research Patient
Data Repository (RPDR) [18]. From these, we included
79,947 patients who were at least 18 years of age and had
at least two clinic visits billed to their PCP between 7/1/05
and 12/31/06, and did not have a height greater than or
equal to seven feet (2.13 meters), weight <70 or >1000
pounds (<31.8 or >453.6 kilograms), systolic BP <50 or
>260 mmHg, or diastolic BP <30 or >150 mmHg. The
study was approved by the Partners Health Care System
Institutional Review Board. g
Obesity is a common problem in the United States that
independently confers risk for chronic disease and early
mortality [1]. Several studies address the importance of
recognition and treatment of obesity in chronic disease
management, but few evaluate body mass index (BMI)
assessment and risk factor documentation and control in
patients who are obese both with and without cardiovas-
cular disease (CVD), diabetes or risk factors [2-6]. Assessments of BMI and CVD or diabetes risk factors are
simple, inexpensive tools that can be used by primary care
physicians (PCPs) to address obesity and its complica-
tions. Little consensus exists in current guidelines con-
cerning the need to screen for BMI and associated risk
factors [7-15]. In 1998 the National Heart, Lung, and
Blood Institute (NHLBI) established guidelines for BMI
and risk factor measurement in all patients under 80 years
of age with the goal of implementation of strategies for
weight loss and risk factor control in patients with a BMI
of ≥ 30 kg/m2 or 25-29.9 kg/m2 and ≥ two risk factors [10]. In 2007 the National Committee for Quality Assurance
(NCQA) organized field tests of a prospective Healthcare
Effectiveness Data and Information Set (HEDIS) measure
to assess the percentage of members 18-74 years of age
with an outpatient visit, and who had BMI documented
and risk factors documented if found to have a BMI of ≥30
kg/m2 [16]. Their work led to the proposed HEDIS 2009
measure BMI Assessment [17], which is proposed for
reporting by commercial, Medicaid, and Medicare plans. BMI Measurement
f We examined risk factor
documentation and control in the clinic population over-
all, and stratified by three BMI categories. Abstract Background: Body mass index (BMI) will be a reportable health measure in the United States (US)
through implementation of Healthcare Effectiveness Data and Information Set (HEDIS) guidelines. We evaluated current documentation of BMI, and documentation and control of associated risk
factors by BMI category, based on electronic health records from a 12-clinic primary care network. Methods: We conducted a cross-sectional analysis of 79,947 active network patients greater than
18 years of age seen between 7/05 - 12/06. We defined BMI category as normal weight (NW, 18-
24.9 kg/m2), overweight (OW, 25-29.9), and obese (OB, ≥ 30). We measured documentation (yes/
no) and control (above/below) of the following three risk factors: blood pressure (BP) ≤130/≤85
mmHg, low-density lipoprotein (LDL) ≤130 mg/dL (3.367 mmol/L), and fasting glucose <100 mg/dL
(5.55 mmol/L) or casual glucose <200 mg/dL (11.1 mmol/L). Results: BMI was documented in 48,376 patients (61%, range 34-94%), distributed as 30% OB, 34%
OW, and 36% NW. Documentation of all three risk factors was higher in obesity (OB = 58%, OW
= 54%, NW = 41%, p for trend <0.0001), but control of all three was lower (OB = 44%, OW =
49%, NW = 62%, p = 0.0001). The presence of cardiovascular disease (CVD) or diabetes modified
some associations with obesity, and OB patients with CVD or diabetes had low rates of control of
all three risk factors (CVD: OB = 49%, OW = 50%, NW = 56%; diabetes: OB = 42%, OW = 47%,
NW = 48%, p < 0.0001 for adiposity-CVD or diabetes interaction). Conclusions: In a large primary care network BMI documentation has been incomplete and for
patients with BMI measured, risk factor control has been poorer in obese patients compared with
NW, even in those with obesity and CVD or diabetes. Better knowledge of BMI could provide an
opportunity for improved quality in obesity care. Page 1 of 13
(page number not for citation purposes) Page 1 of 13
(page number not for citation purposes) http://www.biomedcentral.com/1472-6963/9/236 Page 2 of 13
(page number not for citation purposes) BMI Documentation and Characteristics of Patients with
Documented BMI We defined clinical CVD as coronary artery disease, cere-
brovascular accident, or peripheral vascular disease listed
on the EHR Problem List, or as having two outpatient or
one inpatient International Classification of Diseases,
Ninth Revision (ICD-9) codes for CVD. We defined clini-
cal diabetes as diabetes on the EHR problem list and a dia-
betes medication on the medication list, or as having two
outpatient or one inpatient codes related to diabetes. The
definitions of diabetes, hypertension, hyperlipidemia and
CVD have previously been validated [25,26]. For instance,
the sensitivity of our approach for diabetes or CVD are
>98% and specificity of >97% compared to the gold
standard of trained research nurse chart abstraction. We
used the same approach as for diabetes to define hyper-
tension and hyperlipidemia. Other measurements
included age, race, gender, number of PCP visits during
the study period, insurance type, and clinic site. Of 79,947 patients, 72,633 (90.9%) had an available
weight, 50,345 (63.0%) had an available height, and
48,376 (60.5%) had both height and weight recorded to
calculate BMI (Table 1). Of those with BMI present,
14,290 patients (30%) were classified as obese, 16,402
(34%) overweight, 16,936 (36%) normal weight, and 748
(2%) underweight. Compared to those missing BMI,
patients with BMI documented were younger, had a
higher mean number of PCP visits, were more likely to be
women, and were more likely to have commercial insur-
ance or Medicare than patients without a BMI (p < 0.0001
for all; Table 2). As expected, patients with obesity were
older, less likely to be women, white, or to have private
insurance or Medicare, had a higher mean number of PCP
visits, and were more likely to have a history of CVD, dia-
betes, hypertension, and dyslipidemia than patients of
normal weight (p < 0.0001 for all; Table 3). Setting and Patients Risk Factor, CVD, and Diabetes Measurement
Documentation of a risk factor was defined as being
found in the EHR at least once within the study period. If
a risk factor was documented more than once, we used the
most recently documented value. A risk factor was defined
as controlled if within normal range, defined as: BP ≤130/ We identified 138,933 patients in our primary care
research network of twelve practices that make up the
Massachusetts General Hospital Primary Care Practice
Based Research Network (PBRN). Practices include urban
academic faculty practices, private offices, and commu- Page 2 of 13
(page number not for citation purposes) BMC Health Services Research 2009, 9:236 http://www.biomedcentral.com/1472-6963/9/236 ≤85 mmHg, LDL<130 mg/dL (3.367 mmol/L), and fast-
ing glucose <100 mg/dL (5.55 mmol/L) or casual glucose
<200 mg/dL (11.1 mmol/L) [22-24]. We also measured
documentation and control of all three risk factors com-
bined as an aggregate measure of risk factor management. BMI with risk factor documentation and control. We used
SAS version 9.1 (Cary, NC) for all analyses, and consid-
ered a p value of <0.05 to indicate statistical significance. Documentation and Control of Risk Factors We conducted a cross-sectional analysis. We measured
rates of BMI documentation in each clinic site and overall. Among those with a BMI, we measured risk factor docu-
mentation and control overall, and stratified by the three
BMI categories. We further stratified the analysis by the
presence or absence of CVD or diabetes. We used general-
ized linear models or chi-square tests to test levels or rates
of characteristics by BMI category. We used the Breslow
Day test for homogeneity of odds ratios to test for effect
modification of CVD or diabetes on the association of Overall, less than 78% had at least one risk factor docu-
mented and just half had all three documented (Table 4,
Figure 1). Of these, about half of all patients had all three
risk factors controlled (Figure 1). Documentation of all
three risk factors was higher in obesity than in overweight
or normal weight (p < 0.0001), but control of all three risk
factors was lower, (p < 0.0001), with only 44% of patients
with obesity having all risk factors under control. Table 1: Body Mass Index Documentation in 79,947 Patients in 12 Primary Care Clinics
Clinic
N
BMI
Height Documented
Weight Documented
%
%
%
1
3,027
93.5
94.1
99.1
2
4,559
89.8
95.7
91.6
3
3,274
86.9
87.5
99.1
4
3,989
85.8
94.5
87.4
5
1,344
75.1
75.2
99.5
6
7,747
65.8
66.0
99.0
7
21,299
64.0
65.8
96.1
8
3,962
55.4
55.8
98.7
9
12,919
50.2
55.4
71.1
10
4,781
41.9
44.4
78.4
11
8,052
38.2
38.3
99.0
12
4,994
34.0
35.7
88.9
Total
48,376
60.5
63.0
90.9
Data are ranked by BMI documentation rate, include patients at least 18 years of age with at least two clinic visits billed to their PCP between 7/1/
05 and 12/31/06, and include underweight patients (BMI <18.5 kg/m2) excluded from the main analysis. BMI = Body Mass Index (weight in kilograms/
height in meters2) Table 1: Body Mass Index Documentation in 79,947 Patients in 12 Primary Care Clinics Data are ranked by BMI documentation rate, include patients at least 18 years of age with at least two clinic visits billed to their PCP between 7/1/
05 and 12/31/06, and include underweight patients (BMI <18.5 kg/m2) excluded from the main analysis. BMI = Body Mass Index (weight in kilograms/
height in meters2) Page 3 of 13 http://www.biomedcentral.com/1472-6963/9/236 BMC Health Services Research 2009, 9:236 Table 2: Patient Demographics and Clinical Characteristics in 79,947 Patients in 12 Primary Care Clinics by Documentation or
Missingness of BMI
Characteristic
BMI Documented
48,376
BMI not Documented
31,571
P value
Clinic
n
%
<0.0001
1
2,830
5.9
197
0.6
2
4,093
8.5
466
1.5
3
2,844
5.9
430
1.4
4
3,421
7.1
568
1.8
5
1,009
2.1
335
1.1
6
5,095
10.5
2,652
8.4
7
13,630
28.2
7,669
24.3
8
2,194
4.5
1,768
5.6
9
6,489
13.4
6,431
20.4
10
2,003
4.1
2,778
8.8
11
3,073
6.4
4,979
15.8
12
1,696
3.5
3,298
10.5
Age
mean years
51
53
<0.0001
Women
n
%
31,412
64.9
15,986
50.6
<0.0001
Race
n
%
<0.0001
White
37,625
77.8
25,264
80.0
Asian
2,057
4.3
1,426
4.5
Black
2,908
6.0
1,464
4.6
Hispanic
4,123
8.5
2,397
7.6
Number of PCP visits
mean
range
4.2
(2-61)
3.9
(2-56)
<0.0001
Commercial Insurance or Medicare
n
%
42,740
88.4
27,522
87.2
<0.0001
History of CVD
n
%
7,298
15.1
4,989
15.8
0.0060
History of diabetes
n
%
5,872
12.1
3,453
10.9
<0.0001
History of hypertension
n
%
22,121
45.7
14,698
46.6
0.02
History of dyslipidemia
n
%
25,698
53.1
17,133
54.3
0.002
HbA1c
%
6.7
6.8
0.0001
Blood pressure
mean mmHg
122/75
123/74
<0.0001
Total cholesterol
mean mg/dL (mmol/L)
188.1 (4.87)
188.5 (4.88)
0.29
LDL
mean mg/dL (mmol/L)
104.1 (2.70)
105 (2.72)
0.001
HDL
mean mg/dL (mmol/L)
59.3 (1.54)
57.8 (1.50)
<0.0001
Triglyceride level
mean mg/dL (mmol/L)
127.8 (1.44)
132.6 (1.50)
<0.0001
Casual glucose
mean mg/dL (mmol/L)
101.8 (5.65)
100.8 (5.59)
0.0007
Fasting glucose
mean mg/dL (mmol/L)
102.4 (5.68)
100.9 (5.60)
0.0007
Risk factor documentation
Total n
%
Blood Pressure
64,204
80.3
37,489
77.5
26,715
84.6
<0.0001
LDL
55,210
69.1
33,788
69.8
21,422
67.9
<0.0001
Casual glucose
56,984
71.3
35,125
72.6
21,859
69.2
<0.0001
Fasting glucose
21,533
26.9
13,533
28.0
8,000
25.3
<0.0001
HbA1c
14,252
17.8
9,367
19.4
4,885
15.5
<0.0001
Total cholesterol
58,951
73.7
36,247
74.9
22,704
71.9
<0.0001
HDL
58,373
73.0
35,874
74.2
22,499
71.3
<0.0001
Triglycerides
55,948
70.0
34,247
70.8
21,701
68.7
<0.0001
Risk factor control in those with
documented risk factor
Total n
%
Blood Pressure <130 and < 85
mmHg
46,748
72.8
27,212
72.6
19,536
73.1
0.13
LDL <130 mg/dL
43,369
78.6
26,656
78.9
16,713
78.0
0.015
Casual glucose < 200 mg/dL
55,642
97.6
34,231
97.5
21,411
98.0
0.0001
Fasting glucose < 100 mg/dL
13,782
64.0
8,574
63.4
5,208
65.1
0.01
HbA1c <7%
9,638
67.6
6,463
69.0
3,175
65.0
<0.0001
Total cholesterol <200 mg/dL
37,279
63.2
22,952
63.3
14,327
63.1
0.59
HDL >35 mg/dL (male) and >40
mg/dL (female)
52,971
90.8
32,742
91.3
20,229
89.9
<0.0001
Triglycerides <150 mg/dL
48,188
86.1
29,675
86.7
18,513
85.3
<0.0001
P-value compares BMI documented with BMI not documented. http://www.biomedcentral.com/1472-6963/9/236 betes had lower rates of risk factor documentation and
control than may be ideal given their high absolute risk of
adverse health outcomes. The presence of CVD or diabetes modified some associa-
tions of BMI category with risk factor documentation and
control. In patients with CVD or diabetes, the rates of doc-
umentation were generally less different comparing
patients with obesity with those of normal weight, while
in patients without CVD or diabetes, rates of documenta-
tion were strikingly higher in patients with obesity than
normal weight (Figure 1, Table 5, p = 0.26 to <0.0001 for
CVD- or diabetes-by-BMI category interaction). Likewise,
in patients with CVD or diabetes the rates of control were
generally less different comparing patients with obesity
with those with normal weight, but in patients without
CVD or diabetes, rates of control were strikingly lower in
patients with obesity than normal weight (Figure 1, Table
6). Overall 50% or fewer of the patients with obesity and
CVD or diabetes had all three risk factors under control
(Figure 1, Tables 5 and 6). Our study builds upon findings from previous studies. Lemay et al audited medical records from 465 adult
patients seen at a community health center during one
week in February of 1999 to evaluate height and weight
documentation and obesity diagnosis by the practitioner
over a prior six-month period, and found that only 63%
of their patient cohort had a height and weight docu-
mented [27]. Similar to our study, Lemay looked a group
of patients in normal clinical practice; however, we were
able to expand upon this study in terms of a far larger
study sample as well as a longer sampling frame to assess
provision of services (18 months versus 6 months),
potentially providing a more accurate picture of risk factor
evaluation. Six years after this study and seven years after
the establishment of the NHLBI guidelines, we found the
percentage of BMI documentation to be essentially the
same (63% versus 60.5%). Rifas-Shiman et al studied
5,025 members of the same insurance plan and group
practice who were a subset of participants from a cohort
study and were continuously enrolled since 1999, had a
visit in 2000, had a BMI measurement between January 1,
2000 and December 31, 2000, and who did not have
medical conditions related to weight loss or CVD. http://www.biomedcentral.com/1472-6963/9/236 They
found that higher BMI was an independent predictor of
increased fasting glucose, triglyceride, LDL cholesterol,
and HDL cholesterol screening [28]. Rifas-Shiman identi-
fied lipid and glucose abnormalities over a two-year
period, comparable in time to our study. Neither we nor
Rifas-Shiman could evaluate attempts at management, so
reasons for increased documentation such as guideline
adherence could not be assessed. Our analysis extends
prior studies by evaluating whether those with documen-
tation of risk factors had those risk factors in control g
g
y
(
g
g
Physician, and CVD = Cardiovascular Disease. *Designation "Other" for race deleted from set. g
g
y
(
g
g
g
)
ician, and CVD = Cardiovascular Disease. *Designation "Other" for race deleted from set. P-value is for general association across BMI categories. BMI = Body Mass Index (weight in kilograms/he s for general association across BMI categories. BMI = Body Mass Index (weight in kilograms/height in meters2), PCP = Page 3 of 13 BMI = Body Mass Index (weight in kilograms/height in meters2), PCP = Primary Care
Physician, CVD = Cardiovascular Disease, HbA1c = Hemoglobin A1C, LDL=low density lipoprotein cholesterol, HDL = high density lipoprotein
cholesterol. LDL <130 mg/dL = <3.367 mmol/L; casual glucose <200 mg/dL = <5.55 mmol/L; fasting glucose <100 mg/dL = <11.1 mmol/L Table 2: Patient Demographics and Clinical Characteristics in 79,947 Patients in 12 Primary Care Clinics by Documentation or
Missingness of BMI graphics and Clinical Characteristics in 79,947 Patients in 12 Primary Care Clinics by Documentation or P-value compares BMI documented with BMI not documented. BMI = Body Mass Index (weight in kilograms/height in meters2), PCP = Primary Care
Physician, CVD = Cardiovascular Disease, HbA1c = Hemoglobin A1C, LDL=low density lipoprotein cholesterol, HDL = high density lipoprotein
cholesterol. LDL <130 mg/dL = <3.367 mmol/L; casual glucose <200 mg/dL = <5.55 mmol/L; fasting glucose <100 mg/dL = <11.1 mmol/L Page 4 of 13
(page number not for citation purposes) BMC Health Services Research 2009, 9:236 http://www.biomedcentral.com/1472-6963/9/236 http://www.biomedcentral.com/1472-6963/9/236 g
y
(
g
g
g
)
y
ardiovascular Disease. *Designation "Other" for race deleted from set. Discussion
d Molenaar et al studied rates of treatment and control of
hypercholesterolemia, hypertension, and diabetes in nor-
mal weight, overweight, and obese subjects with a history
of these conditions utilizing a CVD-free subset of the
Framingham Heart Study. They found that subjects with
hypercholesterolemia and hypertension who were obese
were more likely to have these conditions treated than
normal weight subjects. Rates of control of hypertension
and hypercholesterolemia were uniformly poor and did
not differ between weight groups. Rates of control of dia-
betes were poor among all three weight groups, but sub-
jects who were obese were less likely to have control of
fasting blood glucose than normal weight subjects. The
goal of our study, however, was different from that of
Molenaar. Molenaar studied rates of treatment and con-
trol of hypercholesterolemia, hypertension, and diabetes
in normal weight, overweight, and obese subjects with a
history of these conditions who were free of CVD, a sub-
group of patients with a clear indication for BMI screen-
ing. Our goal was to evaluate in all patients, both with and
without obesity-associated risk factors, the current state of
BMI screening and subsequent screening for associated
risk factors by BMI group, according to HEDIS and NHLBI
guidelines. Molenaar et al examined a well-known, stand-
ardized study population, where only 196 subjects had
missing BMI data. Our population was a non-standard-
ized data source, and our subjects were a mixture of both
those with and without CVD and its risk factors, with fur-
ther subanalysis in patients with a history of CVD and dia-
betes. Despite these differences, findings from the The rapidly growing prevalence of obesity has pushed BMI
assessment to appropriate prominence as a newly pro-
posed reportable HEDIS measure. BMI assessment will be
made by several means, including survey of EHRs in
health care networks. Our results suggest that the HEDIS
BMI Assessment measure has potential to provide a timely
quality and safety foundation to improve care for patients
with obesity. At least in our large primary care network,
there clearly is substantial room for improvement in doc-
umentation of BMI and documentation and control of
BMI-associated risk factors. Discussion
d BMC Health Services Research 2009, 9:236 http://www.biomedcentral.com/1472-6963/9/236 http://www.biomedcentral.com/1472-6963/9/236 Table 4: Documentation and Control of Risk Factors among 47,628 Primary Care Patients, by BMI Category
Risk Factor Documentation
Total
n = 47,628
Obese
n = 14,290
Overweight
n = 16,402
Normal
n = 16,936
P value
n
%
n
%
n
%
n
%
BP
36,934
77.6
10,900
76.3
12,890
78.6
13,144
77.6
<0.0001
LDL
33,427
70.2
11,239
78.7
12,013
73.2
10,175
60.1
<0.0001
Casual Glucose
34,589
72.6
11,216
78.5
11,901
72.6
11,472
67.7
<0.0001
Fasting Glucose
13,421
28.2
5,347
37.4
4,804
29.3
3,270
19.3
<0.0001
Risk Factor Control
Total
Obese
Overweight
Normal
P value
n
%
n
%
n
%
n
%
BP <130 and < 85 mmHg
26,736
72.4
6,801
62.4
9,202
71.4
10,733
81.7
<0.0001
LDL <130 mg/dL
26,328
78.8
8,777
78.1
9,273
77.2
8,278
81.4
<0.0001
Casual glucose < 200 mg/dL
33,698
97.4
10,707
95.5
11,635
97.8
11,356
99.0
<0.0001
Fasting glucose < 100 mg/dL
8,482
63.2
2,753
51.5
3,146
65.5
2,583
79.0
<0.0001
P-value is for general association across BMI categories. BMI=Body Mass Index (weight in kilograms/height in meters²); LDL <130 mg/dL=<3.367
mmol/L; casual glucose <200 mg/dL=<5.55 mmol/L; fasting glucose <100 mg/dL=<11.1 mmol/L. entation and Control of Risk Factors among 47,628 Primary Care Patients, by BMI Category P-value is for general association across BMI categories. BMI=Body Mass Index (weight in kilograms/height in meters²); LDL <130 mg/dL=<3.367
mmol/L; casual glucose <200 mg/dL=<5.55 mmol/L; fasting glucose <100 mg/dL=<11.1 mmol/L. P-value is for general association across BMI categories. BMI=Body Mass Index (weight in kilograms/height in me
mmol/L; casual glucose <200 mg/dL=<5.55 mmol/L; fasting glucose <100 mg/dL=<11.1 mmol/L. according to Adult Treatment Panel III (ATP III) metabolic
syndrome guidelines for a patient with average cardiovas-
cular risk. structured Framingham population and from our analysis
of usual clinical care are strikingly similar. This minimizes
to a large degree the concern that high rates of missing
BMI information could have distorted our findings, and
may explain why our percentages of control were higher
than those found by Molenaar, albeit still low overall
[29]. structured Framingham population and from our analysis
of usual clinical care are strikingly similar. This minimizes
to a large degree the concern that high rates of missing
BMI information could have distorted our findings, and
may explain why our percentages of control were higher
than those found by Molenaar, albeit still low overall
[29]. Discussion
d We examined the current state of BMI documentation and
documentation and control of associated risk factors by
BMI category, based on EHR data from almost 80,000
adult patients seen in a 12-clinic primary care network
during 18 months in 2005-2006. Our findings demon-
strate variations in risk factor recognition and control for
obese persons compared to those of normal weight, as
well as for those with and without CVD or type 2 diabetes. We found that documentation of BMI varied widely by
clinic site and was overall low. Among patients with a BMI
recorded, documentation of most risk factors was higher
in patients with obesity compared with normal weight
patients; however, control of risk factors was poorer in
obesity than normal weight. Patients without a docu-
mented history of CVD or diabetes had strikingly more
dissimilar rates of documentation and control between
weight categories than patients with CVD or diabetes. Overall, patients with obesity with or without CVD or dia- Table 3: Characteristics of 47,628 Primary Care Patients by BMI Category Table 3: Characteristics of 47,628 Primary Care Patients by BMI Category
Characteristic
Obese
n = 14,290
Overweight
n = 16,402
Normal
n = 16,936
P value
Age
(mean years)
53
53
48
<0.0001
Number of PCP visits
(mean)
4.7
4.2
3.9
<0.0001
Women
n (%)
8,565
59.9
8,899
54.3
13,272
78.4
<0.0001
Race*
n (%)
<0.0001
White
10,712
75.0
12,674
77.3
13,655
80.6
Asian
179
1.3
567
3.5
1,229
7.3
Black
1,244
8.7
989
6.0
649
3.8
Hispanic
1,722
12.1
1,561
9.5
811
4.8
Private Insurance or Medicare
n (%)
12,104
84.7
14,502
88.4
15,467
91.3
<0.0001
History of CVD
n (%)
2,647
18.5
2,699
16.5
1,862
11.0
<0.0001
History of diabetes
n (%)
3,238
22.7
1,789
10.9
824
4.9
<0.0001
History of hypertension
n (%)
9,116
63.8
7,895
48.1
4,931
29.1
<0.0001
History of dyslipidemia
n (%)
9,235
64.6
9,495
57.9
6,769
40.0
<0.0001
P-value is for general association across BMI categories. BMI = Body Mass Index (weight in kilograms/height in meters2), PCP = Primary Care
Physician, and CVD = Cardiovascular Disease. *Designation "Other" for race deleted from set. ese
n = 14,290
Overweight
n = 16,402
Normal
n = 16,936
P value P-value is for general association across BMI categories. BMI = Body Mass Index (weight in kilograms/height in meters2), PCP = Primary Care
Physician, and CVD = Cardiovascular Disease. *Designation "Other" for race deleted from set. Discussion
d While height and weight and
BMI documentation may reflect individual physician
practice styles, by speaking with medical directors we
found that lack of height and weight appeared to be a
clinic-level and not an individual PCP issue, with height
and weight recording often performed or not performed
before the PCP sees the patient. It is expected that intro-
duction of the HEDIS BMI Assessment measure will
improve this state of affairs, although the effectiveness of
BMI documentation alone to improve care remains to be
demonstrated. According to our study, patients with already documented
CVD or diabetes are more likely to have risk factor docu-
mentation and control regardless of BMI category. Those
without a documented history of CVD or diabetes demon- Page 6 of 13
(page number not for citation purposes) Page 6 of 13
(page number not for citation purposes) BMC Health Services Research 2009, 9:236 http://www.biomedcentral.com/1472-6963/9/236 ocumentation and control of all three risk factors by BMI category
igure 1
Documentation and control of all three risk factors by BMI category. A. Documentation of all 3 risk factors by BMI
ategory; B. Documentation of all 3 risk factors by BMI category, stratified by history of CVD; C. Documentation of all 3 risk
ctors by BMI category, stratified by history of diabetes. D. Control of all 3 risk factors by BMI category; E. Control of all 3 risk
ctors by BMI category, stratified by history of CVD; F. Control of all 3 risk factors by BMI category, stratified by history of
abetes. Discussion
d A
0
10
20
30
40
50
60
70
80
Obese
Overweight
Normal Weight
Percentage
B
0
10
20
30
40
50
60
70
80
Obese
Overweight
Normal Weight
Percentage
CVD
No CVD
E
0
10
20
30
40
50
60
70
80
Obese
Overweight
Normal Weight
Percentage
C
0
10
20
30
40
50
60
70
80
Obese
Overweight
Normal Weight
Percentage
DM
No DM
F
0
10
20
30
40
50
60
70
80
Obese
Overweight
Normal Weight
Percentage A
0
10
20
30
40
50
60
70
80
Obese
Overweight
Normal Weight
Percentage
B
0
10
20
30
40
50
60
70
80
Obese
Overweight
Normal Weight
Percentage
CVD
No CVD E
0
10
20
30
40
50
60
70
80
Obese
Overweight
Normal Weight
Percentage B E C
0
10
20
30
40
50
60
70
80
Obese
Overweight
Normal Weight
Percentage
DM
No DM F
0
10
20
30
40
50
60
70
80
Obese
Overweight
Normal Weight
Percentage F Documentation and control of all three risk factors by BMI category
Figure 1
Documentation and control of all three risk factors by BMI category. A. Documentation of all 3 risk factors by BMI
category; B. Documentation of all 3 risk factors by BMI category, stratified by history of CVD; C. Documentation of all 3 risk
factors by BMI category, stratified by history of diabetes. D. Control of all 3 risk factors by BMI category; E. Control of all 3 risk
factors by BMI category, stratified by history of CVD; F. Control of all 3 risk factors by BMI category, stratified by history of
diabetes. Discussion
d Page 7 of 13
(page number not for citation purposes) http://www.biomedcentral.com/1472-6963/9/236 BMC Health Services Research 2009, 9:236 BMC Health Services Research 2009, 9:236
http://www.biomedcentral.com/1472-6963/9/236
Table 5: Documentation of Risk Factors in 47,628 Patients in 12 Primary Care Clinics Stratified by BMI Category and History of CVD
or Diabetes
Risk Factors
Total
Obese
n = 14,290
Overweight
n = 16,402
Normal
Weight
n = 16,936
P value
History of
CVD
n
%
n
%
n
%
n
%
H/O CVD n
= 7,208
H/O CVD N = 2,647
H/O CVD N = 2,699
H/O CVD N = 1,862
NO H/O
CVD n =
40,420
No H/O CVD N = 11,643
NO H/O CVD N = 13,703
NO H/O CVD N = 15,074
BP
n = 10,900
n = 12,890
n = 13,144
H/O CVD
5,297
73.5
1,931
73.0
1,991
73.8
1,375
73.9
0.26
NO H/O
CVD
31,637
78.3
8,969
77.0
10,899
79.5
11,769
78.1
LDL
n = 11,239
n = 12,013
n = 10,175
H/O CVD
6,199
86.0
2,393
90.4
2,358
87.4
1,448
77.8
0.15
NO H/O
CVD
27,228
67.4
8,846
76.0
9,655
70.5
8,727
57.9
Casual glucose
n = 11,216
n = 11,901
n = 11,472
H/O CVD
6,305
87.5
2,355
89.0
2,322
86.0
1,628
87.4
0.0001
NO H/O
CVD
28,284
70.0
8,861
76.1
9,579
69.9
9,844
65.3
Fasting
glucose
n = 5,347
n = 4,804
n = 3,270
H/O CVD
2,741
38.0
1,158
43.8
1,023
37.9
560
30.1
<0.0001
NO H/O
CVD
10,680
26.4
4,189
36.0
3,781
27.6
2,710
18.0
All 3 risk
factors
n = 8,247
n = 8,797
n = 6,986
H/O CVD
4,417
61.3
1,696
64.1
1,693
62.7
1,028
55.2
<0.0001
NO H/O
CVD
19,613
48.5
6,551
56.3
7,104
51.8
5,958
39.5
History of DM
n
%
n
%
n
%
H/O
diabetes n =
5,851
H/O diabetes N = 3,238
H/O diabetes N = 1,789
H/O diabetes N = 824
NO H/O
diabetes
n = 41,777
NO H/O diabetes N =
11,052
NO H/O diabetes N = 14,613
NO H/O diabetes N =
16,112
BP
n = 10,900
n = 12,890
n = 13,144
H/O
diabetes
4,398
75.2
2,447
75.6
1,366
76.4
585
71.0
0.002
NO H/O
diabetes
32,536
77.9
8,453
76.5
11,524
78.9
12,559
78.0
LDL
n = 11,239
n = 12,013
n = 10,175
H/O
diabetes
5,285
90.3
2,963
91.5
1,617
90.4
705
86.2
0.51
NO H/O
diabetes
28,142
67.4
8,276
74.9
10,396
71.1
9,470
58.8
Casual glucose
n = 11,216
n = 11,901
n = 11,472
H/O
diabetes
5,225
89.3
2,884
89.1
1,596
89.2
745
90.4
<0.0001
NO H/O
diabetes
29,634
70.9
8,332
75.4
10,305
70.5
10,727
66.6
Fasting
glucose
n = 5,347
n = 4,804
n = 3,270 f Risk Factors in 47,628 Patients in 12 Primary Care Clinics Stratified by BMI Category and History of CVD http://www.biomedcentral.com/1472-6963/9/236 BMC Health Services Research 2009, 9:236 H/O
diabetes
2,736
46.8
1,619
50.0
812
45.4
305
37.0
0.003
NO H/O
diabetes
10,685
25.6
3,728
33.7
3,992
27.3
2,965
18.4
All 3 risk
factors
n = 8,247
n = 8,797
n = 6,986
H/O
diabetes
3,936
67.3
2,197
67.9
1,240
69.3
499
60.6
0.001
NO H/O
diabetes
20,094
48.1
6,050
54.7
7,557
51.7
6,487
40.3
P-value compares homogeneity of odds ratios across groups. Discussion
d Furthermore, despite its limitations,
evaluation of clinical data is a strength of this study, in
that it provides a glimpse into current obesity care and
insights into improving this practice. Although our data
were derived from a single academic health care network,
the sites included a representative mixture of urban, sub-
urban, and hospital-based practices, making our findings
generalizable and potentially replicable. Another impor-
tant strength of our study is the use of a long-standing,
widely used EHR encompassing all aspects of patient care
from a large network of diverse clinics and patient popu-
lations. EHRs, while not a perfect tool due to the potential
for improperly entered or overlooked data, have great
potential for research, with studies showing that EHRs
have potential for increasing documentation and treat-
ment of obese patients [33]. Finally while there were dif-
ferences seen in the percentages of documentation of risk
factors, this may be due mainly to test indication, whereby
certain tests such as cholesterol levels are more likely to be
ordered on most patients than fasting glucose. However,
our overall documentation numbers were large enough to
yield consistent results across BMI categories. strated more variation in risk factor documentation and
control by BMI category. At least two recent studies cor-
roborate these data. Melamed et al measured BMI in 289
patients in seven family practice clinics in Israel, and
found that BMI was documented in only half of obese
patients and 39% of overweight patients, and that patients
with other chronic medical conditions were more likely to
have BMI documented than those without documented
comorbidities [30]. Waring et al looked at 2,330 over-
weight and obese patients included in the Cholesterol
Education and Research Trial, and found increased odds
of overweight or obesity management in relation to
weight-related comorbidities for those with moderate or
severe obesity [31]. Risk factor control appears to be related to a previously
diagnosed risk factor and not to obesity. These findings
become even more relevant in light of recent studies that
demonstrate increased CVD risk in patients who are over-
weight and obese compared to normal weight patients,
independent of hypertension and hypercholesterolemia
[32]. This implies an important need to recognize over-
weight and obesity, ideally using a simple technique such
as BMI, in order to enhance CVD and diabetes complica-
tions prevention. Discussion
d CVD = Cardiovascular Disease, BMI = Body Mass Index (weight in kilograms/height in
meters2), BP = Blood Pressure, LDL = low density lipoprotein cholesterol, HDL = high density lipoprotein cholesterol. All 3 risk factors
documented = documentation of BP + LDL + casual OR fasting glucose. Numbers and percentages in table reflect percentage of risk factor
documented by BMI type in patients either with or without a history of CVD or DM (i.e., 1,931 = 73% of 2,647 patients who are obese with BP
documented with a history of CVD). Table 5: Documentation of Risk Factors in 47,628 Patients in 12 Primary Care Clinics Stratified by BMI Category and History of CVD
or Diabetes (Continued) f Risk Factors in 47,628 Patients in 12 Primary Care Clinics Stratified by BMI Category and History of CVD Table 5: Documentation of Risk Factors in 47,628 Patients in 12 Primary Care Clinics Stratified by BMI C
or Diabetes (Continued) Documentation of Risk Factors in 47,628 Patients in 12 Primary Care Clinics Stratified by BMI Category an
tes (Continued) P-value compares homogeneity of odds ratios across groups. CVD = Cardiovascular Disease, BMI = Body Mass Index (weight in kilograms/height in
meters2), BP = Blood Pressure, LDL = low density lipoprotein cholesterol, HDL = high density lipoprotein cholesterol. All 3 risk factors
documented = documentation of BP + LDL + casual OR fasting glucose. Numbers and percentages in table reflect percentage of risk factor
documented by BMI type in patients either with or without a history of CVD or DM (i.e., 1,931 = 73% of 2,647 patients who are obese with BP
documented with a history of CVD). P-value compares homogeneity of odds ratios across groups. CVD = Cardiovascular Disease, BMI = Body Mass Index (weight in kilograms/height in
meters2), BP = Blood Pressure, LDL = low density lipoprotein cholesterol, HDL = high density lipoprotein cholesterol. All 3 risk factors
documented = documentation of BP + LDL + casual OR fasting glucose. Numbers and percentages in table reflect percentage of risk factor
documented by BMI type in patients either with or without a history of CVD or DM (i.e., 1,931 = 73% of 2,647 patients who are obese with BP
documented with a history of CVD). a goal of our study. Conclusions It is well-accepted that intentional weight loss mitigates
many of the risk factors associated with obesity. Despite
rising rates of obesity, physicians appear not to routinely
assess BMI during office visits [34]. According to the
NCQA, multiple organizations recommend measuring
BMI as part of the routine physical examination
[16,9,10,14,15]. Treatment recommendations for obesity
depend on ascertainment of BMI and complications of
obesity. Therefore, screening for BMI and comorbidities
could change patient management. In light of new studies
implicating overweight and obesity as independent risk
factors for CVD, recognition of BMI becomes even more
important in the primary care setting. We examined a
large, diverse primary care network to evaluate current
care and found that EHRs will be a useful tool to evaluate Discussion
d Our findings suggest that PCPs are
aware of CVD, diabetes, and obesity as strongly tied risk
factors, but that they may not recognize obesity as a risk
factor for morbidity and mortality independent of these
other comorbidities. Page 9 of 13
(page number not for citation purposes) Limitations Our results must be interpreted with some limitations in
mind. This is clinical, not research, data, which naturally
suffers from information that is missing and inconsistent
in its recording. We carefully addressed missingness with
multiple methods of ascertaining exposures, and
addressed inconsistencies in data recording by removal of
clinically illogical extreme outliers. Limitations Evaluation of BMI
documentation rates, with the inherent missing data, was Page 9 of 13
(page number not for citation purposes) Page 9 of 13
(page number not for citation purposes) atients in 12 Primary Care Clinics Stratified by BMI Category and History of CVD or Diabetes
Total Controlled
Obese
Overweight
Normal
P value
%
n
%
n
%
n
%
n
%
H/O CVD N = 1,931
H/O CVD N = 1,991
H/O CVD N = 1,375
NO H/O CVD 8,969
NO H/O CVD N = 10,899
NO H/O CVD N = 11,769
n = 6,801
n = 9,202
n = 10,733
73.5
3,338
63.0
1,155
59.8
1,256
63.1
927
67.4
<0.0001
78.3
23,399
74.0
5,647
63.0
7,946
72.9
9,806
83.3
H/O CVD N = 2,393
H/O CVD N = 2,358
H/O CVD N = 1,448
NO H/O CVD N = 8,846
NO H/O CVD N = 9,655
NO H/O CVD N = 6,989
n = 8,777
n = 9,273
n = 8,278
86.0
5,515
89.0
2,152
89.9
2,074
88.0
1,289
89.0
0.002
67.4
20,813
76.4
6,625
74.9
7,199
74.6
6,989
80.1
H/O CVD N = 2,355
H/O CVD N = 2,322
H/O CVD N = 1,628
NO H/O CVD N = 8,861
NO H/O CVD N = 9,579
NO H/O CVD N = 9,844
n = 10,707
n = 11,635
n = 11,356
87.5
5,979
94.8
2,173
92.3
2,218
95.5
1,588
97.5
0.13
70.0
27,719
98.0
8,534
96.3
9,417
98.3
9,768
99.2
H/O CVD N = 1,158
H/O CVD N = 1,023
H/O CVD N = 560
NO H/O CVD N = 4,189
NO H/O CVD N = 3,781
NO H/O CVD N = 2,710
n = 2,753
n = 3,146
n = 2,583
38.0
1,325
48.3
415
35.8
561
54.8
349
62.3
0.0005
26.4
7,157
67.0
2,338
55.8
2,585
68.4
2,234
82.4
H/O CVD N = 829
H/O CVD N = 1,693
H/O CVD N = 1,028
NO H/O CVD N = 2,767
NO H/O CVD N = 7,104
NO H/O CVD N = 5,958
n = 3,596
n = 4,344
n = 4,327
61.3
2,252
51.0
829
48.9
850
50.2
573
55.7
<0.0001
48.5
10,015
51.1
2,767
42.2
3,494
49.2
3,754
63.0 02
3
005
001 7
5
4
01
Low
ect
tory
w the 01
7
5
4
01
Low
ect
tory
w the 01
7
5
4
01
Low
ect
tory
w the H/O diabetes N = 2,447
H/O diabetes N = 1,366
H/O diabetes N = 585
NO H/O diabetes 8,453
NO H/O diabetes N = 11,524 NO H/O diabetes N = 12,559
n = 6,801
n = 9,202
n = 10,733
75.2
2,775
63.1
1,476
60.3
919
67.3
380
65.0
<0.00
77.9
23,961
73.6
5,325
63.0
8,283
71.9
10,353
82
H/O diabetes N = 2,963
H/O diabetes N = 1,617
H/O diabetes N = 705
NO H/O diabetes N = 8,276
NO H/O diabetes N = 10,396
NO H/O diabetes N = 9,470
n = 8,777
n = 9,273
n = 8,278
90.3
4,746
89.8
2,635
88.9
1,462
90.4
649
92.1
0.5
67.4
21,583
76.7
6,143
74.2
7,811
75.1
7,629
80.6
H/O diabetes N = 2,884
H/O diabetes N = 1,596
H/O diabetes N = 745
NO H/O diabetes N = 8,332
NO H/O diabetes N = 10,305 NO H/O diabetes N = 10,727
n = 10,707
n = 11,635
n = 11,356
89.3
4,383
83.9
2,400
83.2
1,342
84.1
641
86.0
0.0
70.9
29,315
98.9
8,307
99.7
10,293
99.9
10,715
99.9
H/O diabetes N =
1,619
H/O diabetes N = 812
H/O diabetes N =
305
0.2
NO H/O diabetes N =
3,728
NO H/O diabetes N =
3,992
NO H/O diabetes
2,965
n = 2,753
n = 3,146
n = 2,583
46.8
554
20.2
285
17.6
170
20.9
99
32.5
25.6
7,928
74.2
2,468
66.2
2,976
74.6
2,484
83.8
H/O diabetes N = 2,197
H/O diabetes N = 1,240
H/O diabetes N = 499
<0.00
NO H/O diabetes N = 6,050
NO H/O diabetes N = 7,557
NO H/O diabetes N = 6,487
n = 3,596
n = 4,344
n = 4,327
67.3
1,754
44.6
929
42.3
587
47.3
238
47.7
48.1
10,513
52.3
2,667
44.1
3,757
49.7
4,089
63.0
ratios across groups. Authors' contributions y
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ysis, and Daniel Singer, M.D., for review and comment on an earlier draft
of the manuscript. Dr. Rose was supported by an Institutional National
Research Service Award #5 T32 HP11001-19. Dr. Grant was supported by
an NIDDK Career Development Award (K23 DK067452). Dr. Meigs was
supported by NIDDK K24 DK080140. Dr. Meigs currently has research
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here: http://www.biomedcentral.com/1472-6963/9/236/pre
b http://www.biomedcentral.com/1472-6963/9/236/pre
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https://openalex.org/W3186115997
|
https://www.frontiersin.org/articles/10.3389/fendo.2021.642698/pdf
|
English
| null |
Polymorphisms in Lysyl Oxidase Family Genes Are Associated With Intracranial Aneurysm Susceptibility in a Chinese Population
|
Frontiers in endocrinology
| 2,021
|
cc-by
| 8,661
|
Polymorphisms in Lysyl Oxidase
Family Genes Are Associated With
Intracranial Aneurysm Susceptibility
in a Chinese Population Chun Luo 1†, Chongyu Hu 2†, Bingyang Li 1,3, Junyu Liu 4, Liming Hu 1, Rui Dong 1,
Xin Liao 1,5, Jilin Zhou 4, Lu Xu 4, Songlin Liu 4, Yifeng Li 4, Dun Yuan 4, Weixi Jiang 4
and Junxia Yan 1,6* 1 Department of Epidemiology and Health Statistics, XiangYa School of Public Health, Central South University,
Changsha, China, 2 Department of Neurology, Hunan People’s Hospital, Changsha, China, 3 Department of Information
Statistics, Changsha Hospital of Traditional Chinese Medicine (Changsha Eight Hospital), Changsha, China, 4 Department of
Neurosurgery, XiangYa Hospital, Central South University, Changsha, China, 5 Department of Scientific Research, The
People’s Hospital of Guangxi Zhuang Autonomous Region, Nanning, China, 6 Hunan Provincial Key Laboratory of Clinical
Epidemiology, XiangYa School of Public Health, Central South University, Changsha, China Keywords: intracranial aneurysm, lysyl oxidase family genes, LOX gene, LOXL2, polymorphism Reviewed by: Reviewed by:
Fernando Rodriguez-Pascual,
Consejo Superior de Investigaciones
Cientı´ficas (CSIC), Spain
Philip C. Trackman,
Boston University, United States Fernando Rodriguez-Pascual,
Consejo Superior de Investigaciones
Cientı´ficas (CSIC), Spain
Philip C. Trackman,
Boston University, United States
*Correspondence:
Junxia Yan
20457456@qq.com
†These authors have contributed
equally to this work *Correspondence:
Junxia Yan
20457456@qq.com Methods: This case-control study included 384 patients with IA and 384 healthy
individuals without IA (controls). We genotyped 27 single nucleotide polymorphisms
(SNPs) of LOX family genes using the Sequenom MassARRAY
® platform. These SNPs
were adjusted for known risk factors and then, odds ratios (OR) and 95% confidence
intervals (CI) were evaluated using binary logistic regression analysis. †These authors have contributed
equally to this work Specialty section:
This article was submitted to
Systems Endocrinology,
a section of the journal
Frontiers in Endocrinology Results: The result showed that LOX rs10519694 was associated with the risk of IA in
recessive (OR, 3.88; 95% CI, 1.12–13.47) and additive (OR, 1.56; 95%CI, 1.05–2.34)
models. Stratified analyses illustrated that LOX rs10519694 was associated with the risk
of single IA in the recessive (OR, 3.95; 95%CI, 1.04–15.11) and additive (OR, 1.64; 95%
CI, 1.04–2.56) models. The LOXL2 rs1010156 polymorphism was associated with
multiple IA in the dominant model (OR, 1.92; 95%CI, 1.02–3.62). No associations were
observed between SNPs of LOXL1, LOXL3, and LOXL4 and risk of IA. Received: 18 December 2020
Accepted: 29 June 2021
Published: 28 July 2021 ORIGINAL RESEARCH
published: 28 July 2021
doi: 10.3389/fendo.2021.642698 Edited by: Edited by:
Marcus M. Seldin,
University of California, Irvine,
United States Purpose: Intracranial aneurysms (IA) comprise a multifactorial disease with unclear
physiological mechanisms. The lysyl oxidase (LOX) family genes (LOX, LOX–like 1–4)
plays important roles in extracellular matrix (ECM) reconstruction and has been
investigated in terms of susceptibility to IA in a few populations. We aimed to determine
whether polymorphisms in LOX family genes are associated with susceptibility to IA in a
Chinese population. Study Population y
p
We recruited 384 consecutive patients with IA at Xiangya
Hospital of Central South University and the Hunan Provincial
People’s Hospital (Chang Sha, Hunan province, China) since
January 2016. The diagnosis and phenotype information of IA
(number, location, and rupture status) was confirmed by
magnetic resonance imaging (MRI), computed tomography
(CTA), or digital subtraction (DSA) angiography. Other
neurological and vascular conditions such as arteriovenous
malformation and Marfan syndrome were excluded. We also
randomly selected 384 healthy individuals (control), who visited
a district community health service center for routine annual
health check-up and had no personal or family history of IA,
SAH, or other known related vascular diseases. Demographic
information and clinical data were collected using a
questionnaire and from medical records. None of the
participants were consanguineous. Peripheral venous blood
sample was collected from all participants into 5 mL EDTA–
K2 anticoagulant tubes (Sanli Medical Technology Co., Ltd.,
Liuyang, China). All participants provided written, informed
consent before the study was initiated. The study was performed
under the approval of the Ethics Committee at Central South
University (Permit No: CTXY–150002–1). Furthermore, to test
the robust of the association results identified in this study, an
additional population control was adopted (208 normal Chinese
Han individuals in the 1000 Genomes Project, the genotype
information was download from http://grch37.ensembl.org/
Homo_sapiens/Info/Index). In addition to the established acquired risk factors of cigarette
smoking and hypertension, genetic factors also play significant
roles in IA (3). Among patients with IA, 4% and 8% have first-
and second-degree relatives with IA, respectively (11). The
predicted probability of IA is higher among individuals with
hereditary pathologies such as autosomal dominant polycystic
kidney disease (ADPKD) and Marfan syndrome (12). The
extracellular matrix (ECM) is an indispensable element for
maintaining arterial wall stability. A disrupted balance between
ECM synthesis and degradation may be the pathophysiological
basis for IA formation (13, 14). For example, some single
nucleotide polymorphisms (SNPs) of endothelin receptor type
A (EDNRA), alpha 1 type III collagen (COL3A1), transcription
factor SOX-17 (SOX17), and lysyl oxidase-like 2 (LOXL2), which
are mainly involved in ECM reconstruction, are associated with
IA (15–18). The LOXL2 gene belongs to the lysyl oxidase (LOX)
family that comprises LOX and lysyl oxidase-like 1–4 (LOXL1,
LOXL2, LOXL3, and LOXL4) (19). INTRODUCTION polymorphisms in LOX family genes are associated with
susceptibility to IA in a Chinese population. Intracranial aneurysms (IA) are cystic bulges caused by local
defects or increased pressure in the intracranial artery wall (1). Unruptured intracranial aneurysms (UIA) occur in 3–5% of the
global population and in 7% of Chinese persons aged 35–75 years
(2–5). The annual risk of IA rupture is 0.5–1.8% (6, 7). The
rupture of an IA can result in aneurysmal subarachnoid
hemorrhage (aSAH), which can be fatal in 50% of patients and
cause 33% of severe sequelae (8, 9). Costs associated with aSAH
supervision exceed £510 million annually in the UK (10). Therefore, risk factors linked to IA should be determined as
soon as possible to screen and treat IA more effectively. Citation: Luo C, Hu C, Li B, Liu J, Hu L,
Dong R, Liao X, Zhou J, Xu L,
Liu S, Li Y, Yuan D, Jiang W and
Yan J (2021) Polymorphisms in
Lysyl Oxidase Family Genes Are
Associated With Intracranial Aneurysm
Susceptibility in a Chinese Population. Front. Endocrinol. 12:642698. doi: 10.3389/fendo.2021.642698 Conclusion: LOX and LOXL2 polymorphisms were associated with risk of single IA and
multiple IA in a Chinese population, suggesting potential roles of these genes in IA. The
effects of these genes on IA require further investigation. July 2021 | Volume 12 | Article 642698 1 Frontiers in Endocrinology | www.frontiersin.org LOX Family Genes and IA Luo et al. Study Population Lysyl oxidase can initiate
covalent crosslinking between soluble collagen and elastin
monomers that are converted into insoluble, stable fibers in
the ECM, thereby ensuring the structural and functional stability
of the arterial wall (20). Dysregulated LOX activity may play
significant roles in cardiovascular diseases such as atherosclerosis
and aneurysms (21), which are mediated by cross-linked
structural ECM proteins. Thus, the LOX family of genes might
be associated with IA. Frontiers in Endocrinology | www.frontiersin.org SNP Selection and Genotyping yp
g
We selected SNPs based on tag SNP and determined by Genome
Variation Server 150 (http://gvs.gs.washington.edu/GVS150/
index.jsp). The threshold of the LD parameter r2 was set at 0.8. The minor allele frequency (MAF) of all selected SNPs was > 5%. After tag SNP screening by gene name, priority was given to
variants with a proven relationship to IA or SNPs located in the
functional exonic region or that covered many other sites. We
finally selected 27 SNPs of LOX family genes into this study
(Table 1). Among them, LOX rs2303656, rs763497, and
rs3900446 SNPs were positively related to IA in a Korean
population (25), while LOXL2 rs1010156 and rs142252012
SNPs were positive in Japanese and Chinese families,
respectively (18, 26). So far, few population studies have explored associations
between polymorphisms in LOX family genes and IA, and the
results were inconsistent. For instance, associations between LOX
gene polymorphisms and IA have not been found in Japanese,
Central European, and South Indian populations (22–24). However, a recent population study in Korea found three SNPs
of LOX (rs2303656, rs3900446, and rs763497) were significantly
associated with IA (maximum OR, 20.15; P = 4.8 × 10–5) (25). Akagawa et al. (18) examined associations between LOXL1–4
gene polymorphisms and susceptibility to familial intracranial
aneurysms (FIA) in Japan and found that rs1010156 of LOXL2
was associated with FIA (OR, 1.49; P = 0.023). Using whole
exome sequencing (WES), Wu et al. (26) also discovered that the
rare variant of LOXL2, c.133C>T (rs142252012), is related to the
FIA susceptibility in a Chinese population. Considering genetic
heterogeneity among ethnic populations and that the genetic
background of FIA might differ from that of sporadic
intracranial aneurysms (SIA), we aimed to determine whether Genomic DNA was extracted using TIANamp Blood
Genomic DNA Isolation kits (TIANGEN Biotech Co., Ltd.,
Beijing, China) and stored at -80°C. Primers were designed
according to the SNP loci using Assign Design 3.1 software
(http://agenacx.com) and their sequences are detailed in Table 1. Target SNPs were genotyped using the MassARRAY iPlex
platform (Agena Bioscience Inc., San Diego, CA, USA). July 2021 | Volume 12 | Article 642698 Frontiers in Endocrinology | www.frontiersin.org 2 ms. SNP Selection and Genotyping Gene
(Genbank
accession
number)
SNPs
Position
Role
Variant
MAFd
Forward primer sequence
(5'-3')
Reverse primer sequence
(5'-3')
Length
(bp)
SNPs covered by tag SNPs
cDNA
Amino
Acid
LOXL3
(NM_032603)
rs715407
(T>G)a
Chr2:74538566
Intron
c.693-
1638T>G
–
0.19
GTCCCCTTTGGAACCTTTAC
AAGCTTCCCACTTCGAGTTC
133
–
rs6707302
(C>T)a
Chr2:74534295
Intron
c.1939
+21C>T
–
0.14
CACTATGATATCCTCACCCC
AAGGCTGTGCAATGGATACC
136
–
rs17010021
(T>A)a
Chr2:74534412
Missense
c.1843T>A
p.I615F
0.36
ACTCAGTGTCTTCGAGACAG
CTTCTCCCCACAGGCATTAC
120
–
rs17010022
(C>G)a
Chr2:74536131
Synonymous
c.1113C>G
p.L371L
0.32
ACTTCCTGATCTTTGCCATC
GTGAACAATCCTCAGCTGTG
128
–
LOXL4
(NM_032211)
rs3793692
(G>A)a
Chr10:98248679
3'UTR
c.242G>A
–
0.47
GGATGACTGGGTTTCCTTAC
GATGGCAAGATCACCAATCC
140
rs737656/rs737657
rs1983864
(G>T)a
Chr10:98257696
Missense
c.1214G>T
p.D406A
0.39
GATATGAGCGGACCCTCAG
ATGCTCAGACCCAAACTCAC
136
rs878177/rs1983866
rs7077266
(G>T)a
Chr10:98259282
Intron
c.702-
54G>T
–
0.19
TGGCATGAAGGGCCTCTATC
GGAGTTCTTATTCGTCAGGC
151
rs3763688 SNP, Single nucleotide polymorphisms; OR, odds ratio; CI, confidence interval; MAF, Minor allele frequency −, not available. aTag SNPs; bPositive variants associated with IAs or aSAH in previous studies; cThe second allele is the minor allele; dThe MAF in the HapMap-HCB population. SNP Selection and Genotyping e
Variant
MAFd
Forward primer sequence
(5'-3')
Reverse primer sequence
(5'-3')
Length
(bp)
SNPs covered by tag SNPs
cDNA
Amino
Acid
se
c.473C>T
p.R158L
0.17
AGAAGTTCCTGCGCTCAGTA
TGGGCCTTTCATAAGTATCG
134
rs2288393/rs10059661
c.1035
+528G>C
–
0.21
TTCACCTGTGAAACCATTCC
GAAATGGTGTCCTTCTGCTC
152
–
c.346-
935C>T
–
0.05
ATGCCACATCACTCCACTTG
CTGAGGAAACTTCTCTAGAC
135
–
c.1132-
113G>T
–
0.02
CTGGGCAACACAAAGAGTTC
TTTCCATAACGTCTCCAGAG
141
–
nic
–
–
0.13
ACATCTAGGCCTACATCGAG
TAAATGGCCCCCAACACAAG
129
–
nic
–
–
0.12
AGGAAGCAAAGCTCAGGTGG
CTTGAAGTTTCCCAGTAAGG
120
–
c.1102
+1976C>T
–
0.07
AAACTGAGCTCTCAAATGCC
CTCTCAATCAACTGGCTTCC
131
rs750460/rs4243042/ rs4886782/
rs12440667
se
c.458G>A
p.G153D
0.13
ACCTCCGTCTCCCAGCAAC
TAGTTCTCGTACTGGCTGAC
143
rs1078967/rs8041642/ rs8042039/
rs8041685
c.1602
+111C>A
–
0.23
TGTTCATGTCCAATGTCCCC
CTGAGACCTAAATCTTCGGC
140
rs1440101/rs12594472/rs16958494
se
c.1103-
293T>C
p.T446R
0.29
AGCTTACATCTCGAGCTCTG
TTCATGCTGTTTTCCCTGCC
143
rs2028386/rs4337252
mous
c.954C>T
p.A318A
0.14
TGCCAAGTGGCCACACCTC
CATGAAGAATGTCACCTGCG
146
rs2294127/rs2294126/rs2294129/
rs3779895/rs9792317/rs10503724/
rs11775841/rs11987443/ rs17089043/
rs17089055
c.355
+1496A>G
–
0.22
GTTGGAAGGGAGGATAACAG
AGAATAGCGCAGACCTCAAC
140
rs4872112/rs7835142/rs10096530/
rs10099318/rs10112621/rs11135728/
rs11992138/rs12674548/ rs12677717/
rs17089161
c.1151-
1948A>G
–
0.49
ATAGACGTTCAGCCACAAGG
AGCCAACTTAAGAGCCTAGC
126
rs7829632/rs11135726/rs12676713/
rs13248926/rs13259317/rs13272473/
rs13282766/rs17088178
c.-83-
5076G>A
–
0.43
CAAGAGATCCTCCTACTCAG
ACCTTTGGCAATTCATTGGC
148
rs7009281//rs11782783/rs4285498/
rs6983061/rs13252670/rs13254155
se
c.1708G>T
p.M570L
0.27
TCTCTTGCCTTGTTGACCAG
AGTTCTCCTCCATGGCACAC
136
rs3765215/rs3808521/ rs4273857/
rs4278162/rs4872103
c.219C>G
–
0.11
AGCAGCTCTGTGGACAAACC
CTACAGCTGTGTCTAAGCTC
119
rs1051157/rs7813349/rs7834641
mous
c.120C>T
p.P40P
0.23
CATTACCCCGAGTACTTCCA
CATCATAGTACACCTCCACC
139
rs17089187/rs4871870/rs6557667
c.426A>C
–
0.37
GGAGGGTTTCATTGGAAGAG
TGACACGTGGACAAATGCGG
127
rs2280937/rs11990784
mous
c.939T>C
p.S313S
0.45
CATGAAGAATGTCACCTGCG
GTCCTCACCTCTGGCTTGTA
120
–
se
c.133G>A
p.H45Y
0.01
CATCATAGTACACCTCCACC
ATTACCCCGAGTACTTCCAG
138
–
(Continued) Luo et al. LOX Family Genes and IA Polymerase chain reactions (PCR) were performed as described
(17) in 5 mL reaction mixtures containing 1.0 mL template DNA
(20–50 ng), PCR Primer mix (1 mL), 10 × PCR buffer (0.5 mL),
dddH2O (1.8 mL), 25 mM MgCl2 (0.4 mL), 25 mM dNTP
(0.1 mL), and 0.2 mL Taq polymerase (5 U/mL) (HotStarTaq®;
Qiagen GmbH, Hilden, Germany). The amplification cycles were
as follows: pre-denaturation for 2 min at 95°C, followed by 45
cycles of 30 sec at 9°C, 30 sec at 56°C, 60 sec at 72°C, and a final
elongation step of 5 min at 72°C. The amplified products were
stored at 25°C. When the PCR endpoint was reached, excess
dNTPs were removed using shrimp alkaline phosphatase
followed by single nucleotide extension and resin desalination
steps. We identified SNP genotypes and alleles using MALDI–
TOF–MS, mass spectrum peaks and MassArray TYPER 4.0
software. Finally, target genotypes were interpreted according
to the peaks. Statistical Analysis y
Data were analyzed using SPSS 23.0 (IBM Corp., Armonk, NY,
USA). Continuous variables are described as means ± standard
deviation (SD) and categorical variables are expressed as ratios
(%). Pairs of normally distributed continuous variables were
compared using Student t-tests, and distribution differences
between two categorical variables were compared using chi-
square or Fisher exact tests. The Hardy-Weinberg equilibrium
of each SNP in the control group was assessed using chi-square
tests. Pairwise LD was evaluated using the mean value of the
squared correlation coefficient (r2) and the standardized
disequilibrium coefficient (D’) in Haploview v.4.2 (https://
www.broadinstitute.org/haploview/haploview) (27). A pair of
SNPs was determined in strong LD under the criterion r2 ≥
80%. Complete LD was determined when D’ = 1 (28). We
calculated OR and 95% CI using binary logistic regression in
different genetic models after adjusting for known risk factors
(dominant, recessive, and additive models). The threshold of
significance was set at p < 0.05. Statistical power (SP) was
calculated according to the used significance level, sample size,
obtained OR and the corresponding genotype carrier rate in the
control group under two-sided Z test by using PASS 11 software
(NCSS LLC, Kaysville, Utah, USA). Frontiers in Endocrinology | www.frontiersin.org Characteristics of Study Population y
p
Females comprised 69.5% of the 384 patients with IA and the 384
controls and ~50% participants of both groups had hypertension
(P > 0.05). Among the patients, 48.7% had IA located in the
internal carotid arteries and 64.5% and 35.5% had single and
multiple IA, respectively. The distribution of gender and
hypertension did not differ significantly between patients with
single or multiple IA and the controls. The mean age was lower
and smoking, diabetes, and hyperlipidemia were less frequent in
the patients than in the controls (P < 0.05). In single or multiple
IA groups, approximately 50% and 20% of aneurysms were
located in the internal carotid and middle cerebral arteries, July 2021 | Volume 12 | Article 642698 4 LOX Family Genes and IA Luo et al. respectively. Table 2 lists the detailed characteristics of
the participants. hyperlipidemia. The SNP of LOX rs10519694 was associated
with an increased risk of single IA (recessive and additive models:
OR, 3.95; 95%CI, 1.04–15.11, P = 0.044; SP, 100% and OR, 1.64;
95%CI, 1.04–2.56, P = 0.032, SP, 42%, respectively, Table 4). The
association was verified when used the 208 additional population
control under additive model (OR, 1.97; 95%CI, 1.26–3.09,
P = 0.003) (Table S3). Another SNP of LOX rs2303656 was
found to be associated with a decreased risk of single IA
(dominant and additive models: OR, 0.44; 95%CI, 0.19–0.96; P
= 0.041 and OR, 0.44, 95%CI, 0.20–0.96; P = 0.04, respectively,
Table 4). However, the statistical power was relatively low (60%)
and the association was not verified in the additional population
control. Another gene polymorphism LOXL2 rs1010156 SNP
was found to be positively associated with risk of multiple IA in
the dominant model after adjusting for potential confounders
(OR, 1.92; 95% CI, 1.02–3.62, P = 0.044; Table 5) and the SP was
90% although the association was not replicated in the additional
control (Table S4). DISCUSSION We aimed to determine associations between polymorphisms of
LOX family genes and risk of IA in a Chinese population. We
found that the LOX rs10519694 and LOXL2 rs1010156
polymorphism was associated with the risk of single and
multiple IA respectively. As far as we can ascertain, this is the
first systematic exploration of associations between LOX family
genes polymorphisms and the risk of IA in a Chinese population,
and the findings offer a new perspective for the prevention and
treatment of IA. Associations Between LOX Family Genes
Polymorphisms and IA Susceptibility y
p
p
y
Table 3 lists the genotypes of 27 SNP of LOX family genes and
their associations with IA. The results of univariate logistic
regression analyses associated LOX rs10519694, LOXL1
rs3825942, and LOXL3 rs715407 polymorphisms with risk of
total IA (Table S1). However, only the association of LOX
rs10519694 and IA remained significant after adjusting for age,
smoking status, diabetes mellitus, and hyperlipidemia (recessive
and additive models: OR, 3.88; 95%CI, 1.12–13.47; p = 0.033; and
OR, 1.56; 95%CI, 1.05–2.34; p = 0.030, respectively; Table 3). The
calculated statistical power of the association of LOX rs10519694
and risk of IA under recessive and additive model was 100% and
41%, respectively. The association was verified when used the
208 Chinese Han populations in the 1000 Genome Project as
additional controls under additive model (OR, 1.89; 95%CI,
1.24–2.89; p = 0.003) (Table S2). Hardy-Weinberg Equilibrium and Linkage
Disequilibrium Analysis q
y
The detection rate of all SNP genotypes was 100%. Except for the
allele frequency and genotype distribution of LOXL2 rs2280935,
all SNPs in the controls were distributed in Hardy-Weinberg
equilibrium (P > 0.05; Table 3). Figure 1 shows the linkage
disequilibrium (LD) block of the targeted SNPs of the LOX
family genes in all participants. SD, standard deviation; Bold font indicates p < 0.05 compared with control group −, not available. Stratification Analysis The LOX family genes comprise five members which encode
LOX and LOX–like isoenzymes (LOXL1, LOXL2, LOXL3, and
LOXL4), which are copper-dependent oxidases with a conserved
C-terminal region that corresponds to the catalytic domain and a
different N-termini (29). Therefore, each isoform has similar
amine oxidase activities in which the ϵ-amino group of peptidyl Considering that single IA and multiple IA might be formed via
different mechanisms, we stratified the patients according to the
number of IA to assess associations between variants of LOX
family genes and risk of single or multiple IA. Only the LOX gene
polymorphism was associated with the risk of single IA after
adjusting for age, smoking status, diabetes mellitus, and TABLE 2 | Characteristics of the study population. Characteristics
IA
Control (n = 384)
Single IA (n = 248)
Multiple IA (n = 136)
Total (n = 384)
Age, years (Mean±SD)
57.3±10.4
56.7±10.9
57.1±10.6
66.5±2.1
Female, n (%)
166 (66.9)
101 (74.3)
267 (69.5)
267 (69.5)
Smoking, n (%)
30 (12.1)
15 (11.0)
59 (15.4)
111 (28.9)
Alcohol use, n (%)
24(9.7)
12 (8.8)
36 (9.4)
48 (12.5)
Hypertension, n (%)
127 (51.2)
81 (59.5)
208 (54.2)
200 (52.1)
Diabetes mellitus, n (%)
15 (6.0)
9 (6.6)
24 (6.3)
65 (16.9)
Hyperlipidemia, n (%)
13 (5.2)
9 (6.6)
22 (5.7)
8 (2.1)
Site of intracranial aneurysm, n (%)
Internal carotid artery
113 (45.6)
174 (51.0)
287 (48.7)
–
Middle cerebral artery
45 (18.1)
69 (20.2)
114 (19.4)
–
Anterior cerebral artery
15 (6.0)
24 (7.0)
39 (6.6)
–
Posterior cerebral artery
2 (0.8)
13 (3.8)
15 (2.5)
–
Anterior communicating artery
41 (16.5)
20 (5.9)
61 (10.4)
–
Posterior communicating artery
18 (7.3)
16 (4.7)
34 (5.8)
–
Basilar/Vertebral artery
14 (5.6)
25 (7.3)
39 (5.1)
–
SD, standard deviation; Bold font indicates p < 0.05 compared with control group −, not available. TABLE 2 | Characteristics of the study population. July 2021 | Volume 12 | Article 642698 5 LOX Family Genes and IA Luo et al. TABLE 3 | Multivariate logistic regression analysis of association of polymorphisms in LOX family genes and risk of IA in Chinese population. Stratification Analysis another polymorphism of LOX (rs2303656) with decreased risk
of IA (P = 8.2 × 10-4) in Korean population, we also identified
that this variant was associated with the decreased risk of single
IA in Chinese, even a definite conclusion couldn’t be drawn
considering the fact of that the statistical power was relatively low
and the association was not replicated in the additional control. Hong et al. also found that the C allele of LOX rs3900446 and
the G allele of rs763497 with increased risk of IA (OR, 20.15;
P = 4.8 × 10-5 and OR, 2.26; P = 4.8 × 10-5) in Korean population,
respectively (25). However, the polymorphisms of LOX gene is
not associated with IA in Japanese and Dutch (23), central
European (22) and South Indian (24) populations. Lan Ma
et al. found that another SNP of LOX (rs1800449) was
associated with the susceptibility to coronary artery diseases in
Chinese population (37) and a recent mouse model also
suggested that the missense mutation (p.G473A) caused by
rs1800449 could lead to loss tumor suppressor function of the
LOX propeptide and thus accelerate carcinogen-induced tumor
formation (38). However, this SNP was not found to be
associated with IA in our study. We speculate that these results
are associated with the population genetic heterogeneity or
limited statistical power of previous studies. These lines of
evidence illustrated that the polymorphisms of LOX may
perform roles in IA susceptibility in particular populations. The detail functions of the identified variants were not clear,
considering the fact of that rs10519694 and rs2303656 are both lysine is oxidized to generate peptidyl aldehyde (20, 30, 31). Collagen and elastin are the specific physiological substrates of
LOX family oxidases. Cross-linked collagen and elastic fibers are
essential for ECM stability and provide most of the tensile
strength and structural integrity of connective tissue (32). The
abnormal expression of LOX enzymes is related to changes in
ECM composition characteristic of atherosclerosis and aortic
aneurysms (21). The LOX located at 5q23.1, which was the first identified
subtype and represent of the LOX family genes.LOX catalyzes the
translation of peptidyl lysine and oxidizes it to peptidyl aldehyde
and a–aminohexyl–d semialdehyde. The collagen and elastin
crosslinking initiated by this chemical reaction assures
deposition of these fiber proteins in the ECM (33). Stratification Analysis Gene
SNP
Genotypea
Dominant model
Recessive model
Additive model
Case (n)
Control (n)
PHWE
b
OR (95%CI)
P value
OR (95%CI)
P value
OR (95%CI)
P value
LOX
rs1800449(C>T)
237/125/22
247/124/13
0.867
1.15(0.80-1.65)
0.450
1.52(0.65-3.56)
0.336
1.16(0.86-1.58)
0.328
rs2956540(G>C)
195/147/42
210/146/28
0.931
1.28(0.90-1.83)
0.164
1.34(0.71-2.51)
0.365
1.23(0.93-1.61)
0.142
rs10519694(C>T)
313/50/21
336/44/4
0.194
1.54(0.94-2.51)
0.084
3.88(1.12-13.47)
0.033
1.56(1.05-2.34)
0.030
rs2303656(G>T)
348/36/0
342/41/1
0.981
0.72(0.40-1.30)
0.274
–
–
0.71(0.39-1.28)
0.250
rs763497(A>G)
273/97/14
270/100/14
0.472
1.06(0.72-1.57)
0.753
0.95(0.39-2.34)
0.913
1.04(0.75-1.43)
0.824
rs3900446(A>G)
305/72/7
313/66/5
0.778
1.01(0.65-1.58)
0.954
2.37(0.63-8.90)
0.200
1.09(0.74-1.61)
0.673
LOXL1
rs2165241(C>T)
303/76/5
313/66/5
0.780
1.06(0.68-1.65)
0.786
1.09(0.26-4.50)
0.909
1.06(0.71-1.57)
0.784
rs3825942(G>A)
284/90/10
304/78/2
0.450
1.17(0.77-1.79)
0.458
1.85(0.30-11.28)
0.504
1.18(0.80-1.75)
0.398
rs2304721(C>A)
223/133/28
208/147/29
0.910
1.11(0.77-1.58)
0.571
0.92(0.47-1.77)
0.792
1.05(0.80-1.38)
0.739
rs12441130(T>C)
180/154/50
149/178/57
0.950
0.79(0.56-1.13)
0.202
0.94(0.57-1.54)
0.818
0.91(0.71-1.16)
0.907
LOXL2
rs2294128(C>T)
298/80/6
279/95/10
0.856
0.77(0.51-1.16)
0.209
0.62(0.17-2.26)
0.472
0.78(0.54-1.13)
0.186
rs7818494(A>G)
241/121/22
236/129/19
0.969
0.97(0.68-1.40)
0.887
0.83(0.38-1.80)
0.632
0.96(0.71-1.28)
0.765
rs4323477(A>G)
97/197/90
87/188/109
0.942
1.08(0.71-1.62)
0.731
0.67(0.45-1.01)
0.056
0.88(0.69-1.13)
0.327
rs7818416(G>A)
119/190/75
122/178/84
0.458
0.92(0.63-1.33)
0.644
0.84(0.54-1.30)
0.428
0.91(0.72-1.16)
0.458
rs1063582(G>T)
236/126/22
226/134/24
0.790
1.02(0.71-1.46)
0.922
1.08(0.51-2.29)
0.840
1.02(0.77-1.37)
0.875
rs2280936(C>G)
241/125/18
245/124/15
0.990
0.98(0.68-1.41)
0.920
1.41(0.61-3.22)
0.420
1.03(0.76-1.39)
0.837
rs2294133(C>T)
236/116/32
215/147/22
0.892
0.82(0.57-1.17)
0.276
1.39(0.69-2.78)
0.352
0.93(0.70-1.24)
0.627
rs2280935(A>C)
155/177/52
128/212/44
0.007
0.77(0.53-1.10)
0.144
1.56(0.92-2.66)
0.102
0.96(0.74-1.26)
0.787
rs1010156(T>C)
103/197/84
114/198/72
0.693
1.18(0.80-1.75)
0.401
1.07(0.69-1.66)
0.777
1.10(0.85-1.42)
0.473
rs142252012(G>A)
373/11/0
372/12/0
0.953
1.05(0.36-3.05)
0.927
–
–
1.05(0.36-3.05)
0.927
LOXL3
rs715407(T>G)
252/121/11
278/98/8
0.983
1.29(0.89-1.89)
0.183
0.95(0.29-3.06)
0.931
1.22(0.87-1.72)
0.246
rs6707302(C>T)
265/109/10
284/93/7
0.982
1.20(0.81-1.77)
0.360
0.92(0.26-3.25)
0.894
1.15(0.81-1.63)
0.431
rs17010021(T>A)
166/176/42
167/178/39
0.702
1.04(0.73-1.48)
0.831
0.87(0.49-1.56)
0.642
0.99(0.76-1.30)
0.958
rs17010022(C>G)
174/167/43
161/175/48
0.999
0.99(0.69-1.42)
0.982
0.83(0.48-1.45)
0.516
0.96(0.74-1.24)
0.742
LOXL4
rs3793692(G>A)
86/206/92
91/198/95
0.828
0.84(0.57-1.26)
0.410
0.97(0.65-1.47)
0.894
0.93(0.72-1.19)
0.555
rs1983864(G>T)
126/192/66
124/183/77
0.818
1.05(0.72-1.52)
0.817
0.73(0.46-1.15)
0.172
0.93(0.72-1.19)
0.549
rs7077266(G>T)
271/104/9
273/97/14
0.358
1.02(0.70-1.50)
0.911
0.81(0.31-2.16)
0.682
0.99(0.72-1.37)
0.964
SNP, single nucleotide polymorphism; OR, odds ratio; CI, confidence interval; −, not available. aGenotype presented as wild type/heterozygous/homozygous; bHardy-Weinberg equilibrium test; Bold font indicates p < 0.05. SNP, single nucleotide polymorphism; OR, odds ratio; CI, confidence interval; −, not available. aGenotype presented as wild type/heterozygous/homozygous; bHardy-Weinberg equilibrium test; Bold font indicates p < 0.05. Frontiers in Endocrinology | www.frontiersin.org Stratification Analysis Changes in
the ECM disrupt the vascular wall, as evidenced in
atherosclerosis, a cardiovascular disease that features vigorous
destruction of the ECM (34, 35). Some morphological changes
caused by atherosclerosis can result in the deposition of fibrous
tissues that ultimately develop into IA (13, 36). Thus, we
hypothesis that the disrupted LOX expression might be
associated with IA that is characterized by ECM destruction. The present study found that LOX rs10519694 was associated
with the increased risk of single IA in recessive and additive
models and the association was verified in the additional
population control. Furthermore, similar to the findings of a
case-control study by Hong et al., who associated the A allele of July 2021 | Volume 12 | Article 642698 Frontiers in Endocrinology | www.frontiersin.org 6 Luo et al. LOX Family Genes and IA FIGURE 1 | Graphical representation of the SNP locations and LD structure of each isoform of LOX family genes. The SNP distribution and haplotype block
structure across LOX family genes are shown. The measures of LD (r2) among all possible pairs of SNPs are shown graphically according to the shade of color,
where white represents very low r2 and scarlet represents very high r2. The numbers in squares are r2 values (r2 × 100). The graphics (A–C) represent the SNP
locations and LD structures of LOX gene in total, IA and control group, respectively. Accordingly, graphics (D–F) were for LOXL1, graphics (G–I) were for LOXL2
graphics (J–L) were for LOXL3 and graphic (M–O) were for LOXL4, respectively. | Graphical representation of the SNP locations and LD structure of each isoform of LOX family genes. The SNP distribution and haplotype bloc
across LOX family genes are shown. The measures of LD (r2) among all possible pairs of SNPs are shown graphically according to the shade of c
te represents very low r2 and scarlet represents very high r2. The numbers in squares are r2 values (r2 × 100). The graphics (A–C) represent the
and LD structures of LOX gene in total, IA and control group, respectively. Accordingly, graphics (D–F) were for LOXL1, graphics (G–I) were for L
J–L) were for LOXL3 and graphic (M–O) were for LOXL4, respectively. FIGURE 1 | Graphical representation of the SNP locations and LD structure of each isoform of LOX family genes. The SNP distribution and haplotype block
structure across LOX family genes are shown. Stratification Analysis The measures of LD (r2) among all possible pairs of SNPs are shown graphically according to the shade of color,
where white represents very low r2 and scarlet represents very high r2. The numbers in squares are r2 values (r2 × 100). The graphics (A–C) represent the SNP
locations and LD structures of LOX gene in total, IA and control group, respectively. Accordingly, graphics (D–F) were for LOXL1, graphics (G–I) were for LOXL2,
graphics (J–L) were for LOXL3 and graphic (M–O) were for LOXL4, respectively. FIGURE 1 | Graphical representation of the SNP locations and LD structure of each isoform of LOX family genes. The SNP distribution and haplotype block
structure across LOX family genes are shown. The measures of LD (r2) among all possible pairs of SNPs are shown graphically according to the shade of color,
where white represents very low r2 and scarlet represents very high r2. The numbers in squares are r2 values (r2 × 100). The graphics (A–C) represent the SNP
locations and LD structures of LOX gene in total, IA and control group, respectively. Accordingly, graphics (D–F) were for LOXL1, graphics (G–I) were for LOXL2,
graphics (J–L) were for LOXL3 and graphic (M–O) were for LOXL4, respectively. FIGURE 1 | Graphical representation of the SNP locations and LD structure of each isoform of LOX family genes. The SNP distribution and haplotype block
structure across LOX family genes are shown. The measures of LD (r2) among all possible pairs of SNPs are shown graphically according to the shade of color,
where white represents very low r2 and scarlet represents very high r2. The numbers in squares are r2 values (r2 × 100). The graphics (A–C) represent the SNP
locations and LD structures of LOX gene in total, IA and control group, respectively. Accordingly, graphics (D–F) were for LOXL1, graphics (G–I) were for LOXL2,
graphics (J–L) were for LOXL3 and graphic (M–O) were for LOXL4, respectively. July 2021 | Volume 12 | Article 642698 7 Frontiers in Endocrinology | www.frontiersin.org LOX Family Genes and IA Luo et al. TABLE 4 | Multivariate logistic regression analysis of associations of polymorphisms in LOX family genes and risk of single IA in a Chinese population. Stratification Analysis GENE
SNP
Genotypea
Dominant model
Recessive model
Additive model
Case (n)
Control (n)
OR (95%CI)
P value
OR (95%CI)
P value
OR (95%CI)
P value
LOX
rs1800449(C>T)
163/72/13
247/124/13
0.99(0.65-1.51)
0.972
1.31(0.48-3.58)
0.594
1.03(0.72-1.47)
0.876
rs2956540(G>C)
137/87/24
210/146/28
1.11(0.74-1.67)
0.615
1.21(0.58-2.51)
0.617
1.10(0.80-1.51)
0.546
rs10519694(C>T)
201/32/15
336/44/4
1.64(0.95-2.83)
0.078
3.95(1.04-15.11)
0.044
1.64(1.04-2.56)
0.032
rs2303656(G>T)
233/15/0
342/41/1
0.44(0.19-0.96)
0.041
–
–
0.44(0.20-0.96)
0.040
rs763497(A>G)
177/64/7
270/100/14
0.79(0.27-2.35)
0.672
1.01(0.65-1.58)
0.954
0.98(0.68-1.42)
0.919
rs3900446(A>G)
203/40/5
313/66/5
0.86(0.51-1.44)
0.560
3.64(0.92-14.34)
0.065
1.01(0.64-1.59)
0.969
LOXL1
rs2165241(C>T)
201/43/4
313/66/5
0.98(0.59-1.65)
0.952
1.30(0.28-6.16)
0.739
1.01(0.64-1.59)
0.966
rs3825942(G>A)
186/56/6
304/78/2
1.07(0.66-1.73)
0.792
2.29(0.32-16.38)
0.410
1.11(0.71-1.73)
0.656
rs2304721(C>A)
141/84/23
208/147/29
1.18(0.79-1.78)
0.420
1.18(0.58-2.41)
0.652
1.14(0.83-1.55)
0.420
rs12441130(T>C)
116/97/35
156/171/57
0.85(0.56-1.27)
0.424
1.19(0.69-2.06)
0.525
0.97(0.73-1.28)
0.820
LOXL2
rs2294128(C>T)
198/54/4
279/95/10
0.70(0.44-1.14)
0.152
0.43(0.08-2.21)
0.309
0.71(0.46-1.09)
0.117
rs7818494(A>G)
158/74/16
236/129/19
0.91(0.60-1.38)
0.664
0.99(0.42-2.33)
0.976
0.94(0.67-1.32)
0.718
rs4323477(A>G)
60/129/59
89/186/109
1.13(0.70-1.83)
0.616
0.72(0.45-1.14)
0.159
0.92(0.69-1.22)
0.559
rs7818416(G>A)
92/110/46
125/175/84
0.89(0.58-1.36)
0.586
0.72(0.43-1.21)
0.216
0.86(0.65-1.14)
0.297
rs1063582(G>T)
154/85/9
226/134/24
1.10(0.73-1.66)
0.647
0.84(0.34-2.09)
0.714
1.01(0.74-1.46)
0.816
rs2280936(C>G)
153/85/10
245/124/15
1.03(0.68-1.56)
0.880
1.11(0.39-3.13)
0.848
1.04(0.73-1.48)
0.846
rs2294133(C>T)
148/74/26
215/147/22
0.88(0.58-1.32)
0.530
1.88(0.89-3.96)
0.096
1.03(0.75-1.42)
0.839
rs2280935(A>C)
101/111/36
140/200/44
0.88(0.58-1.33)
0.531
1.58(0.86-2.91)
0.141
1.04(0.76-1.42)
0.795
rs1010156(T>C)
75/118/55
114/198/72
0.94(0.60-1.46)
0.780
0.95(0.57-1.60)
0.855
0.96(0.72-1.28)
0.772
rs142252012(G>A)
241/7/0
372/12/0
1.20(0.34-4.27)
0.780
–
–
1.20(0.34-4.27)
0.780
LOXL3
rs715407(T>G)
161/79/8
278/98/8
1.22(0.79-1.90)
0.366
1.18(0.32-4.36)
0.800
1.19(0.81-1.75)
0.381
rs6707302(C>T)
168/73/7
284/93/7
1.18(0.75-1.85)
0.475
1.12(0.26-4.69)
0.880
1.15(0.77-1.72)
0.494
rs17010021(T>A)
112/111/25
167/178/39
0.87(0.58-1.30)
0.493
0.78(0.39-1.54)
0.468
0.87(0.64-1.19)
0.392
rs17010022(C>G)
110/112/26
161/175/48
0.11(0.74-1.68)
0.609
0.85(0.45-1.61)
0.614
1.02(0.75-1.38)
0.892
LOXL4
rs3793692(G>A)
53/135/60
94/195/95
0.98(0.61-1.57)
0.930
0.99(0.62-1.59)
0.962
0.99(0.84-1.32)
0.933
rs1983864(G>T)
73/127/48
129/178/77
1.06(0.69-1.63)
0.791
0.77(0.45-1.31)
0.334
0.95(0.71-1.26)
0.717
rs7077266(G>T)
170/73/5
273/97/14
1.02(.66-1.59)
0.917
0.73(0.22-2.45)
0.615
0.99(0.68-1.44)
0.938
SNP, single nucleotide polymorphism; OR, odds ratio; CI, confidence interval; −, not available. aGenotype presented as wild type/heterozygous/homozygous; Bold font indicates p < 0.05. SNP, single nucleotide polymorphism; OR, odds ratio; CI, confidence interval; −, not available. aGenotype presented as wild type/heterozygous/homozygous; Bold font indicates p < 0.05. (R257G) could prevent LOXL2 from proteolytic cleavage by
serine protease, resulting in LOXL2 couldn’t remove the first
two SRCR domains and subsequently can’t bind to type IV
collagen, finally disturb the structural and functional stability of
ECM (29, 41). LOXL3 is expressed abundantly in the retina and
central nervous system; abnormal LOXL3 expression is
associated with cleft palate and spinal deformities (43). These
anomalies may be associated with the inability of LOXL3 to
effectively crosslink collagen (30). Stratification Analysis The LOXL4 is the latest
discovery in the LOX family and limited information is
available regarding this isoform. However, LOXL4 plays
specific roles in the proliferation and metastasis of cells in
certain malignancies, such as gastric and liver cancer (44, 45). Considering the role of LOXL4 in tumorigenesis, it may serve as
a potential independent prognostic marker and therapeutic
target for these cancers. Further detailed investigations are
necessary to fully understand the biological functions of LOX
family gene members. intronic variants, as a tagSNP, the associations between them and
IA might exist different possibilities: the polymorphism has a
causal role or the polymorphism has no causal role but is
associated with a nearby causal variant. The detailed
mechanism of LOX polymorphisms on IA should be
further explored. LOXL1–4 are located at 15q24.1, 8p21.3, 2p13.1, and 10q24.2,
respectively. Similar with LOX, LOXL1 also contains an N-
terminal propeptide, while the N-terminal of LOXL2-4 consists
of four scavenger receptor cysteine-rich (SRCR) domains (29). The biological effects of LOXL1 are similar to those of LOX, and
are significant not only for elastogenesis but also for supporting
the deposition of elastin (39). For example, a large-scale GWAS
study found that LOXL1 variants can increase the deposition of
elastin and fibrillin-1 that stabilizes the ECM, thereby protecting
against exfoliation syndrome (40). Since LOXL2 affects the
pathophysiological processes of cancer, such as cell adhesion
and invasion (30) and promotes endothelial tube formation by
cross–linking collagen IV in the vascular system, its expression
may be related to tumor angiogenesis and progression (21). The
glycosylation and proteolytic processing of extracellular LOXL2
are essential for cross–linking basement membrane type IV
collagen in the ECM (41, 42). A recent crystal structure study
found that a point mutation of LOXL2 (N455Q) could affect the
activity of the catalytic domain by eliminating the glycosylation
in the fourth SRCR domains of LOXL2, and another mutation We found that one SNP of LOXL2 rs1010156 associated with
the risk of multiple IA after adjusting for potential confounders
(OR, 1.92; 95% CI, 1.02–3.62, P = 0.044). Even the result was not
verified in the additional control, we thought that the previous
analysis was more reasonable due to that we just performed
univariate logistic regression analysis in the additional controls
and potential confounders were not adjusted due to these
informations was not available. Frontiers in Endocrinology | www.frontiersin.org Stratification Analysis Our findings were similar to July 2021 | Volume 12 | Article 642698 Frontiers in Endocrinology | www.frontiersin.org 8 LOX Family Genes and IA Luo et al. TABLE 5 | Univariate logistic regression analysis of association of polymorphisms in LOX family genes and risk of multiple IAs in a Chinese population. GENE
SNP
Genotypea
Dominant model
Recessive model
Additive model
Case (n)
Control (n)
OR (95%CI)
P value
OR (95%CI)
P value
OR (95%CI)
P value
LOX
rs1800449(C>T)
74/53/9
247/124/13
1.41(0.84-2.38)
0.198
1.77(0.58-5.38)
0.316
1.37(0.89-2.11)
0.150
rs2956540(G>C)
58/60/18
210/206/46
1.61(0.95-2.70)
0.075
1.59(0.67-3.78)
0.294
1.44(0.97-2.13)
0.070
rs10519694(C>T)
112/18/6
336/44/4
1.48(0.71-3.09)
0.297
4.28(0.76-24.08)
0.099
1.52(0.82-2.80)
0.184
rs2303656(G>T)
115/21/0
342/41/1
-
-
1.22(0.56-2.65)
0.614
1.19(0.56-2.55)
0.647
rs763497(A>G)
96/33/7
270/100/14
1.23(0.3-4.17)
0.741
1.13(0.65-1.98)
0.669
1.12(0.71-1.76)
0.638
rs3900446(A>G)
102/32/2
313/66/5
1.39(0.75-2.59)
0.299
0.65(0.04-9.88)
0.755
1.30(0.73-2.31)
0.374
LOXL1
rs2165241(C>T)
102/33/1
313/66/5
1.12(0.60-2.10)
0.722
0.66(0.07-6.3)
0.711
1.06(0.61-1.86)
0.834
rs3825942(G>A)
98/34/4
304/78/2
1.36(0.74-2.50)
0.315
1.11(0.11-10.41)
0.931
1.31(0.75-2.29)
0.349
rs2304721(C>A)
82/49/5
208/147/29
1.10(0.65-1.85)
0.730
0.52(0.14-1.88)
0.315
0.97(0.64-1.49)
0.904
rs12441130(T>C)
64/57/15
156/171/57
0.84(0.50-1.42)
0.512
0.62(0.28-1.39)
0.250
0.82(0.56-1.19)
0.297
LOXL2
rs2294128(C>T)
100/34/2
279/95/10
0.97(0.54-1.76)
0.922
1.10(0.20-5.96)
0.909
0.99(0.59-1.66)
0.959
rs7818494(A>G)
83/47/6
236/129/19
1.08(0.64-1.92)
0.786
0.66(1.19-2.33)
0.519
0.99(0.64-1.54)
0.984
rs4323477(A>G)
39/66/31
89/186/109
0.92(0.51-1.67)
0.793
0.92(0.51-1.67)
0.793
0.56(0.30-1.06)
0.075
rs7818416(G>A)
51/56/29
125/175/84
1.01(0.58-1.74)
0.977
1.13(0.61-2.10)
0.691
1.04(0.74-1.48)
0.809
rs1063582(G>T)
82/41/13
226/134/24
0.90(0.52-1.53)
0.684
1.80(0.65-5.04)
0.260
1.02(0.66-1.58)
0.928
rs2280936(C>G)
88/40/8
245/124/15
0.85(0.49-1.45)
0.542
2.04(0.72-5.83)
0.182
1.00(0.65-1.55)
1.000
rs2294133(C>T)
88/42/6
215/147/22
0.73(0.43-1.24)
0.248
0.62(1.18-2.14)
0.447
0.75(0.48-1.18)
0.215
rs2280935(A>C)
63/57/16
140/200/44
0.62(0.37-1.05)
0.077
1.47(0.68-3.18)
0.324
0.84(0.56-1.26)
0.398
rs1010156(T>C)
28/29/79
114/198/72
1.92(1.02-3.62)
0.044
1.31(0.69-2.49)
0.403
1.42(0.97-2.10)
0.073
rs142252012(G>A)
132/4
372/12/0
0.94(0.21-4.12)
0.932
–
–
0.94(0.21-4.12)
0.932
LOXL3
rs715407(T>G)
91/42/3
278/98/8
1.46(0.84-2.55)
0.177
0.64(0.09-4.42)
0.649
1.32(0.80-2.16)
0.277
rs6707302(C>T)
97/36/3
284/93/7
1.29(0.73-2.27)
0.376
0.68(0.09-4.92)
0.702
1.20(0.72-1.99)
0.489
rs17010021(T>A)
55/64/17
167/178/39
1.35(0.79-2.30)
0.269
1.12(0.49-2.53)
0.794
1.21(0.82-1.79)
0.339
rs17010022(C>G)
64/55/17
161/175/48
0.74(0.44-1.24)
0.252
0.85(0.38-1.93)
0.705
0.82(0.55-1.21)
0.306
LOXL4
rs3793692(G>A)
36/68/32
94/195/95
0.76(0.44-1.34)
0.347
0.99(0.54-1.85)
0.990
0.90(0.62-1.29)
0.550
rs1983864(G>T)
54/64/18
129/178/77
0.93(0.54-1.59)
0.777
0.71(0.35-1.43)
0.331
0.87(0.61-1.26)
0.471
rs7077266(G>T)
101/31/4
273/97/14
0.88(0.49-1.55)
0.647
1.10(0.30-3.98)
0.887
0.92(0.57-1.48)
0.742
SNP, single nucleotide polymorphism; OR, odds ratio; CI, confidence interval; −, not available. aGenotype presented as wild type/heterozygous/homozygous; Bold font indicates p < 0.05. SNP, single nucleotide polymorphism; OR, odds ratio; CI, confidence interval; −, not available. aGenotype presented as wild type/heterozygous/homozygous; Bold font indicates p < 0.05. population (18). Considering the limited power of the
statistics, larger sample size studies are needed in the future. the results obtained by Akagawa et al. Stratification Analysis (18) who systematically
screened LOXL family genes and reported that the LOXL2
polymorphism was associated with susceptibility to FIA. However, a comparison of SIA and FIA between patients and
controls did not reveal any associations. In addition, using WES,
Wu et al. found that a rare variant of LOXL2 c.133C>T
(rs142252012) can increase susceptibility to FIA (26), however,
we could not verify the association between this variant and
susceptibility to IA in Chinese patients with SIA. We found an
association between LOXL2 and multiple IA but not single IA,
which may be attributed to the involvement of different
pathophysiological pathways in the formation of multiple and
single IA. Unlike single IA, multiple IA were not distributed
randomly in the Circle of Willis, but were rather arranged in
clusters near the index aneurysm, which may predict the
formation of mirror aneurysms. Such aneurysmal clusters may
be affected by genetic and hemodynamic variables (46). Multiple
IA that develop more frequently in patients with familial IA can
be larger and rupture at a higher frequency in younger
individuals (14), which further suggests a genetic
predisposition. This study did not find an association between
LOXL1, LOXL3, and LOXL4 polymorphisms and susceptibility
to IA, which was consistent with the results of Akagawa et al.,
who found no associations between LOXL1, LOXL3, and LOXL4
gene polymorphisms and IA susceptibility in a Japanese the results obtained by Akagawa et al. (18) who systematically
screened LOXL family genes and reported that the LOXL2
polymorphism was associated with susceptibility to FIA. However, a comparison of SIA and FIA between patients and
controls did not reveal any associations. In addition, using WES,
Wu et al. found that a rare variant of LOXL2 c.133C>T
(rs142252012) can increase susceptibility to FIA (26), however,
we could not verify the association between this variant and
susceptibility to IA in Chinese patients with SIA. We found an
association between LOXL2 and multiple IA but not single IA,
which may be attributed to the involvement of different
pathophysiological pathways in the formation of multiple and
single IA. Unlike single IA, multiple IA were not distributed
randomly in the Circle of Willis, but were rather arranged in
clusters near the index aneurysm, which may predict the
formation of mirror aneurysms. Such aneurysmal clusters may
be affected by genetic and hemodynamic variables (46). Frontiers in Endocrinology | www.frontiersin.org REFERENCES 10. Riveroarias O, Gray A, Wolstenholme J. Burden of Disease and Costs of
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(2011) 10(7):626–36. doi: 10.1016/S1474-4422(11)70109-0 12. Caranci F, Briganti F, Cirillo L, Leonardi M, Muto M. Epidemiology and
Genetics of Intracranial Aneurysms. Eur J Radiol (2013) 82(10):1598–605. doi: 10.1016/j.ejrad.2012.12.026 13. Jung KH. New Pathophysiological Considerations on Cerebral Aneurysms. Neurointervention (2018) 13(2):73–83. doi: 10.5469/neuroint.2018.01011 3. Brinjikji W, Zhu YQ, Lanzino G, Cloft HJ, Kallmes D. Risk Factors for
Growth of Intracranial Aneurysms: A Systematic Review and Meta-Analysis. AJNR Am J Neuroradiol (2016) 37(4):615–20. doi: 10.3174/ajnr.A4575 14. Texakalidis P, Sweid A, Mouchtouris N, Peterson EC, Jabbour P. Aneurysm
Formation, Growth and Rupture: The Biology and Physics of Cerebral
Aneurysms. World Neurosurg (2019) 130:277–84. doi: 10.1016/j.wneu. 2019.07.093 4. Jeon TY, Jeon P, Kim KH. Prevalence of Unruptured Intracranial Aneurysm on MR
Angiography. Korean J Radiol (2011) 12(5):547–53. doi: 10.3348/kjr.2011.12.5.547 5. Li M-H, Chen S-W, Li Y-D, Chen Y-C, Cheng Y-S, Hu D-J, et al. Prevalence
of Unruptured Cerebral Aneurysms in Chinese Adults Aged 35 to 75 Years: A
Cross-Sectional Study. Ann Intern Med (2013) 159(8):514–21. doi: 10.7326/
0003-4819-159-8-201310150-00004 15. Theodotou CB, Snelling BM, Sur S, Haussen DC, Peterson EC, Elhammady
MS. Genetic Associations of Intracranial Aneurysm Formation and Sub-
Arachnoid Hemorrhage. Asian J Neurosurg (2017) 12(3):374–81. doi: 10.4103/1793-5482.180972 6. Sonobe M. Small Unruptured Intracranial Aneurysm Verification Study: SUAVe
Study, Japan. Stroke (2010) 41(9):1969–77. doi: 10.1161/STROKEAHA. 110.585059 16. Yasuno K, Bakircioglu M, Low SK, Bilguvar K, Gaal E, Ruigrok YM, et al. Common Variant Near the Endothelin Receptor Type A (EDNRA) Gene Is
Associated With Intracranial Aneurysm Risk. Proc Natl Acad Sci (2011) 108
(49):19707–12. doi: 10.1073/pnas.1117137108 7. AUTHOR CONTRIBUTIONS The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fendo.2021. 642698/full#supplementary-material Conceptualization: CL, CH, and JY. Data curation: CL, BL, and
JL. Formal analysis: CL and BL. Funding acquisition: JY. Conceptualization: CL, CH, and JY. Data curation: CL, BL, and
JL. Formal analysis: CL and BL. Funding acquisition: JY. CONCLUSIONS Investigation: CL, CH, BL, JL, LH, RD, XL, JZ, LX, SL, YL, DY,
and WJ. Methodology: CH, CL, and JY. Project administration:
CL, BL, and JY. Resources: CH, JL, WJ, and JY. Supervision: CL,
CH, and JY. Writing—original draft preparation: CL. Writing—
review and editing: JY. All authors contributed to the article and
approved the submitted version. Investigation: CL, CH, BL, JL, LH, RD, XL, JZ, LX, SL, YL, DY,
and WJ. Methodology: CH, CL, and JY. Project administration:
CL, BL, and JY. Resources: CH, JL, WJ, and JY. Supervision: CL,
CH, and JY. Writing—original draft preparation: CL. Writing—
review and editing: JY. All authors contributed to the article and
approved the submitted version. Investigation: CL, CH, BL, JL, LH, RD, XL, JZ, LX, SL, YL, DY,
and WJ. Methodology: CH, CL, and JY. Project administration:
CL, BL, and JY. Resources: CH, JL, WJ, and JY. Supervision: CL,
CH, and JY. Writing—original draft preparation: CL. Writing—
review and editing: JY. All authors contributed to the article and
approved the submitted version. Our findings showed that LOX and LOXL2 polymorphisms were
associated with risk of single IA and multiple IA in a Chinese
population, suggesting potential roles of these genes in IA. Further investigations are imperative to elucidate the effects of
these genes on IA. FUNDING The original contributions presented in the study are included in
the article/Supplementary Material. Further inquiries can be
directed to the corresponding author. This work was supported by the National Nature Science
Foundation, China [grant numbers: 81502881], and the Graduate
Student Innovative Scientific Research Project of Central South
University, China [grant numbers:1053320192459]. Stratification Analysis Multiple
IA that develop more frequently in patients with familial IA can
be larger and rupture at a higher frequency in younger
individuals (14), which further suggests a genetic
predisposition. This study did not find an association between
LOXL1, LOXL3, and LOXL4 polymorphisms and susceptibility
to IA, which was consistent with the results of Akagawa et al.,
who found no associations between LOXL1, LOXL3, and LOXL4
gene polymorphisms and IA susceptibility in a Japanese g
p
This study has several limitations. First, the controls comprised
individuals who had not been diagnosed with IA or other
cerebrovascular diseases. As some participants were assessed by
imaging modalities, classification might have been biased. However,
this influence was probably small considering that few people had
IA in China. Second, we explored associations between
polymorphisms in LOX family genes and risk of single and
multiple IAs. Due to the sample size was relatively small and the
statistical power was limited, the results may be not stable, further
studies with larger sample size are needed to verify our findings. Third, considering the fact that the identified associated variants
were tagSNP of target genes, we didn’t know whether these variants
were the real causal ones or just the surrogate of nearby causal
variants, the potential functions and the detail pathological
mechanisms of polymorphisms of LOX family genes on IA are
needed to further explored. Furthermore, this study has been
performed in a central south Chinese population and the results
may can’t directly extrapolate to other ethnicities considering the
fact that there may be population genetic heterogeneity. Our results
need to be validated in further studies. Nevertheless, this study is the
first to reveal associations between polymorphisms of the LOX
family genes and susceptibility to IA in the Chinese population and
offers a new perspective for the early diagnosis of IA. July 2021 | Volume 12 | Article 642698 Frontiers in Endocrinology | www.frontiersin.org 9 LOX Family Genes and IA Luo et al. ACKNOWLEDGMENTS The studies involving human participants were reviewed and
approved by The Ethics Committee at Central South University
(Permit No: CTXY–150002–1). The patients/participants
provided their written informed consent to participate in
this study. The authors thank all participants in this study. REFERENCES Matsumoto K, Oshino S, Sasaki M, Tsuruzono K, Taketsuna S, Yoshimine T. Incidence of Growth and Rupture of Unruptured Intracranial Aneurysms
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93. doi: 10.1159/000079940 39. Liu X, Zhao Y, Gao J, Pawlyk B, Starcher B, Spencer JA, et al. REFERENCES Linkage Disequilibrium for Different Scales and Applications. Brief Bioinform (2004) 4):355–64. doi: 10.1093/bib/5.4.355 29. Vallet SD, Ricard-Blum S. Lysyl Oxidases: From Enzyme Activity to
Extracellular Matrix Cross-Links. Essays Biochem (2019) 63(3):349–64. doi: 10.1042/ebc20180050 46. Roethlisberger M, Achermann R, Bawarjan S, Stienen MN, Fung C, Dalonzo
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and Disorders. J Glaucoma (2018) 27(p):S15–S9. doi: 10.1097/IJG. 0000000000000910 Conflict of Interest: The authors declare that the research was conducted in the
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012-0105-z Publisher’s Note: All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated organizations, or those of
the publisher, the editors and the reviewers. Any product that may be evaluated in
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of the Extracellular Matrix of the Arterial Wall to the Development of
Intracranial Aneurysms. Hum Mol Genet (2006) 15(22):3361–8. doi: 10.1093/hmg/ddl412 Copyright © 2021 Luo, Hu, Li, Liu, Hu, Dong, Liao, Zhou, Xu, Liu, Li, Yuan, Jiang
and Yan. This is an open-access article distributed under the terms of the Creative
Commons Attribution License (CC BY). The use, distribution or reproduction in other
forums is permitted, provided the original author(s) and the copyright owner(s) are
credited and that the original publication in this journal is cited, in accordance with
accepted academic practice. REFERENCES Elastic Fiber
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LOX on 5q31 With Intracranial Aneurysm. J Hum Genet (2003) 48(6):309–14. doi: 10.1007/s10038-003-0030-6 40. Tin A, Mineo O, Mei CL, Ursula S-S, Gudmar T, Takanori M, et al. Genetic
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of LOXL4 Promotes Hepatocellular Carcinoma Cell Invasion and Metastasis. Mol Cancer (2019) 18(1):18. doi: 10.1186/s12943-019-0948-8 28. Mueller JC. REFERENCES No use, distribution or reproduction is permitted which
does not comply with these terms. 35. Cristina Rodrı́guez JM-G, Raposo B, Alcudia JF, Guadall A, Badimon L. Regulation of Lysyl Oxidase in Vascular Cells: Lysyl Oxidase as a New Player
in Cardiovascular Diseases. Cardiovasc Res (2008) 79(1):7–13. doi: 10.1093/
cvr/cvn102 July 2021 | Volume 12 | Article 642698 Frontiers in Endocrinology | www.frontiersin.org
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Generalized Orthopair Fuzzy Weighted Power Bonferroni Mean Operator and Its Application in Decision Making
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Citation: Hou, B.; Chen, Y. Generalized Orthopair Fuzzy
Weighted Power Bonferroni Mean
Operator and Its Application in
Decision Making. Symmetry 2023, 15,
2007. https://doi.org/10.3390/
sym15112007 Bowen Hou and Yongming Chen * Bowen Hou and Yongming Chen * College of Applied Mathematics, Chengdu University of Information Technology, Chengdu 610225, China;
3221004010@stu.cuit.edu.cn
* Correspondence: chenym@cuit.edu.cn Abstract: The generalized orthopair fuzzy set is more favored by decision-makers and extensively
utilized in areas like supply chain management, risk investment, and pattern recognition because it
offers a broader decision information boundary than the intuitionistic fuzzy set and Pythagorean
fuzzy set. This enables it to express fuzzy information more comprehensively and accurately in multi-
attribute decision-making problems. To this end, this paper combines the ability of the power average
(PA) operator to eliminate the impact of extreme values and the advantage of the Bonferroni mean
(BMs,t) operator in reflecting the relationships between variables, then incorporates weight indicators
for different attributes to define the generalized orthopair fuzzy weighted power Bonferroni mean
operator. The effectiveness of this operator is demonstrated through aggregation laws for generalized
orthopair fuzzy information. Subsequently, the desirable properties of this operator are discussed. Based on these findings, a novel generalized orthopair fuzzy multi-attribute decision-making method,
with a correlation between attributes, is proposed. Lastly, an investment decision-making example
illustrates the feasibility and superiority of this method. Keywords: generalized orthopair fuzzy set; multiple-attribute decision making; weighted power
Bonferroni mean operator symmetry
S
S symmetry
S
S Citation: Hou, B.; Chen, Y.
Generalized Orthopair Fuzzy
Weighted Power Bonferroni Mean
Operator and Its Application in
Decision Making. Symmetry 2023, 15,
2007. https://doi.org/10.3390/
sym15112007 1. Introduction In practical decision-making problems, certain attribute values can be represented by
definite numerical figures, such as temperature, length, and the speed at which a car travels. However, for some issues, due to the complexity of the problem and the limitations in the
decision-maker’s knowledge, it becomes challenging to provide them in a definitive form. Examples include an individual’s perception of cold or the degree of acceptance towards a
particular item. Such issues often encompass multifaceted considerations and uncertainties,
making it difficult to arrive at an optimal decision using traditional mathematical methods. As a solution, fuzzy set theory, proposed by the cybernetics expert Zadeh, serves as a
potent tool for handling fuzzy and uncertain information in decision-making problems. He extended the classical sets to fuzzy sets and the characteristic functions to membership
functions. This made it possible to mathematically characterize fuzzy concepts, pioneering
a new perspective based on fuzzy sets to study uncertain phenomena [1]. Academic Editors: Jian Zhou,
Ke Wang and Yuanyuan Liu Received: 19 September 2023
Revised: 24 October 2023
Accepted: 26 October 2023
Published: 31 October 2023 p
p
y
y
p
Atanassov first introduced the concept of the intuitionistic fuzzy set (IFS) in 1983 [2]. Intuitionistic fuzzy sets encompass two dimensions, “membership degree u” and “non-
membership degree v”, satisfying the conditions u ∈[0, 1], v ∈[0, 1] and u + v ≤1. Additionally, intuitionistic fuzzy sets incorporated the notion of hesitancy, defining the
hesitancy degree π = 1 −u −v to represent the level of uncertainty regarding a particular
decision. Subsequently, Yager introduced the concept of the Pythagorean fuzzy set (PFS) [3]
and Fermatean fuzzy set (FFS) [4], which broadened the constraints of intuitionistic fuzzy
sets and provided a stronger capability in describing fuzzy phenomena. Copyright:
© 2023 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/). However, as decision-making conditions have become increasingly complex, the
applicability of the Pythagorean fuzzy set and Fermatean fuzzy set has become more https://www.mdpi.com/journal/symmetry Symmetry 2023, 15, 2007. https://doi.org/10.3390/sym15112007 Symmetry 2023, 15, 2007 2 of 15 restricted. For instance, when experts use 0.9 and 0.7 to represent the “membership
degree u” and “non-membership degree v” of their decision opinion, respectively, since
0.92 + 0.72 > 1, 0.93 + 0.73 > 1, the experts’ decision opinion (0.9, 0.7) cannot be represented
by the Pythagorean fuzzy set or Fermatean fuzzy set. To address such issues, Yager
once again introduced the concept of the generalized orthopair fuzzy set (i.e., q-rung
orthopair fuzzy set, q-ROF) [5]. The generalized orthopair fuzzy set not only results in less
information distortion, it also offers people a broader decision-making scope and provides
more possibilities for decision-making. p
g
Information aggregation operators based on intuitionistic fuzzy sets have been ex-
tensively studied. For instance, Zhou et al. investigated the power mean operator of
intuitionistic triangular fuzzy numbers [6], while Xu and colleagues introduced the intu-
itionistic fuzzy hybrid average operator [7]. Research on information aggregation operators
based on Pythagorean fuzzy set and Fermatean fuzzy set has also made some progress:
Akram et al. [8] introduced a series of Pythagorean Dombi fuzzy aggregation operators. Khan et al. [9] extended the prioritized aggregation operators to the Pythagorean fuzzy
environment to address decision-making problems where attributes and decision-makers
have a hierarchical relationship. Wei et al. [10] proposed a series of Pythagorean fuzzy
Hamacher power aggregation operators using Hamacher operation and power aggregation
in the Pythagorean fuzzy environment. Senapati et al. [11] studied the Fermatean fuzzy
weighted averaging and geometric operators. Senapati and Yager [12] introduced subtrac-
tion, division, and Fermatean arithmetic mean operations over the Fermatean fuzzy set. On the other hand, information aggregation operators based on the generalized orthopair
fuzzy set have been attracting a significant amount of scholarly attention in recent years. For example, Jun et al. proposed the generalized orthopair fuzzy Maclaurin symmetric
mean operator [13], Riaz investigated the generalized orthopair fuzzy geometric aggrega-
tion operators [14], Alcantud introduced complemental fuzzy sets and provided semantic
justification for generalized orthopair fuzzy sets [15], and so on. However, most of the current studies tend to aggregate information from a single
perspective; to address this limitation and propose operators that are more comprehensive
and better suited to increasingly complex multi-variable decision-making problems in
reality, this paper selects some of the most representative operators for multi-variable
information aggregation: the power average (PA) operator [16] and the Bonferroni mean
(BMs,t) operator [17]. The PA operator is leveraged for its ability to mitigate the negative
impacts from extreme evaluations made by experts and the BMs,t operator reflects the
correlation among input variables. By merging these operators and incorporating the
significance of weight indicators, we introduce the generalized orthopair fuzzy weighted
power Bonferroni mean operator and demonstrate its superior properties. Finally, we
further illustrate the feasibility and superiority of this operator through practical examples. y
p
y
p
g
p
p
The arrangement for the remainder of this paper is as follows. We introduce the pre-
liminaries such as the definition of GOF set, distance measure, and arithmetic laws firstly
in Section 2. Secondly, we propose the generalized orthopair fuzzy weighted power Bon-
ferroni mean operator and prove its feasibility, then study some of its desirable properties
in Section 3. In Section 4, we introduce a novel multi-attribute decision-making (MADM)
model based on the GOFWPBMs,t operator. After that, we use a practical example to
elucidate the superiority of this novel operator, then conduct some comparative analyses
under different parameters and existing methods in Section 5. Finally, we summarize some
conclusions and point out the application scenarios of this method in Section 6. Definition 1 [5]. Let X be a non-empty general set, then the expression for the generalized orthopair
fuzzy set A defined on X is given by 2. Preliminaries Definition 1 [5]. Let X be a non-empty general set, then the expression for the generalized orthopair
fuzzy set A defined on X is given by (1) A = {< x, uA(x), vA(x) >|x ∈X} A = {< x, uA(x), vA(x) >|x ∈X} Symmetry 2023, 15, 2007 3 of 15 3 of 15 where uA(x): X →[0, 1] and vA(x): X →[0, 1] represent the membership function and non-
membership function of A, respectively; q is a positive integer independent of x and ∀x ∈X,
0 ≤uA(x)q + vA(x)q ≤1. Define the degree of hesitation π(x) =
qq
1 −uA(x)q −vA(x)q. “Orthopair” refers to the simultaneous inclusion of both membership and non-
membership dimensions. Consequently, the intuitionistic fuzzy set, Pythagorean fuzzy set,
Fermatean fuzzy set, and generalized orthopair fuzzy set all fall within the domain of the
orthopair fuzzy set. The constraint condition for the generalized orthopair fuzzy set is that
the sum of the qth power of membership and the qth power of non-membership is less than
or equal to 1, that is, uq + vq ≤1. When q = 1, the generalized orthopair fuzzy set evolves
into an intuitionistic fuzzy set; when q = 2, it morphs into a Pythagorean fuzzy set; and
when q = 3, it evolves into a Fermatean fuzzy set as illustrated in Figure 1. Figure 1. Comparison of an intuitionistic fuzzy set, Pythagorean fuzzy set, Fermatean fuzzy set, and
generalized orthopair fuzzy set. Figure 1. Comparison of an intuitionistic fuzzy set, Pythagorean fuzzy set, Fermatean fuzzy set, and
generalized orthopair fuzzy set. Figure 1. Comparison of an intuitionistic fuzzy set, Pythagorean fuzzy set, Fermatean fuzzy set, and
generalized orthopair fuzzy set. The generalized orthopair fuzzy set allows for a broader boundary condition in
decision-making information, enabling a more comprehensive and accurate representation
of fuzzy information. This aligns more closely with real-life decision-making scenarios,
which Alcantud and colleagues have studied [15]. Moreover, it encompasses the special
cases of q = 1 (intuitionistic fuzzy set), q = 2 (Pythagorean fuzzy set), and q = 3 (Fermatean
fuzzy set), offering greater versatility and a broader range of applications. It serves as an
effective tool for depicting phenomena characterized by uncertainty. 2. Preliminaries It is noteworthy that there are other extensions of intuitionistic fuzzy set and
Pythagorean fuzzy set, which do not further extend the “q” exponent, but instead hybridize
the model through other methods, such as the interval-valued intuitionistic fuzzy set [18],
complex Pythagorean fuzzy set [19], Pythagorean fuzzy soft rough set [20], intuitionistic
fuzzy soft set [21], and so on. Definition 2 [22]. Let ai = (ui, vi)(i = 1, 2), a = (u, v) be three generalized orthopair fuzzy
numbers, λ is any real number greater than or equal to zero. The operational laws are defined
as follows (1)
a1
L a2 = ( qq
uq
1 + uq
2 −uq
1uq
2, v1v2);
(2)
a1
N a2 = (u1u2, qq
vq
1 + vq
2 −vq
1vq
2);
(3)
λa = (
qq
1 −(1 −uq)λ, vλ);
(4)
aλ = (uλ,
qq
1 −(1 −vq)λ). From the above, we can derive the following conclusions From the above, we can derive the following conclusions 4 of 15 Symmetry 2023, 15, 2007 (1)
a1
L a2 = a2
L a1;
(2)
a1
N a2 = a2
N a1;
(3)
λ(a1
L a2) = λa1
L λa2;
(4)
(a1
N a2)λ = aλ
1
N aλ
2;
(5)
λ1a L λ2a = (λ1 + λ2)a;
(6)
aλ1 N aλ2 = aλ1+λ2. (1)
a1
L a2 = a2
L a1;
(2)
a1
N a2 = a2
N a1;
(3)
λ(a1
L a2) = λa1
L λa2;
(4)
(a1
N a2)λ = aλ
1
N aλ
2;
(5)
λ1a L λ2a = (λ1 + λ2)a;
(6)
aλ1 N aλ2 = aλ1+λ2. Definition 3 [22]. Let a = (u, v) be a generalized orthopair fuzzy number, then the score-valued
function of a is defined as S(a) = uq −vq, and the accuracy-valued function of a is defined as
H(a) = uq + vq. Based on the functions S(a) and H(a), for any two generalized orthopair fuzzy
numbers a1 = (u1, v1), a2 = (u2, v2), the comparison method is defined as (1)
if S(a1) > S(a2), then a1 > a2;
(2)
if S(a1) = S(a2) and H(a1) < H(a2), then a1 < a2;
(3)
if S(a1) = S(a2) and H(a1) = H(a2), then a1 = a2. Definition 4 [22]. 2. Preliminaries 5 of 15 Symmetry 2023, 15, 2007 5 of 15 3. Generalized Orthopair Fuzzy Weighted Power Bonferroni Mean Operator
3.1. Definition and Demonstration of GOFWPBM Operator To harness the combined benefits of the BMs,t operator and the power average (PA)
operator, He et al. [32] combined the PA operator with the BMs,t operator, introducing
the power Bonferroni mean
PBMs,t
operator. Subsequently, the PBMs,t operator was
extended to various fuzzy environments, including hesitant fuzzy sets [32], intuitionistic
fuzzy sets [33,34], interval intuitionistic fuzzy sets [35], and linguistic intuitionistic fuzzy
sets [36]. Further, Khan et al. [37], employing Dombi operations, expanded the PBMs,t
operator into the interval-valued intuitionistic setting to tackle multi-attribute decision-
making problems with interval-valued intuitionistic information. However, to date, there
has been no research on how to employ the PBMs,t operator to aggregate generalized
orthopair fuzzy numbers. Hence, to develop an comprehensive method for generalized
orthopair fuzzy numbers and expand the application domain of the PBMs,t operator, we
subsequently introduce the generalized orthopair fuzzy power Bonferroni mean operator. Definition 7 [32]. Let ai(i = 1, 2, · · · , n) be a set of non-negative real numbers, s and t are
non-negative real numbers that are not both 0, then the power Bonferroni mean
PBMs,t
operator
can be defined as (a1, a2, · · · , an) = [
1
n(n −1)∑
n
i,j=1,i̸=j
(
n(1 + T(ai))
∑n
k=1 (1 + T(ak)) ai)s(
n(1 + T(aj))
∑n
k=1 (1 + T(ak)) aj)t
! ]
1
s+t
(5)
where T(ai) = ∑n
j=1,j̸=i Supp
ai, aj
(i = 1, 2, · · · , n), Supp
ai, aj
= 1 −d
ai, aj
represents
the support degree between ai and aj. (5) Definition 8. Let s and t be non negative real numbers that are not both 0, and let ai(i = 1, 2, · · · , n)
be a set of generalized orthopair fuzzy numbers. Then, the generalized orthopair fuzzy power Bonfer-
roni mean
GOFPBMs,t
operator of ai(i = 1, 2, · · · , n) can be defined as PBMs,t(a1, a2, · · · , an) = [
1
n(n −1)
n
L
i, j = 1, i ̸= j
(
n(1 + T(ai))
∑n
k=1 (1 + T(ak)) ai)s O
(
n(1 + T(aj))
∑n
k=1 (1 + T(ak)) aj)t
! 2. Preliminaries Assuming a1 = (u1, v1), a2 = (u2, v2) are any two generalized orthopair
fuzzy numbers, the hamming distance d(a1, a2) between a1 and a2 can be defined as Definition 4 [22]. Assuming a1 = (u1, v1), a2 = (u2, v2) are any two generalized orthopair
fuzzy numbers, the hamming distance d(a1, a2) between a1 and a2 can be defined as d(a1, a2) =
uq
1 −uq
2
+
vq
1 −vq
2
+
πq
1 −πq
2
2
(2) (2) The power average (PA) operator is an aggregation operator that effectively considers
the interrelationships among data information. By taking into account the support relation-
ships among input data to calculate attribute weights, it diminishes the adverse impact of
outlier data on decision-making results, making the decision information processing more
objective and impartial. Accordingly, it has garnered attention from many scholars [23–26]. Definition 5 [16]. Assuming ai(i = 1, 2, · · · , n) to be a set of non-negative real numbers, the
result aggregated by the power average operator of ai(i = 1, 2, · · · , n) is given by PA(a1, a2, · · · , an) = ∑
n
i=1
1 + T(ai)
∑n
i=1 (1 + T(ai)) ai
(3) (3) where PA is termed as the power average operator. Here, T(ai) = ∑n
j=1,j̸=i Supp
ai, aj
(i = 1, 2, · · · , n), Supp
ai, aj
=
1 −d
ai, aj
represents the support degree between
ai and aj. The BMs,t operator is a particular type of aggregation function. The most significant
advantage of this operator is its ability to reflect interrelationships among input variables,
akin to the Heronian mean operator. With the continuous advancement of contemporary
society, many attributes in real-world decision-making scenarios exhibit strong interrela-
tions. Consequently, due to this characteristic, the BMs,t operator is extensively utilized in
information aggregation and multi-attribute decision-making [27–31]. Definition 6 [17]. Let parameters s, t ≥0 and s + t > 0, ai(i = 1, 2, · · · , n) be a series of
non-negative real numbers, if BMs,t(a1, a2, · · · , an) = (
1
n(n −1)∑
n
i,j=1,i̸=j as
i at
j)
1
s+t
(4) (4) then BMs,t is referred to as the Bonferroni mean operator. then BMs,t is referred to as the Bonferroni mean operator. 2. Preliminaries ]
1
s+t
(6)
where T(ai) = ∑n
j=1,j̸=i Supp
ai, aj
(i = 1, 2, · · · , n), Supp
ai, aj
= 1 −d
ai, aj
represents
the support degree between ai and aj. (6) Given the aforementioned formulas, it is evident that while this operator combines the
strengths of both the PA and BMs,t operators, it only contemplates the correlation between
the weight vectors derived from the power average operator and the generalized orthopair
fuzzy numbers pending aggregation. Notably, the intrinsic significance of the data has
not been taken into account, meaning this operator is formulated under the premise that
the significance level of the aggregating variables is uniform. However, in many practical
decision-making problems, the significance of distinct attributes might differ, thereby
leading to non-uniform weights. To incorporate the importance of attribute weights,
we will proceed to define the generalized orthopair fuzzy weighted power Bonferroni
mean operator. Definition 9. Let s and t be non-negative real numbers that are not both 0, and let ai(i = 1,2,· · · , n)
be a set of generalized orthopair fuzzy numbers. Then, the generalized orthopair fuzzy weighted
power Bonferroni mean
GOFWPBMs,t
operator of ai(i = 1, 2, · · · , n) can be defined as GOFWPBMs,t(a1, a2, · · · , an) = [
1
n(n −1)
n
L
i, j = 1, i ̸= j
(
nωi(1 + T(ai))
∑n
k=1 ωk(1 + T(ak)) ai)s O
(
nωj(1 + T(aj))
∑n
k=1 ωk(1 + T(ak)) aj)t
! ]
1
s+t (7) Symmetry 2023, 15, 2007 6 of 15 where ω
=
(ω1, ω2, ω3 . . . ωn)T represents a set of weight vectors and ∑ωi
=
1. T(ai) = ∑n
j=1,j̸=i ωjSupp
ai, aj
(i = 1, 2, · · · , n). where ω
=
(ω1, ω2, ω3 . . . ωn)T represents a set of weight vectors and ∑ωi
=
1. T(ai) = ∑n
j=1,j̸=i ωjSupp
ai, aj
(i = 1, 2, · · · , n). where ω
=
(ω1, ω2, ω3 . . . ωn)T represents a set of weight vectors and ∑ωi
=
1. T(ai) = ∑n
j=1,j̸=i ωjSupp
ai, aj
(i = 1, 2, · · · , n). Theorem 1. GOFWPBMs,t(a1, a2, · · · , an) =
1 −[∏
n
i, j = 1
i ̸= j
(1 −(1 −(1 −uq
i )
nωi(1+T(ai))
∑n
k=1 ωk(1+T(ak)) )s(1 −(1 −uq
j )
nωj(1+T(aj))
∑n
k=1 ωk(1+T(ak)) )t)]
1
n(n−1)
1
s+t
, q
v
u
u
u
u
u
u
t1 −
1 −[∏
n
i, j = 1
i ̸= j
[1 −(1 −v
qωin(1+T(ai))
∑n
k=1 ωk(1+T(ak))
i
)s(1 −v
qωjn(1+T(aj))
∑n
k=1 ωk(1+T(ak))
j
)t]
1
n(n−1)
1
s+t
)
(8) (8) Proof of Theorem 1. 2. Preliminaries Assume s and t are non-negative real numbers that are not simultaneously 0, and
ai(i = 1, 2, · · · , n) are generalized orthopair fuzzy numbers, then use the GOFWPBMs,t operator
which aggregates these generalized orthopair fuzzy numbers and the aggregated value is still a
generalized orthopair fuzzy number, and GOFWPBMs,t(a1, a2, · · · , an) = GOFWPBMs,t(a1, a2, · · · , an) = Proof of Theorem 1. Given And And nωj(1 + T(aj))
∑n
k=1 ωk(1 + T(ak)) aj = (
q
s
1 −(1 −uq
j )
nωj(1+T(aj))
∑n
k=1 ωk(1+T(ak)) , v
qωjn(1+T(aj))
∑n
k=1 ωk(1+T(ak))
j
) It follows that (
nωi(1 + T(ai))
∑n
k=1 ωk(1 + T(ak)) ai)s = ((
q
r
1 −(1 −uq
i )
nωi(1+T(ai))
∑n
k=1 ωk(1+T(ak)) )s,
q
s
1 −(1 −v
qωin(1+T(ai))
∑n
k=1 ωk(1+T(ak))
i
)s),
s
s
(1+T(
)) (
nωi(1 + T(ai))
∑n
k=1 ωk(1 + T(ak)) ai)s = ((
q
r
1 −(1 −uq
i )
nωi(1+T(ai))
∑n
k=1 ωk(1+T(ak)) )s,
q
s
1 −(1 −v
qωin(1+T(ai))
∑n
k=1 ωk(1+T(ak))
i
)s), (
nωi(1 + T(ai))
∑n
k=1 ωk(1 + T(ak)) ai)s = ((
q
r
1 −(1 −uq
i )
nωi(1+T(ai))
∑n
k=1 ωk(1+T(ak)) )s,
q
s
1 −(1 −v
qωin(1+T(ai))
∑n
k=1 ωk(1+T(ak))
i
)s),
(
nωj(1 + T(aj))
∑n
k=1 ωk(1 + T(ak)) aj)t = ((
q
s
1 −(1 −uq
j )
nωj(1+T(aj))
∑n
k=1 ωk(1+T(ak)) )t,
q
s
1 −(1 −v
qωjn(1+T(aj))
∑n
k=1 ωk(1+T(ak))
j
)t) (
nωj(1 + T(aj))
∑n
k=1 ωk(1 + T(ak)) aj)t = ((
q
s
1 −(1 −uq
j )
nωj(1+T(aj))
∑n
k=1 ωk(1+T(ak)) )t,
q
s
1 −(1 −v
qωjn(1+T(aj))
∑n
k=1 ωk(1+T(ak))
j
)t) Hence
(
nωi(1 + T(ai))
∑n
k=1 ωk(1 + T(ak)) ai)s O
(
nωj(1 + T(aj))
∑n
k=1 ωk(1 + T(ak)) aj)t =
(
q
r
1 −(1 −uq
i )
nωi(1+T(ai))
∑n
k=1 ωk(1+T(ak)) )s(
q
s
1 −(1 −uq
j )
nωj(1+T(aj))
∑n
k=1 ωk(1+T(ak)) )t, Hence Hence (
nωi(1 + T(ai))
∑n
k=1 ωk(1 + T(ak)) ai)s O
(
nωj(1 + T(aj))
∑n
k=1 ωk(1 + T(ak)) aj)t = (
q
r
1 −(1 −uq
i )
nωi(1+T(ai))
∑n
k=1 ωk(1+T(ak)) )s(
q
s
1 −(1 −uq
j )
nωj(1+T(aj))
∑n
k=1 ωk(1+T(ak)) )t, 7 of 15 7 of 15 Symmetry 2023, 15, 2007 q
s
2 −(1 −v
qωin(1+T(ai))
∑n
k=1 ωk(1+T(ak))
i
)s −(1 −v
qωjn(1+T(aj))
∑n
k=1 ωk(1+T(ak))
j
)t −[1 −(1 −v
qωin(1+T(ai))
∑n
k=1 ωk(1+T(ak))
i
)s][1 −(1 −v
qωjn(1+T(aj))
∑n
k=1 ωk(1+T(ak))
j
)t])
n
L
i, j = 1, i ̸= j
[(
nωi(1 + T(ai))
∑n
k=1 ωk(1 + T(ak)) ai)s O
(
nωj(1 + T(aj))
∑n
k=1 ωk(1 + T(ak)) aj)t] =
( q
v
u
u
u
t1 −∏
n
i, j = 1
i ̸= j
[1 −(1 −(1 −uq
i )
nωi(1+T(ai))
∑n
k=1 ωk(1+T(ak)) )s(1 −(1 −uq
j )
nωj(1+T(aj))
∑n
k=1 ωk(1+T(ak)) )t],
q
v
u
u
u
u
t∏
n
i, j = 1
i ̸= j
[1 −(1 −v
qnωi(1+T(ai))
∑n
k=1 ωk(1+T(ak))
i
)s(1 −v
qnωj(1+T(aj))
∑n
k=1 ωk(1+T(ak))
j
)t]) n
L
i, j = 1, i ̸= j
[(
nωi(1 + T(ai))
∑n
k=1 ωk(1 + T(ak)) ai)s O
(
nωj(1 + T(aj))
∑n
k=1 ωk(1 + T(ak)) aj)t] = We use mathematical induction to prove the following theorem
(I)
For n = 2, We use mathematical induction to prove the following theorem
(I)
For n = 2, (I)
For n = 2, (I)
For n = 2, 2
L
i, j = 1, i ̸= j
[(
nωi(1 + T(ai))
∑n
k=1 ωk(1 + T(ak)) ai)s O
(
nωj(1 + T(aj))
∑n
k=1 ωk(1 + T(ak)) aj)t] = [(
2ω1(1 + T(a1))
∑2
k=1 ωk(1 + T(ak))
a1)s O
(
2ω2(1 + T(a2))
∑2
k=1 ωk(1 + T(ak))
a2)t]
M
[(
2ω2(1 + T(a2))
∑2
k=1 ωk(1 + T(ak))
a2)s O
(
2ω1(1 + T(a1))
∑2
k=1 ωk(1 + T(ak))
a1)t] = (
q
r
1 −(1 −(1 −(1 −uq
1)
2ω1(1+T(a1))
∑2
k=1 ωk(1+T(ak)) )s(1 −(1 −uq
2)
2ω2(1+T(a2))
∑2
k=1 ωk(1+T(ak)) )t)(1 −(1 −(1 −uq
2)
2ω2(1+T(a2))
∑2
k=1 ωk(1+T(ak)) )s(1 −(1 −uq
1)
2ω1(1+T(a1))
∑2
k=1 ωk(1+T(ak)) )t, q
s
(1 −(1 −v
2qω1(1+T(a1))
∑2
k=1 ωk(1+T(ak))
1
)s(1 −v
2qω2(1+T(a2))
∑2
k=1 ωk(1+T(ak))
2
)t)(1 −(1 −v
2qω2(1+T(a2))
∑2
k=1 ωk(1+T(ak))
2
)s(1 −v
2qω1(1+T(a1))
∑2
k=1 ωk(1+T(ak))
1
)t) v
2qω1(1+T(a1))
∑2
k=1 ωk(1+T(ak))
1
)s(1 −v
2qω2(1+T(a2))
∑2
k=1 ωk(1+T(ak))
2
)t)(1 −(1 −v
2qω2(1+T(a2))
∑2
k=1 ωk(1+T(ak))
2
)s(1 −v
2qω1(1+T(a1))
∑2
k=1 ωk(1+T(ak))
1
)t) We can establish that the statement is true for n = 2. Proof of Theorem 1. (II)
Assume it holds true for some arbitrary n = m, that is m
L
i, j = 1, i ̸= j
[(
mωi(1 + T(ai))
∑m
k=1 ωk(1 + T(ak)) ai)s O
(
mωj(1 + T(aj))
∑m
k=1 ωk(1 + T(ak)) aj)t] = ( q
v
u
u
u
t1 −∏
m
i, j = 1
i ̸= j
[1 −(1 −(1 −uq
i )
mωi(1+T(ai))
∑m
k=1 ωk(1+T(ak)) )s(1 −(1 −uq
j )
mωj(1+T(aj))
∑m
k=1 ωk(1+T(ak)) )t],
q
v
u
u
u
u
t∏
m
i, j = 1
i ̸= j
[1 −(1 −v
qmωi(1+T(ai))
∑m
k=1 ωk(1+T(ak))
i
)s(1 −v
qmωj(1+T(aj))
∑m
k=1 ωk(1+T(ak))
j
)t]) ( q
v
u
u
u
t1 −∏
m
i, j = 1
i ̸= j
[1 −(1 −(1 −uq
i )
mωi(1+T(ai))
∑m
k=1 ωk(1+T(ak)) )s(1 −(1 −uq
j )
mωj(1+T(aj))
∑m
k=1 ωk(1+T(ak)) )t], q
v
u
u
u
u
t∏
m
i, j = 1
i ̸= j
[1 −(1 −v
qmωi(1+T(ai))
∑m
k=1 ωk(1+T(ak))
i
)s(1 −v
qmωj(1+T(aj))
∑m
k=1 ωk(1+T(ak))
j
)t]) Then, for n = m + 1, Then, for n = m + 1, 8 of 15 Symmetry 2023, 15, 2007 m + 1
L
i, j = 1, i ̸= j
[((m + 1)ωi(1 + T(ai))
∑m+1
k=1 ωk(1 + T(ak))
ai)s O
(
(m + 1)ωj(1 + T(aj))
∑m+1
k=1 ωk(1 + T(ak))
aj)t] = (
m
L
i, j = 1, i ̸= j
[( (m+1)ωi(1+T(ai))
∑m+1
k=1 ωk(1+T(ak)) ai)s N(
(m+1)ωj(1+T(aj))
∑m+1
k=1 ωk(1+T(ak)) aj)t]) L(
m
L
i = 1
[( (m+1)ωi(1+T(ai))
∑m+1
k=1 ωk(1+T(ak)) ai)s N( (m+1)ωm+1(1+T(am+1))
∑m+1
k=1 ωk(1+T(ak))
am+1)t])
L(
m
L
j = 1
[( (m+1)ωm+1(1+T(am+1))
∑m
k=1 ωk(1+T(ak))
am+1)s N(
(m+1)ωj(1+T(aj))
∑m
k=1 ωk(1+T(ak)) aj)t]) L(
m
L
j = 1
[( (m+1)ωm+1(1+T(am+1))
∑m
k=1 ωk(1+T(ak))
am+1)s N(
(m+1)ωj(1+T(aj))
∑m
k=1 ωk(1+T(ak)) aj)t]) m
L
i = 1
[((m + 1)ωi(1 + T(ai))
∑m+1
k=1 ωk(1 + T(ak))
ai)s O
((m + 1)ωm+1(1 + T(am+1))
∑m+1
k=1 ωk(1 + T(ak))
am+1)t] = (
q
s
1 −∏
m
i=1 [1 −(1 −(1 −uq
i )
(m+1)ωi(1+T(ai))
∑m+1
k=1 ωk(1+T(ak)) )s(1 −(1 −uq
m+1)
(m+1)ωm+1(1+T(am+1))
∑m+1
k=1 ωk(1+T(ak))
)t],
q
s
∏
m
i=1 [1 −(1 −v
q(m+1)ωi(1+T(ai))
∑m+1
k=1 ωk(1+T(ak))
i
)s(1 −v
q(m+1)ωm+1(1+T(am+1))
∑m+1
k=1 ωk(1+T(ak))
m+1
)t])
m
L
j = 1
[((m + 1)ωm+1(1 + T(am+1))
∑m+1
k=1 ωk(1 + T(ak))
am+1)s O
(
(m + 1)ωj(1 + T(aj))
∑m+1
k=1 ωk(1 + T(ak))
aj)t] = (
q
s
1 −∏
m
j=1 [1 −(1 −(1 −uq
m+1)
(m+1)ωm+1(1+T(am+1))
∑m+1
k=1 ωk(1+T(ak))
)s(1 −(1 −uq
j )
(m+1)ωj(1+T(aj))
∑m+1
k=1 ωk(1+T(ak)) )t],
q
v
u
u
t
∏
m
j=1 [1 −(1 −v
q(m+1)ωm+1(1+T(am+1))
∑m+1
k=1 ωk(1+T(ak))
m+1
)s(1 −v
q(m+1)ωj(1+T(aj))
∑m+1
k=1 ωk(1+T(ak))
j
)t]) (
q
s
1 −∏
m
j=1 [1 −(1 −(1 −uq
m+1)
(m+1)ωm+1(1+T(am+1))
∑m+1
k=1 ωk(1+T(ak))
)s(1 −(1 −uq
j )
(m+1)ωj(1+T(aj))
∑m+1
k=1 ωk(1+T(ak)) )t], q
v
u
u
t
∏
m
j=1 [1 −(1 −v
q(m+1)ωm+1(1+T(am+1))
∑m+1
k=1 ωk(1+T(ak))
m+1
)s(1 −v
q(m+1)ωj(1+T(aj))
∑m+1
k=1 ωk(1+T(ak))
j
)t]) Considering the above equations, it can be concluded m + 1
L
i, j = 1, i ̸= j
[((m + 1)ωi(1 + T(ai))
∑m+1
k=1 ωk(1 + T(ak))
ai)s O
(
(m + 1)ωj(1 + T(aj))
∑m+1
k=1 ωk(1 + T(ak))
aj)t] = −∏
m+1
i,j=1,i̸=j [1 −(1 −(1 −uq
i )
(m+1)ωi(1+T(ai))
∑m+1
k=1 ωk(1+T(ak)) )s(1 −(1 −uq
j )
(m+1)ωj(1+T(aj))
∑m+1
k=1 ωk(1+T(ak)) )t],
q
v
u
u
t
∏
m+1
i,j=1,i̸=j [1 −(1 −v
q(m+1)ωi(1+T(ai))
∑m+1
k=1 ωk(1+T(ak))
i
)s(1 −v
q(m+1)ωj(1+T(aj))
∑m+1
k=1 ωk(1+T(ak))
j
)t]) 1 −∏
m+1
i,j=1,i̸=j [1 −(1 −(1 −uq
i )
(m+1)ωi(1+T(ai))
∑m+1
k=1 ωk(1+T(ak)) )s(1 −(1 −uq
j )
(m+1)ωj(1+T(aj))
∑m+1
k=1 ωk(1+T(ak)) )t], q
v
u
u
t
∏
m+1
i,j=1,i̸=j [1 −(1 −v
q(m+1)ωi(1+T(ai))
∑m+1
k=1 ωk(1+T(ak))
i
)s(1 −v
q(m+1)ωj(1+T(aj))
∑m+1
k=1 ωk(1+T(ak))
j
)t]) Consequently, the statement holds true for n = m + 1. (
m
L
i, j = 1, i ̸= j
[ Proof of Theorem 1. By the principle of mathematical
induction, the statement is true for all positive integers n. 9 of 15 Symmetry 2023, 15, 2007 Symmetry 2023, 15, 2007
9 of 15
Thus
1
n(n −1)
n
L
i, j = 1, i ̸= j
(
(
nωi(1 + T(ai))
∑n
k=1 ωk(1 + T(ak)) ai)s O
(
nωj(1 + T(aj))
∑n
k=1 ωk(1 + T(ak)) aj)t
)
]
1
s+t =
( q
v
u
u
u
t1 −[∏
n
i, j = 1
i ̸= j
(1 −(1 −(1 −uq
i )
nωi(1+T(ai))
∑n
k=1 ωk(1+T(ak)) )s(1 −(1 −uq
j )
nωj(1+T(aj))
∑n
k=1 ωk(1+T(ak)) )t)]
1
n(n−1) ,
1
qn(n−1)
v
u
u
u
u
t∏
n
i, j = 1
i ̸= j
[1 −(1 −v
qωin(1+T(ai))
∑n
k=1 ωk(1+T(ak))
i
)s(1 −v
qωjn(1+T(aj))
∑n
k=1 ωk(1+T(ak))
j
)t])
GOFWPBMs,t(a1, a2, · · · , an) = [
1
n(n −1)
n
L
i, j = 1, i ̸= j
(
nωi(1 + T(ai))
∑n
k=1 ωk(1 + T(ak)) ai)s O
(
nωj(1 + T(aj))
∑n
k=1 ωk(1 + T(ak)) aj)t
! 3.2. Properties of GOFWPBM Operator 3.2. Properties of GOFWPBM Operator 3.2. Properties of GOFWPBM Operator Some basic properties of the generalized orthopair fuzzy weighted power Bonferroni
mean operator are discussed below. Property 1. When
(1+T(ai))
∑n
k=1 ωk(1+T(ak)) = 1
n then, Property 1. When
(1+T(ai))
∑n
k=1 ωk(1+T(ak)) = 1
n then, GOFWPBMs,t(a1, a2, · · · , an)
= [
1
n(n−1)
n
L
i, j = 1, i ̸= j
(
nωi(1+T(ai))
∑n
k=1 ωk(1+T(ak)) ai)s N(
nωj(1+T(aj))
∑n
k=1 ωk(1+T(ak)) aj)t
]
1
s+t
= [
1
n(n−1)
n
L
i, j = 1, i ̸= j
(ωiai)s N(ωjaj)t]
1
s+t
= GOFWBMs,t(a1, a2, · · · , an) · · , an)
= [
1
n(n−1)
n
L
i, j = 1, i ̸= j
(
nωi(1+T(ai))
∑n
k=1 ωk(1+T(ak)) ai)s N(
nωj(1+T(aj))
∑n
k=1 ωk(1+T(ak)) aj)t
]
1
s+t = [
1
n(n−1)
n
L
i, j = 1, i ̸= j
(ωiai)s N(ωjaj)t]
1
s+t
= GOFWBMs,t(a1, a2, · · · , an) , j
, ̸
j
= GOFWBMs,t(a1, a2, · · · , an) At this time, the GOFWPBMs,t operator degenerates into the generalized orthopair
fuzzy weighted Bonferroni mean
GOFWBMs,t
operator. This operator does not possess
the advantages of the power mean operator, which emphasizes the support among data
and excludes outliers, thereby ensuring the overall coherence among data. Property 2. When ω = (ω1, ω2, . . . , ωn)T = ( 1
n, 1
n, 1
n . . . 1
n)T then, Property 2. When ω = (ω1, ω2, . . . , ωn)T = ( 1
n, 1
n, 1
n . . . 1
n)T then, GOFWPBMs,t(a1, a2, · · · , an)
= [
1
n(n−1)
n
L
i, j = 1, i ̸= j
(
nωi(1+T(ai))
∑n
k=1 ωk(1+T(ak)) ai)s N(
nωj(1+T(aj))
∑n
k=1 ωk(1+T(ak)) aj)t
]
1
s+t
= [
1
n(n−1)
n
L
i, j = 1, i ̸= j
(
n(1+T(ai))
∑n
k=1 (1+T(ak)) ai)s N(
n(1+T(aj))
∑n
k=1 (1+T(ak)) aj)t
]
1
s+t
= GOFPBMs,t(a1, a2, · · · , an) , j
, ̸
j
= GOFPBMs,t(a1, a2, · · · , an) At this point, the GOFWPBMs,t operator degenerates into the generalized orthopair
fuzzy power Bonferroni mean
GOFPBMs,t
operator. Proof of Theorem 1. ]
1
s+t =
(
1
n(n −1)
q
v
u
u
u
t1 −∏
n
i, j = 1
i ̸= j
(1 −(1 −(1 −uq
i )
nωi(1+T(ai))
∑n
k=1 ωk(1+T(ak)) )s(1 −(1 −uq
j )
nωj(1+T(aj))
∑n
k=1 ωk(1+T(ak)) )t),
q
v
u
u
u
u
t∏
n
i, j = 1
i ̸= j
[1 −(1 −v
qωin(1+T(ai))
∑n
k=1 ωk(1+T(ak))
i
)s(1 −v
qωjn(1+T(aj))
∑n
k=1 ωk(1+T(ak))
j
)t]
)
1
s+t =
( q
v
u
u
u
t1 −(∏
n
i, j = 1
i ̸= j
(1 −(1 −(1 −uq
i )
nωi(1+T(ai))
∑n
k=1 ωk(1+T(ak)) )s(1 −(1 −uq
j )
nωj(1+T(aj))
∑n
k=1 ωk(1+T(ak)) )t))
1
n(n−1) ,
q
v
u
u
u
u
u
u
t
(
n
∏
i, j = 1
i ̸= j
[1 −(1 −v
qωin(1+T(ai))
∑n
k=1 ωk(1+T(ak))
i
)s(1 −v
qωjn(1+T(aj))
∑n
k=1 ωk(1+T(ak))
j
)t])
1
n(n−1) )
1
s+t =
(
q
v
u
u
u
u
u
u
t
1 −[∏
n
i, j = 1
i ̸= j
(1 −(1 −(1 −uq
i )
nωi(1+T(ai))
∑n
k=1 ωk(1+T(ak)) )s(1 −(1 −uq
j )
nωj(1+T(aj))
∑n
k=1 ωk(1+T(ak)) )t]
1
n(n−1)
1
s+t
,
q
v
u
u
u
u
u
u
t
1 −[∏
n
i, j = 1
i ̸= j
(1 −(1 −v
qωin(1+T(ai))
∑n
k=1 ωk(1+T(ak))
i
)s(1 −v
qωjn(1+T(aj))
∑n
k=1 ωk(1+T(ak))
j
)t]
1
n(n−1)
1
s+t
)
□ Thus 1
n(n −1)
n
L
i, j = 1, i ̸= j
(
(
nωi(1 + T(ai))
∑n
k=1 ωk(1 + T(ak)) ai)s O
(
nωj(1 + T(aj))
∑n
k=1 ωk(1 + T(ak)) aj)t
)
]
1
s+t =
1 −[∏
n
i, j = 1
i ̸= j
(1 −(1 −(1 −uq
i )
nωi(1+T(ai))
∑n
k=1 ωk(1+T(ak)) )s(1 −(1 −uq
j )
nωj(1+T(aj))
∑n
k=1 ωk(1+T(ak)) )t]
1
n(n−1)
1
s+t
, q
v
u
u
u
u
u
u
t
1 −[∏
n
i, j = 1
i ̸= j
(1 −(1 −v
qωin(1+T(ai))
∑n
k=1 ωk(1+T(ak))
i
)s(1 −v
qωjn(1+T(aj))
∑n
k=1 ωk(1+T(ak))
j
)t]
1
n(n−1)
1
s+t
)
□
1 −[∏
n
i, j = 1
i ̸= j
(1 −(1 −v
qωin(1+T(ai))
∑n
k=1 ωk(1+T(ak))
i
)s(1 −v
qωjn(1+T(aj))
∑n
k=1 ωk(1+T(ak))
j
)t]
1
n(n−1)
1
s+t
) Symmetry 2023, 15, 2007 10 of 15 3.2. Properties of GOFWPBM Operator Although this operator combines
the benefits of the power average operator and the Bonferroni mean operator, it cannot
assign weights to each variable indicator. Property 3. (Boundedness) Let ai(i = 1, 2, · · · , n) be a set of generalized orthopair fuzzy numbers. Define a+ = max
1≤i≤nai, a−= min
1≤i≤nai. Then Property 3. (Boundedness) Let ai(i = 1, 2, · · · , n) be a set of generalized orthopair fuzzy numbers. Define a+ = max
1≤i≤nai, a−= min
1≤i≤nai. Then a−≤GOFWPBMs,t(a1, a2, · · · , an) ≤a+ 4. The MADM Model Based on the GOFWPBMs,t Operator The multi-attribute decision-making model is a current research focus in the field of
decision science, and is extensively utilized to address decision-making issues in real-life
scenarios. For instance, Garg [38] proposed a multi-attribute decision-making algorithm
based on trigonometric generalized orthopair fuzzy numbers. Xu et al. [39] introduced
a multi-attribute decision-making method based on fuzzy soft sets. Chen et al. [40] put
forward a multi-attribute decision-making model based on the intuitionistic trapezoidal
fuzzy generalized Heronian OWA operator, and Li et al. [41] proposed a series of multi-
attribute decision-making methods based on the Pythagorean fuzzy power Muirhead
mean operators. From the above analysis, it is evident that the GOFWPBMs,t operator combines the
advantages of numerous operators in information aggregation. Based on this, we propose a
multi-attribute decision-making model based on the GOFWPBMs,t operator. This decision-
making model will be applied to an investment attraction project in a certain city to verify
the feasibility and superiority of the method proposed in this paper. In the multi-attribute decision-making problem with generalized orthopair fuzzy
information, suppose there are n candidate schemes X = (x1, x2, . . . xn), and m decision
attributes C =(c1, c2, c3 . . . cm). The weight vector corresponding to each decision attribute
is ω = (ω1, ω2, ω3 . . . ωm)T. Experts provide generalized orthopair fuzzy evaluation
information. We denote the attribute value of scheme xi under attribute cj as aij, where
aij is a generalized orthopair fuzzy number. Thus, a generalized orthopair fuzzy decision
matrix M =
aij
mn is obtained, where aij =
uij, vij
, uij represents the membership value
and vij represents the non-membership value of candidate scheme i regarding attribute j. The specific steps are as follows. p
p
Step 1. Based on the actual situation, establish the generalized orthopair fuzzy matrix
M =
aij
mn. Convert each decision attribute value in it to the benefit-type, resulting in the
if C i
b
fit
t
tt ib t standard matrix [42] M =
aij
mn, where aij =
aij, if Cj is a benefit −type attribute
vij, uij
, if Cj is a cost −type attribute. j
j
j
Step 2. Using the information aggregation (GOFWPBMs,t) operator, compute the
attribute values of each solution in the generalized orthopair fuzzy standard matrix
M =
aij
mn. a−≤GOFWPBMs,t(a1, a2, · · · , an) ≤a+ a−≤GOFWPBMs,t(a1, a2, · · · , an) ≤a+ 4. The MADM Model Based on the GOFWPBMs,t Operator Then, determine the score function for each solution based on these at-
tribute values. Step 3. Rank the score functions from high to low and select the optimal solution. 5. Case Analysis 5.1. Decision-Making Process □
Therefore, by combining the two inequalities, we have □
Therefore, by combining the two inequalities, we have Proof of Property 3. GOFWPBMs,t(a1, a2, a3, · · · , an)
= [
1
n(n−1)
n
L
i, j = 1, i ̸= j
(
nωi(1+T(ai))
∑n
k=1 ωk(1+T(ak)) ai)s N(
nωj(1+T(aj))
∑n
k=1 ωk(1+T(ak)) aj)t
]
1
s+t
≤[
1
n(n−1)
n
L
i, j = 1, i ̸= j
(a+)s N(a+)t]
1
s+t = [
1
n(n−1)
n
L
i, j = 1, i ̸= j
(a+)s+t]
1
s+t = a+ 11 of 15 Symmetry 2023, 15, 2007 GOFWPBMs,t(a1, a2, a3, · · · , an)
= [
1
n(n−1)
n
L
i, j = 1, i ̸= j
(
nωi(1+T(ai))
∑n
k=1 ωk(1+T(ak)) ai)s N(
nωj(1+T(aj))
∑n
k=1 ωk(1+T(ak)) aj)t
]
1
s+t
≥[
1
n(n−1)
n
L
i, j = 1, i ̸= j
(a−)s N(a−)t]
1
s+t = [
1
n(n−1)
n
L
i, j = 1, i ̸= j
(a−)s+t]
1
s+t = a−
□
Therefore, by combining the two inequalities, we have 5.1. Decision-Making Process In this case study, to show the complete results, the value of q is set to be greater
than or equal to 2. Here, q = 3. The comprehensive attribute values for each scheme are: q
q
p
x1 = < 0.352, 0.536 >, x2 = < 0.549, 0.414 >, x3 = < 0.453, 0.453 >, x4 = < 0.540, 0.483 >,
x5 = < 0.464, 0.335 >. Based on these comprehensive values, the score function values for
each scheme are: Sx1 = −0.1103, Sx2 = 0.0941, Sx3 = −0.0002, Sx4 = 0.0449, Sx5 = 0.0621. q
q
p
x1 = < 0.352, 0.536 >, x2 = < 0.549, 0.414 >, x3 = < 0.453, 0.453 >, x4 = < 0.540, 0.483 >,
x5 = < 0.464, 0.335 >. Based on these comprehensive values, the score function values for
each scheme are: Sx1 = −0.1103, Sx2 = 0.0941, Sx3 = −0.0002, Sx4 = 0.0449, Sx5 = 0.0621. x1
x
x3
x4
x5
Step 3. According to the aforementioned score function values and ranking them from
highest to lowest, we have the order Sx2 > Sx5 > Sx4 > Sx3 > Sx1. Thus, the optimal scheme
is x2 (Chain catering company). 5.1. Decision-Making Process Since the outbreak of the COVID-19 pandemic, the economic development in various
regions has been consistently sluggish. Responding to the call for national economic re-
covery, a particular city government took the lead in allocating funds to stimulate local
economic recovery. They planned to invest in some privately-owned enterprise in the city. But, due to reasons such as financial difficulties and policy restrictions, the government Symmetry 2023, 15, 2007 12 of 15 12 of 15 aimed to raise fiscal revenue, increase employment opportunities, and reduce investment
risks by seeking partners in the market. After rigorous market research, five potential
partners were identified: x1 Daily goods manufacturing, x2 Chain catering, x3 Internet
company, x4 Civil and building materials, x5 Civil and building materials. Taking into con-
sideration its own situation, the decision-making group composed of government officials
and experts decided to inspect the candidates based on the following four factors. C1: Fiscal
Assessment (including company’s financial reserves, investment orientation evaluation,
etc.), C2: Developmental Assessment (including enterprise scale, future development plan,
etc.), C3: Social Influence Assessment (including the company’s social status and public
reputation, etc.), C4: Green Development Assessment (including sustainable development
strategies and environment-friendly levels of the enterprise). g
y
p
Following the assessment by the opinion group, a generalized orthopair fuzzy de-
cision matrix M =
aij
5×4 was established as shown in Table 1, where the evaluation
value aij =
uij, vij
is a generalized orthopair fuzzy number, and the weight vector
ω = (ω1, ω2, ω3 . . . ωn)T is considered equal weight. Based on the decision matrix pro-
vided by the opinion group, we will evaluate the five prospective partner companies. Table 1. Generalized orthopair fuzzy decision matrix M. Table 1. Generalized orthopair fuzzy decision matrix M. Schemes
C1
C2
C3
C4
x1
<0.4, 0.5>
<0.5, 0.4>
<0.2, 0.7>
<0.2, 0.5>
x2
<0.6, 0.4>
<0.6, 0.3>
<0.6, 0.3>
<0.3, 0.6>
x3
<0.5, 0.5>
<0.4, 0.5>
<0.4, 0.4>
<0.5, 0.4>
x4
<0.7, 0.2>
<0.5, 0.4>
<0.2, 0.5>
<0.6, 0.7>
x5
<0.5, 0.3>
<0.3, 0.4>
<0.6, 0.2>
<0.4, 0.4> Table 1. Generalized orthopair fuzzy decision matrix M. Step 1. Establishing the generalized orthopair fuzzy standard matrix M based on
Table 1. Since all attributes in this case are of benefit type, the standard matrix M is equal
to M. Step 2. Using Formula (7), we derived the comprehensive attribute values for each
scheme. 5.2. Parameters Analysis The analysis and conclusion above were drawn under the condition of s, t = 1. To
ensure generality and robustness in the decision-making process, it is pertinent to discuss
the ranking of the best candidate schemes under different parameter values for s and t, as
shown in Table 2. Table 2. Composite attribute values of each scheme under different parameters. Table 2. Composite attribute values of each scheme under different parameters. s,t
x1
x2
x3
x4
x5
1
<0.352, 0.536>
<0.549, 0.414>
<0.453, 0.453>
<0.540, 0.483>
<0.464, 0.335>
2
<0.389, 0.532>
<0.571, 0.411>
<0.458, 0.453>
<0.574, 0.477>
<0.485, 0.335>
3
<0.408, 0.528>
<0.581, 0.408>
<0.464, 0.452>
<0.590, 0.470>
<0.499, 0.334> Table 2 shows the composite attribute values of each scheme under different parame-
ters. Based on this table, the score rankings under different parameters are calculated in
Table 3. 13 of 15 13 of 15 Symmetry 2023, 15, 2007 Table 3. Score and ranking of each scheme. Table 3. Score and ranking of each scheme. Table 3. Score and ranking of each scheme. s,t
Score
Ranking
1
S = (−0.1103, 0.0941, −0.0002, 0.0449, 0.0621)
Sx2 > Sx5 > Sx4 > Sx3 > Sx1
2
S = (−0.0916, 0.1164, 0.0035, 0.0805, 0.0765)
Sx2 > Sx4 > Sx5 > Sx3 > Sx1
3
S = (−0.0790, 0.1283, 0.0072, 0.1013, 0.0871)
Sx2 > Sx4 > Sx5 > Sx3 > Sx1 Table 3 presents the ranking results under different parameters using the general-
ized orthopair fuzzy weighted power Bonferroni mean (GOFWPBMs,t) operator. Due to
the novel operator having two parameters, s and t, it allows for greater flexibility and
convenience in information aggregation. Therefore, during the decision-making process,
the expert group can select appropriate parameter values based on the complexity of the
problem combined with their risk preferences. Moreover, the comparison results from
the Table 3 above show that the scores of each candidate will increase as the values of the
parameters s and t increase. However, due to the stability of the GOFWPBMs,t operator, it
does not distort information with excessively large values of s and t like other operators. This avoids influencing the final judgment results, making it more suitable for today’s
increasingly complex decision-making environment. 5.3. Comparative Analysis To further demonstrate the superiority of the operator proposed in this paper, the
following text will compare this operator with existing operators. Specifically, we selected
the generalized orthopair fuzzy power average (GOFPA) operator and the generalized
orthopair fuzzy Bonferroni mean
GOFBMs,t
operator for comparison. For convenience of
calculation, parameter values in various information aggregation operators are uniformly
taken as one. The comparison results are shown in Table 4. Table 4. Best solution ranking under different operators. Operators
Score
Ranking
GOFPA
S = (0.2251, 0.5930, 0.3767, 0.6091, 0.4259)
Sx4> Sx2> Sx5> Sx3> Sx1
GOFBMs,t
S = (−0.4886, −0.2530, −0.3233, −0.3823, −0.1440)
Sx5> Sx2> Sx3> Sx4> Sx1
GOFWPBMs,t
S = (−0.1103, 0.0941, −0.0002, 0.0449, 0.0621)
Sx2> Sx5> Sx4> Sx3> Sx1 Table 4. Best solution ranking under different operators. Table 4. Best solution ranking under different operators. Based on the comparative outcomes presented in the Table 4, it is discernible that score
values and the resultant rankings exhibit slight differences across various methodologies. The underlying reason for these divergences can be attributed to the distinct information
aggregation methodologies employed by each of the emblematic information aggregation
operators. Even though all these operators are grounded in the average-based paradigm,
their focal points during the information aggregation process vary notably. It can be
observed that the candidate scheme x4 consistently exhibits extreme values across various
attributes. As a result, using the GOFPA operator, the outcome points to x4. This is
primarily because the GOFPA operator can negate the adverse impact brought about by
extreme values encountered in expert evaluations. Additionally, it emphasizes the support
degree between data, ensuring cohesion among them. However, it does not take into
consideration potential inter-relationships between attributes. In this particular case, it
is evident that the attribute C2 influences the development of C3. On the other hand, the
GOFBMs,t operator primarily captures the interrelationships between pieces of information. Yet, its vulnerability to extreme values can lead to distorted outcomes. Consequently, in this
instance, after being influenced by the extreme value, the result derived from GOFBMs,t
operator is x5. p
The newly defined GOFWPBMs,t operator in this study combines the advantages of
both: it not only takes into account the overall balance among the data but also considers
the weights of each attribute and the potential correlations that may exist between different Symmetry 2023, 15, 2007 14 of 15 attributes. 5.3. Comparative Analysis This helps prevent biased decision-makers from skewing the results with outlier
preference values (i.e., exceptionally high or low values in the original data), ensuring a
more fair and objective decision-making process. Furthermore, the GOFWPBMs,t operator
retains the parameters s and t, allowing decision-makers to adjust the parameters based on
their risk preferences, making the decision-making process more flexible. Additionally, the
GOFWPBMs,t operator introduces the concept of weights, providing an avenue to account
for the intrinsic importance of the data, thereby rendering the decision-making process
more rational and in line with real-world multi-attribute decision-making problems. 6. Conclusions In response to the growing scholarly interest in generalized orthopair fuzzy multi-
attribute decision-making problems, this study delves into the theory of fuzzy sets and
information aggregation operators, introducing a novel generalized orthopair fuzzy ag-
gregation operator, termed the generalized orthopair fuzzy weighted power Bonferroni
mean operator. This novel operator combines the commendable attributes of both the
power average operator and the Bonferroni mean operator. It not only takes into account
the overall balance among various data but also considers the weights of individual at-
tributes and potential correlations that might exist between different attributes. Moreover,
the paper explores some admirable properties and specific scenarios pertaining to this
novel operator. Building on this, a multi-attribute decision-making method based on the
generalized orthopair fuzzy weighted power Bonferroni mean operator is elucidated, with
its effectiveness and superiority validated through empirical instances and comparative
analysis. The decision-making methodology proposed herein holds potential for further
application in areas such as supplier selection assessment, product scheme evaluation, and
talent recruitment recommendations in human resource departments, showcasing both
theoretical and practical significance. Author Contributions: Conceptualization, B.H.; methodology, B.H.; formal analysis, B.H.; data
curation, B.H.; writing—original draft preparation, B.H.; writing—review and editing, Y.C. All
authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Funding: This research received no external funding. Data Availability Statement: The data presented in this study are contained within the article. Data Availability Statement: The data presented in this study are contained within the article. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References 1. Zadeh, L.A. Fuzzy sets. Inf. Control 1965, 8, 338–353. [CrossRef] 2. Atanassov, K.T. Intuitionistic fuzzy sets. Fuzzy Sets Syst. 1986, 20, 87–96. [CrossRef] y
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people or property resulting from any ideas, methods, instructions or products referred to in the content.
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Assessment of Multilayered Plates with Hyperelastic Coatings Subjected to Dynamic Loadings by Impact at Low Velocities
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Materiale plastice
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Assessment of Multilayered Plates with Hyperelastic Coatings
Subjected to Dynamic Loadings by Impact at Low Velocities FLORINA BUCUR, LIVIU-CRISTIAN MATACHE* RINA BUCUR, LIVIU-CRISTIAN MATACHE*
ry Technical Academy „Ferdinand I”, 39-49 G. Coșbuc Av., 040531, Bucharest, Romania ,
Military Technical Academy „Ferdinand I”, 39-49 G. Coșbuc Av., 040531, Bucharest, Romania Abstract: This paper describes the use of video and digital image processing in investigation of the
impact between a rigid hemispherical shape impactor and Hybrid Polyurea-Polyurethane-MWCNTs
Nanocomposite Coatings. An experimental study was performed for six sample configurations: single
aluminum plates (reference test), multilayer plates with 4 types of coatings and double aluminum plates. The impact phenomenon was recorded with a high-speed video camera and the variation of the
projectile's velocity during the impact was obtained through digital analysis. Additionally, the test was
instrumented using a force sensor specially designed and mounted on the impactor. The video processing
was used to draw the velocity curves and to estimate the evolution of the contact forces between the
impactor and the multilayer structures, the results obtained being compared with the force sensor data. Some differences between these two types of measurements are observed, so in order to analyze the
configurations behavior, a numerical study of the phenomena was performed in LS-DYNA software
using a 2D axial symmetric model. The simulations showed that the profile of the force evolution
measured with the sensor is affected by the chosen constructive solution and the data obtained based on
the video images are more accurate. The deformations were analyzed, the maximum deformation based
on image processing and the residual deformation based on 3D Scan post-test. The video technique
combined with 3D Scan are precise enough to study the impact at low velocities and the numerical
simulations provide results according to reality. The hyperelastic coatings contribute to a better
resistance of the aluminum plates. Keywords: polyurea-polyurethane, multiwall carbon nanotubes, coatings, 3D Scan, video image
processing, impact, numerical simulation MATERIALE PLASTICE
https://revmaterialeplastice.ro
https://doi.org/10.37358/Mat.Plast.1964 *email: liviu.matache@mta.ro 1. Introduction The possibility to evaluate the behavior of polyurea in different configurations and for different
loadings was investigated through numerical simulations with materials models available in
computational codes, being obtained results similar to the real ones [11, 13, 25, 30-32]. The present study refers to an experimental and numerical evaluation of a hyperelastic material,
namely polyurea-polyurethane-MWCNTs nanocomposite. Aluminum specimens (0.5 mm thickness
plates) were coated with an additional layer with 1 mm thickness of simple polyurea-polyurethane and
reinforced with multiwall carbon nanotubes in different concentrations (back-side – opposite to impact). The method for obtaining the polyurea-polyurethane-multiwall carbon nanotubes (MWCNTs)
nanocomposites evaluated in this paper is presented in [5, 20]. Also, their potential to be used in impact
application due to their improved mechanical properties is demonstrated (the integrity of the aluminum
plate coated with nanocomposite layers is maintained at a force value almost 200% higher than for the
uncoated standard plates). The possibility of using 3D scans and video images processing, corroborated with classical force
transducer to estimate parameters of interest that characterize the behavior of the multilayer plates, is
investigated. The use of experimental video images processing in order to understand how phenomena
unfold in laboratory conditions at low velocity, for foam type materials, was already explored [23]. The numerical approach is used to clarify the differences between each type of measurement tools
that is used. Using the LS-DYNA software, a 2D axis-symmetric model was created and numerical
simulations were performed for each configuration experimentally tested. The results obtained through numerical simulation validated the experimental ones, the results being
consistent. 1. Introduction Lately, the development of lightweight configurations based on metal – hyperelastic material
combinations used in ballistic protection applications has been intensively studied. The scientific literature presents many studies where is highlighted the potential of polyurea, as the
most common hyperplastic material, to enhance the mitigation of dynamic and impulsive loads by
different materials and structures when is used in various combinations [1-5]. A large number of papers refers to the polyurea coatings on structures used to mitigate blast and
impact effects, especially for decreasing the damaging effect on structures used for individual or
collective ballistic protection [6-13]. Moreover, the research also extended towards the study of different polyurea synthesis with
improved physical and mechanical properties and, also, location combinations (in the front, in the middle
or on the back of tested configurations) [5, 11-12]. Besides the study of coated steel plates, the potential of polyurea to be used as protection layer for
aluminum plates and its behavior when is subjected to low velocities and high velocities impact loadings
was approached [13-17]. At the same time, the polyurea properties improvement was investigated and, namely, polyurea-
polyurethane-nanocomposite coatings for ballistic protection have been the subject of several papers
[4, 18-20]. Mater. Plast., 61 (1), 2024, 170-184 https://doi.org/10.37358/MP.24.1.5712 170 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 Another exploited direction refers to the testing methods and also test instrumentation procedures for
specific parameters measurement and the results processing. In this regard, for a better understanding of materials behavior, during the experimental tests
(mechanical testing, ultrasonic testing), different types and configurations of samples and measurement
tools have been tested (simple/bilayer/multilayer configurations, force or pressure transducers, video-
speed cameras, scanning electron microscopy, computed tomography etc.), in static, dynamic or
impulsive regime, corroborated with numerical simulation, in order to obtain valid outcomes and results
that can be extrapolated to large-scale experiments and applications [13, 20-29]. Regarding the laboratory testing of aluminum-polyurea composite plates, punch indentation [29],
impact of low velocity blunt-nosed projectiles [16] or impact of a blunt-nosed hammer [28] were
investigated by experiments and numerical simulations. Also, in terms of test instrumentation, besides classical sensors (transducers), the potential of high-speed
photography, digital image correlation methods and X-ray computed tomography (CT), the comparison
between experimental data and numerical simulations [13, 19, 21, 26-27], has been investigated. 2. Materials and methods
2.1. Experimental testing The experimental study is performed at a standard temperature of 25°C, in dynamic regime, in order
to analyze the behavior of multilayer configurations at loadings produced at low velocities. The laboratory testing follows a simple methodology that implies using a pneumatic propulsion
system for determining the resistance force resulting from the loading of specimen on impact with a
hemispherical shape impactor. Specimens with the same lateral dimension but different structures,
single-layer and multilayer, were tested. The 3D experimental set-up is presented in Figure 1. Mater. Plast., 61 (1), 2024, 170-184 171 https://doi.org/10.37358/MP.24.1.5712 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
Figure 1. Experimental set-up
The rigid hemispherical impactor (non-deformable) has the head diameter of 40 mm and a mass of
800 g (Figure 2a). The reference sample used for these tests is represented by an aluminum plate with 2.78 g/cm3
density, having a thickness of 0.5 mm and a free diameter of 100 mm (Figure 2b). MATERIALE PLASTICE https://revmaterialeplastice.ro https://revmaterialeplastice.ro https://doi.org/10.37358/Mat.Plast.1964 Figure 1. Experimental set-up Figure 1. Experimental set-up Figure 1. Experimental set-up Figure 1. Experimental set-up The rigid hemispherical impactor (non-deformable) has the head diameter of 40 mm and a mass of
800 g (Figure 2a). 800 g (Figure 2a). The reference sample used for these tests is represented by an aluminum plate with 2.78 g/cm3
density, having a thickness of 0.5 mm and a free diameter of 100 mm (Figure 2b). The reference sample used for these tests is represented by an aluminum plate with 2.78 g/cm3
density, having a thickness of 0.5 mm and a free diameter of 100 mm (Figure 2b). Figure 2. a) hemispherical shape impactor, b) sample prepared for testing,
c) polymeric films: PU-simple polyurea-polyurethane,
PU-NC-polyurea-polyurethane with nanocomposites Figure 2. a) hemispherical shape impactor, b) sample prepared for testing,
c) polymeric films: PU-simple polyurea-polyurethane,
PU-NC-polyurea-polyurethane with nanocomposites experimental tests included six series of tests executed at different low velocities impact The experimental tests included six series of tests executed at different low velocities impact:
• simple aluminum plate of 0.5 mm thickness –S.A. 2. Materials and methods
2.1. Experimental testing – 4 tests; p
p
• multilayer plate: aluminum plate (0.5 mm) + simple polyurea-polyurethane with 1 mm thickness –
SA+PU – 5 tests; • multilayer plate: aluminum plate (0.5 mm) + polyurea-polyurethane-MWCNTs nanocomposite
(1 mm black polyurea + carbon nanotubes in a percentage of 0.1%) –SA+ PU-NC2 – 6 tests; multilayer plate: aluminum plate (0.5 mm) + polyurea polyurethane MWCNTs nanocomposite
(1 mm black polyurea + carbon nanotubes in a percentage of 0.1%) –SA+ PU-NC2 – 6 tests; p y
p
g
er plate: aluminum plate (0.5 mm) + polyurea-polyurethane-MWCNTs nanocomposi
polyurea + carbon nanotubes in a percentage of 0.2%) – SA+PU-NC3 – 6 tests; • multilayer plate: aluminum plate (0.5 mm) + polyurea-polyurethane-MWCNTs nanocomposite
(1 mm black polyurea + carbon nanotubes in a percentage of 0.2%) – SA+PU-NC3 – 6 tests;
• multilayer plate: aluminum plate (0.5 mm) + polyurea-polyurethane-MWCNTs nanocomposite
(1 mm black polyurea + carbon nanotubes in a percentage of 0.3%) – SA+PU-NC4 – 6 tests; p y
p
g
• multilayer plate: aluminum plate (0.5 mm) + polyurea-polyurethane-MWCNTs nanocomposite
(1 mm black polyurea + carbon nanotubes in a percentage of 0.3%) – SA+PU-NC4 – 6 tests;
• multilayer plate: double aluminum plates (2 x 0.5 mm) – DA – 6 tests. y
p
p
(
)
p
y
p
y
p
(1 mm black polyurea + carbon nanotubes in a percentage of 0.3%) – SA+PU-NC4 – 6 tests;
• multilayer plate: double aluminum plates (2 x 0.5 mm) – DA – 6 tests. The four types of polyurea-polyurethane nanocomposites tested were obtained through a facile
synthesis route: multiwall carbon nanotubes (prior pre-dispersed in a polyester polyol matrix) were
mixed with two component of amino functional groups and a component of isocyanate functional
groups. To ensure the homogenous and uniform dispersion of the nanofiller inside the polymetric matrix
the solution was subjected to ultrasound [20]. The synthetized polyurea-polyurethane nanocomposite films (polymeric films, Figure 2c) were
subjected to uniaxial tensile tests. The mechanical properties and the description of each type of
hyperelastic coating layers used on the aluminum plates are presented in detail in [20]. Mater. Plast., 61 (1), 2024, 170-184 https://doi.org/10.37358/MP.24.1.5712 172 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 According to those information and the true-stress curves evolution drawn from it, is point out that
the polyurea-polyurethane nanocomposites with 0,2 % MWCNTs has the best properties (Figure 3). 2. Materials and methods
2.1. Experimental testing According to those information and the true-stress curves evolution drawn from it, is point out that
the polyurea-polyurethane nanocomposites with 0,2 % MWCNTs has the best properties (Figure 3). Figure 3. True stress-true strain curves for the polyurea-polyurethane nanocomposite films Figure 3. True stress-true strain curves for the polyurea-polyurethane nanocomposite films igure 3. True stress-true strain curves for the polyurea-polyurethane nanocomposite film To launch the impactor in the direction of the sample center a pneumatic gun was used. Due to the
rapid impact and resulting stress, the sample plates suffer permanent deformations, cracks and even
fracture (Figure 4). To launch the impactor in the direction of the sample center a pneumatic gun was used. Due to the
rapid impact and resulting stress, the sample plates suffer permanent deformations, cracks and even
fracture (Figure 4). To launch the impactor in the direction of the sample center a pneumatic gun was used. Due to the
rapid impact and resulting stress, the sample plates suffer permanent deformations, cracks and even
fracture (Figure 4). Figure 4. Aspects from testing a simple aluminum plate [5]
A PCB Piezotronics 200B05 force transducer, attached to a rigid steel support, along with a PCB
signal conditioner and a PicoScope®6 series were used. This location was chosen in order to emphasize
the resistance of the sample to dynamic loads in the case of using multilayer structures (Figure 5a). In the same testing configuration, a high-speed camera PHOTRON FASTCAM SA-Z (30,000 fps
setting) was used to record experimental tests (Figure 5b). Figure 4. Aspects from testing a simple aluminum plate [5] Figure 4. Aspects from testing a simple aluminum plate [5] A PCB Piezotronics 200B05 force transducer, attached to a rigid steel support, along with a PCB
signal conditioner and a PicoScope®6 series were used. This location was chosen in order to emphasize
the resistance of the sample to dynamic loads in the case of using multilayer structures (Figure 5a). In the same testing configuration, a high-speed camera PHOTRON FASTCAM SA-Z (30,000 fps
setting) was used to record experimental tests (Figure 5b). A PCB Piezotronics 200B05 force transducer, attached to a rigid steel support, along with a PCB
signal conditioner and a PicoScope®6 series were used. This location was chosen in order to emphasize
the resistance of the sample to dynamic loads in the case of using multilayer structures (Figure 5a). 2. Materials and methods
2.1. Experimental testing In the same testing configuration, a high-speed camera PHOTRON FASTCAM SA-Z (30,000 fps
setting) was used to record experimental tests (Figure 5b). A PCB Piezotronics 200B05 force transducer, attached to a rigid steel support, along with a PCB
signal conditioner and a PicoScope®6 series were used. This location was chosen in order to emphasize
the resistance of the sample to dynamic loads in the case of using multilayer structures (Figure 5a). In the same testing configuration, a high-speed camera PHOTRON FASTCAM SA-Z (30,000 fps
setting) was used to record experimental tests (Figure 5b). p
y
g
y
In the same testing configuration, a high-speed camera PHOTRON FASTCAM S
setting) was used to record experimental tests (Figure 5b). Figure 5. Measuring tools: a) piezoelectric force
sensor, b) high-speed video camera Figure 5. Measuring tools: a) piezoelectric force
sensor, b) high-speed video camera Figure 5. Measuring tools: a) piezoelectric force
sensor, b) high-speed video camera Mater. Plast., 61 (1), 2024, 170-184 https://doi.org/10.37358/MP.24.1.5712 173 MATERIALE PLASTICE Plast., 61 (1), 2024, 170-184 https://doi.org/10.37358/MP.24.1.5712 174 https://doi.org/10.37358/MP.24.1.5712 MATERIALE PLASTICE
https://revmaterialeplastice.ro
https://doi.org/10.37358/Mat.Plast.1964 Figure 8. Video image processing Figure 8. Video image processing Figure 8. Video image processing 2.2. Numerical study MATERIALE PLASTICE https://revmaterialeplastice.ro https://revmaterialeplastice.ro
https://doi.org/10.37358/Mat.Plast.1964 https://revmaterialeplastice.ro
https://doi.org/10.37358/Mat.Plast.1964 In order to analyze the performed tests, the sensor recorded force, the videos from high-speed camera
and 3D scans of tested plates are used. After laboratory testing, each plate deformation pattern is investigated with a professional-grade 3D
scanner, Portable 3D ScannerGo! (a Creaform ACADEMIA™ solution) [33]. This device and its
technology allow the capture of high-quality geometries and accurate measurements. Some sequences
from the 3D scanning operation are presented in Figure 6. Figure 6. Sequences from 3D scanning operation
The 3D scans are evaluated by VXmodel, a post-processing software that integrates VXelements, a 3D
scanning platform that allows the captured 3D scan data to be investigated for further measurements [34]. In Figure 7, the program interface and a model of deformation measurement are illustrated. All tested plates, had been evaluated to determine de residual deformation after impact, and the data
obtained are presented in Section 3. Figure 6. Sequences from 3D scanning operation Figure 6. Sequences from 3D scanning operation The 3D scans are evaluated by VXmodel, a post-processing software that integrates VXelements, a 3D
scanning platform that allows the captured 3D scan data to be investigated for further measurements [34]. In Figure 7, the program interface and a model of deformation measurement are illustrated. All tested plates, had been evaluated to determine de residual deformation after impact, and the data
obtained are presented in Section 3. The 3D scans are evaluated by VXmodel, a post-processing software that integrates VXelements, a 3D
scanning platform that allows the captured 3D scan data to be investigated for further measurements [34]. In Figure 7, the program interface and a model of deformation measurement are illustrated. All tested plates, had been evaluated to determine de residual deformation after impact, and the data
obtained are presented in Section 3. Figure 7. 3D scan processing
The videos recorded are investigated with Tracker software (Figure 8). The displacement for three
distinct points is recorded and the data are exported and processed through Mathcad software, using an
algorithm to determine the velocity, the acceleration and the maximum force. Figure 7. 3D scan processing The videos recorded are investigated with Tracker software (Figure 8). The displacement for three
distinct points is recorded and the data are exported and processed through Mathcad software, using an
algorithm to determine the velocity, the acceleration and the maximum force. Mater. 2.2. Numerical study The target plates were constrained on the exterior contours in order to replicate the test set-up. The target plates were constrained on the exterior contours in order to replicate the test set-up. The contact between the component elements of the model was achieved by imposing the
"CONTACT_2D_AUTOMATIC_SURFACE_TO_SURFACE" contact condition. The contact between the component elements of the model was achieved by imposing the
"CONTACT_2D_AUTOMATIC_SURFACE_TO_SURFACE" contact condition. The material models introduced in the program for simulated structures are: for aluminum plate, a
material model of type *MAT_PLASTIC_KINEMATIC, and for the polyurea-polyurethane coated on
the back of the aluminum plate (for all 4 cases), a material model of type *MAT_OGDEN_RUBBER. No erosion or failure criteria were used for either material types. The material models introduced in the program for simulated structures are: for aluminum plate, a
material model of type *MAT_PLASTIC_KINEMATIC, and for the polyurea-polyurethane coated on
the back of the aluminum plate (for all 4 cases), a material model of type *MAT_OGDEN_RUBBER. material model of type *MAT_PLASTIC_KINEMATIC, and for the polyurea-polyurethane coated on
the back of the aluminum plate (for all 4 cases), a material model of type *MAT_OGDEN_RUBBER. No erosion or failure criteria were used for either material types. No erosion or failure criteria were used for either material types. In both cases, were used the material constants presented in [20] and the true stress-true strain curves
presented in Figure 3. 2.2. Numerical study The numerical study of the experimental tests was performed through LS-DYNA software
specialized in modeling the transient nonlinear phenomena [35]. The testing conditions and the geometry
allow the use of a simplified model, namely, the problem is formulated 2D axis-symmetric. In Figure 9
the virtual model geometry (2D and 3D) for impactor and plate is illustrated. The numerical study of the experimental tests was performed through LS-DYNA software
specialized in modeling the transient nonlinear phenomena [35]. The testing conditions and the geometry
allow the use of a simplified model, namely, the problem is formulated 2D axis-symmetric. In Figure 9
the virtual model geometry (2D and 3D) for impactor and plate is illustrated. The simulation was carried out in six configurations, as follows: impact on an aluminum plate, impact
on an aluminum plate with polyurea-polyurethane coating (4 configurations) and impact on two
aluminum plates arranged together. Figure 9. Virtual model geometry: a) specific dimensions, b) 3D model Figure 9. Virtual model geometry: a) specific dimensions, b) 3D model The geometry of the virtual model is in accordance with the real ones: the impactor has the
dimensions presented in Figure 9a and for the aluminum plate a disk with a diameter of 100 mm
representing the central circular area of the tested plate was considered. The geometry of the virtual model is in accordance with the real ones: the impactor has the
dimensions presented in Figure 9a and for the aluminum plate a disk with a diameter of 100 mm
representing the central circular area of the tested plate was considered. For the modeled cases, axial symmetric plane elements of SHELL 15 type were used, with weighting of
the functions by volume. The discretized domain, with detail in the impact area is illustrated in Figure 10. Mater. Plast., 61 (1), 2024, 170-184 175 https://doi.org/10.37358/MP.24.1.5712 MATERIALE PLASTICE https://revmaterialeplastice.ro https://revmaterialeplastice.ro
https://doi.org/10.37358/Mat.Plast.1964 https://doi.org/10.37358/Mat.Plast.1964 Figure 10. Shape-headed impactor discretization with a detail on the impact area Figure 10. Shape-headed impactor discretization with a detail on the impact area The mesh is structured with a total number of 12778 elements and 13389 nodes, having a finer
discretization in the impact area of the hemispherical shape impactor head. p
p
p
p
The impact is simulated by imposing the values of the impact velocities on all the nodes
corresponding to the impactor geometry. 3. Results and discussions All the results obtained from the laboratory test analysis (force sensor, 3D Scan and video processing)
are presented in relation with the results from the numerical study. From the experimental tests, the maximum force is obtained for both approaches, and in terms of
deformation, from the 3D Scan processing the residual deformation is obtained and from the video
processing the maximum deformation. Two representative examples of the maximum force evolution obtained by force sensor and by
analytically processed data from video processing (velocity time derivation and second principle of
mechanics) are presented in Figure 11. It is observed that the curve of force evolution has an ascending
trend with oscillations. Figure 11. Force evolution (standard plate): a) registered by force sensor,
b) processed with the Mathcad software from the video footage Figure 11. Force evolution (standard plate): a) registered by force sensor,
b) processed with the Mathcad software from the video footage Mater. Plast., 61 (1), 2024, 170-184 https://doi.org/10.37358/MP.24.1.5712 176 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 Regarding the tested plates behavior, it is observed that an identical deformation pattern is obtained
in the following cases (Table 1): • 0.2 bar pressure on SA plate – deformation but no fissures; • 0.2 bar pressure on SA plate – deformation but no fissures ar pressure on the multilayer plate, SA+PU – deformation but no fissures; • 0.4 bar pressure on the multilayer plate, SA+PU – deformation but no fissures; 6 bar pressure on the multilayer plate, SA+PU-NC3 – deformation but no fissures. • 0.6 bar pressure on the multilayer plate, SA+PU-NC3 – deformation but no fiss From the images with the deformation patterns, it can be observed that for the multilayer
configurations the damage of the aluminum plate is reduced and the polyurea-polyurethane layer absorbs
the kinetic energy of the impactor and reduce the degree of plates deformation. Furthermore, aluminum plates coated with polyurea-polyurethane-MWCNTs
configuration allows the metal plate to withstand a load two times higher in terms of ki It can be noticed the behavior of multilayer plates when higher deformation and the cracking process
and fracture appears. Both aluminum plate coated with polyurea-polyurethane 0.2% carbon nanotubes
and double aluminum plate suffered a complete perforation at an approximate velocity value of 14 m/s. It can be noticed the behavior of multilayer plates when higher deformation and the cracking process
and fracture appears. 3. Results and discussions Both aluminum plate coated with polyurea-polyurethane 0.2% carbon nanotubes
and double aluminum plate suffered a complete perforation at an approximate velocity value of 14 m/s. In Table 2 are given the main parameters measured: velocity, deformation and force. The same data
are presented in a graphical manner in Figures 12-14. The curves are divided in two groups: SA, SA+PU
and DA, and SA + PU, SA + PU-NC2, SA + PU-NC3 and SA + PU-NC4, for each type of measurement
and parameter of interest. It can be seen that the maximum force increases along with the impact velocity until the cracks occur. No significant increase is observed after this threshold, regardless of the type of configuration subjected
to the impact. p
Moreover, the measured results are in good accordance with data obtained from image processing
until the above-mentioned threshold is reached. Mater. Plast., 61 (1), 2024, 170-184 177 https://doi.org/10.37358/MP.24.1.5712
Table 1. Deformation patterns after impact loading [5]
Aluminum plate
(SA)
Al. plate + PU
Al. plate +
PU-NC2
Al. plate +
PU-NC3
Al. plate +
PU-NC4
Double
Al. plate (DA)
0.2 bar
0.3 bar
0.4 bar
0.5 bar Double
Al. plate (DA) Mater. Plast., 61 (1), 2024, 170-184 Mater. Plast., 61 (1), 2024, 170-184 https://doi.org/10.37358/MP.24.1.5712 https://doi.org/10.37358/MP.24.1.5712 177 MATERIALE PLASTICE
https://revmaterialeplastice.ro
https://doi.org/10.37358/Mat.Plast.1964
Mater. Plast., 61 (1), 2024, 170-184 178 https://doi.org/10.37358/MP.24.1.5712
0.6 bar
0.7 bar
0.8 bar
0.9 bar
Table 2. Important parameters values at the impact loading from experimental study
Parameters
Aluminum
plate (SA)
Al. plate +
PU
Al. plate +
PU-NC2
Al. plate +
PU-NC3
Al. plate +
PU-NC4
Double Al. Mater. Plast., 61 (1), 2024, 170-184 178 https://doi.org/10 184 178 https://doi.org/10.37358/MP.24.1.5712 3. Results and discussions plate (DA)
0.2 bar
Velocity (m/s)
5.63
5.81
5.62
Deformation
(mm)
3D
Scan
9.87
9.64
9.35
Video
9.98
9.85
9.69
Force
(KN)
Sensor
2
2
2.1
Video
2.38
2.43
2.39
0.3 bar
Velocity (m/s)
7.33
7.29
7.1
6.93
7.00
7.39
Deformation
(mm)
3D Scan
13.4
12.75
12.25
12.1
12.06
8.75
Video
13.57
13.09
12.54
12.5
12.62
9.48
Force
(KN)
Sensor
2.6
3.0
2.8
2.3
2
3.22
Video
2.92
3.1
3.02
2.78
3.07
3.7
0.4 bar
Velocity (m/s)
8.99
9.03
9.2
9.03
8.97
9.02
Deformation
(mm)
3D Scan
20.19
15.31
15.43
14.54
14.93
13.04
Video
20.55
15.92
15.79
16.16
15.97
13.26
Force
(KN)
Sensor
2.5
3.6
3.8
3.8
4.0
4.28
Video
2.61
3.78
3.96
3.88
4.14
4.74
0.5 bar
Velocity (m/s)
9.23
10.67
10.82
10.73
10.63
10.66
Deformation
(mm)
3D Scan
22.38
17.18
17.02
18.06
17.02
13.13
Video
23
18.24
17.48
18.26
17.31
14.87
Force
(KN)
Sensor
1.8
4.1
4.3
5.0
4.4
4.97
Video
2.7
4.29
4.5
4.64
4.58
5.66 MATERIALE PLASTICE 0.6 bar
0.7 bar
0.8 bar
0.9 bar 0.6 bar
0.7 bar
0.8 bar
0.9 bar Table 2. Important parameters values at the impact loading from experimental study
Parameters
Aluminum
plate (SA)
Al. plate +
PU
Al. plate +
PU-NC2
Al. plate +
PU-NC3
Al. plate +
PU-NC4
Double Al. 3. Results and discussions plate (DA)
0.2 bar
Velocity (m/s)
5.63
5.81
5.62
Deformation
(mm)
3D
Scan
9.87
9.64
9.35
Video
9.98
9.85
9.69
Force
(KN)
Sensor
2
2
2.1
Video
2.38
2.43
2.39
0.3 bar
Velocity (m/s)
7.33
7.29
7.1
6.93
7.00
7.39
Deformation
(mm)
3D Scan
13.4
12.75
12.25
12.1
12.06
8.75
Video
13.57
13.09
12.54
12.5
12.62
9.48
Force
(KN)
Sensor
2.6
3.0
2.8
2.3
2
3.22
Video
2.92
3.1
3.02
2.78
3.07
3.7
0.4 bar
Velocity (m/s)
8.99
9.03
9.2
9.03
8.97
9.02
Deformation
(mm)
3D Scan
20.19
15.31
15.43
14.54
14.93
13.04
Video
20.55
15.92
15.79
16.16
15.97
13.26
Force
(KN)
Sensor
2.5
3.6
3.8
3.8
4.0
4.28
Video
2.61
3.78
3.96
3.88
4.14
4.74
0.5 bar
Velocity (m/s)
9.23
10.67
10.82
10.73
10.63
10.66
Deformation
(mm)
3D Scan
22.38
17.18
17.02
18.06
17.02
13.13
Video
23
18.24
17.48
18.26
17.31
14.87
Force
(KN)
Sensor
1.8
4.1
4.3
5.0
4.4
4.97
Video
2.7
4.29
4.5
4.64
4.58
5.66 https://doi.org/10.37358/MP.24.1.5712 178 MATERIALE PLASTICE p
p
https://doi.org/10.37358/Mat.Plast.1964 0.6 bar
Velocity (m/s)
11.93
11.86
10.7
11.68
11.63
Deformation
(mm)
3D Scan
21.01
20.4
17.64
19.03
15.03
Video
21.13
20.8
18.59
19.45
16.22
Force
(KN)
Sensor
3.6
3.9
4.1
4.8
5.47
Video
4.39
4.46
4.43
4.95
6.11
0.7 bar
Velocity (m/s)
12.98
13.16
12.9
13.12
Deformation
(mm)
3D Scan
18.99
20.88
18.19
17.67
Video
20.66
21.37
19.97
18.57
Force
(KN)
Sensor
4.03
4.54
4.0
6.45
Video
4.75
5.16
4.9
6.35
0.8 bar
Velocity (m/s)
14.23
13.98
14.1
Deformation
(mm)
3D
Scan
22.81
P
20.95
Video
23.97
P
21.77
Force
(KN)
Sensor
4.4
4.1
6.19
Video
4.9
4.66
5.88
0.9 bar
Velocity (m/s)
14.41
Deformation
(mm)
3D
Scan
P
Video
P
Force
(KN)
Sensor
6.32
Video
5.97
P – Complete perforation. Figure 12. 3. Results and discussions Comparative evolution of deformation – velocity curve 0.6 bar
Velocity (m/s)
11.93
11.86
10.7
11.68
11.63
Deformation
(mm)
3D Scan
21.01
20.4
17.64
19.03
15.03
Video
21.13
20.8
18.59
19.45
16.22
Force
(KN)
Sensor
3.6
3.9
4.1
4.8
5.47
Video
4.39
4.46
4.43
4.95
6.11
0.7 bar
Velocity (m/s)
12.98
13.16
12.9
13.12
Deformation
(mm)
3D Scan
18.99
20.88
18.19
17.67
Video
20.66
21.37
19.97
18.57
Force
(KN)
Sensor
4.03
4.54
4.0
6.45
Video
4.75
5.16
4.9
6.35
0.8 bar
Velocity (m/s)
14.23
13.98
14.1
Deformation
(mm)
3D
Scan
22.81
P
20.95
Video
23.97
P
21.77
Force
(KN)
Sensor
4.4
4.1
6.19
Video
4.9
4.66
5.88
0.9 bar
Velocity (m/s)
14.41
Deformation
(mm)
3D
Scan
P
Video
P
Force
(KN)
Sensor
6.32
Video
5.97
P – Complete perforation. 0.6 bar
Velocity (m/s)
11.93
11.86
10.7
11.68
11.63
Deformation
(mm)
3D Scan
21.01
20.4
17.64
19.03
15.03
Video
21.13
20.8
18.59
19.45
16.22
Force
(KN)
Sensor
3.6
3.9
4.1
4.8
5.47
Video
4.39
4.46
4.43
4.95
6.11
0.7 bar
Velocity (m/s)
12.98
13.16
12.9
13.12
Deformation
(mm)
3D Scan
18.99
20.88
18.19
17.67
Video
20.66
21.37
19.97
18.57
Force
(KN)
Sensor
4.03
4.54
4.0
6.45
Video
4.75
5.16
4.9
6.35
0.8 bar
Velocity (m/s)
14.23
13.98
14.1
Deformation
(mm)
3D
Scan
22.81
P
20.95
Video
23.97
P
21.77
Force
(KN)
Sensor
4.4
4.1
6.19
Video
4.9
4.66
5.88
0.9 bar
Velocity (m/s)
14.41
Deformation
(mm)
3D
Scan
P
Video
P
Force
(KN)
Sensor
6.32
Video
5.97
P – Complete perforation. Mater. Plast., 61 (1), 2024, 170-184 179 https://doi.org/10.37358/MP.24.1.5712
Deformation
(mm)
Scan
P
Video
P
Force
(KN)
Sensor
6.32
Video
5.97
P – Complete perforation. Figure 12. Comparative evolution of deformation – velocity curve P – Complete perforation. Figure 12. Comparative evolution of deformation – velocity curve Figure 12. Comparative evolution of deformation – velocity curve Mater. Plast., 61 (1), 2024, 170-184 179 https://doi.org/10.37358/MP.24.1.5712 179 Mater. Plast., 61 (1), 2024, 170-184 Mater. Plast., 61 (1), 2024, 170-184 179 https://doi.org/10.37358/MP.24.1.5712 MATERIALE PLASTICE p
p
https://doi.org/10.37358/Mat.Plast.1964 https://doi.org/10.37358/Mat.Plast.1964
Figure 13. Comparative evolution of maximum force – velocity curve
Figure 14. Comparative evolution of maximum force – deformation curve Fi
13 C
i
l i
f
i
f
l
i Figure 13. Comparative evolution of maximum force – velocity curve
Figure 14. Comparative evolution of maximum force – deformation curve Figure 13. Comparative evolution of maximum force – velocity curve Figure 13. 3. Results and discussions Impactor velocity curve (Al. plate + PU-NC3 – 7 m/s)
Figure 17. Impactor acceleration curve (Al. plate + PU-NC3 – 7 m/s) Figure 16. Impactor velocity curve (Al. plate + PU-NC3 – 7 m/s) Figure 16. Impactor velocity curve (Al. plate + PU-NC3 – 7 m/s)
Figure 17. Impactor acceleration curve (Al. plate + PU-NC3 – 7 m/s)
Figure 18. Deformation of plate central point (Al. plate + PU-NC3 – 7 m/s)
Table 3. Important parameters values at the impact from numerical study
Parameters
Aluminum
plate
Al. plate +
PU
Al. plate +
PU-NC2
Al. plate +
PU-NC3
Al. plate +
PU-NC4
Double
Al. plate
elocity (m/s)
5.6
6
5.6
ual Deform. (mm)
11.5
9.85
9.78
um Deform. (mm)
12
10.2
10.1
mum Force (kN)
2.6
2.82
2.7
elocity (m/s)
7.3
7.29
7.1
7
7.0
7.0
ual Deform. (mm)
14.2
13.1
12.95
10
12.5
10.1
um Deform. (mm)
14.7
13.5
13.2
12.3
12.85
10.6
mum Force (kN)
3.6
3.9
3.4
3.0
2.88
4.2
elocity (m/s)
9.0
9.2
9.0
9.0
9
ual Deform. (mm)
15.86
15.43
15.06
14.93
13.53
um Deform. (mm)
16.21
15.79
16.8
15.97
13.81
mum Force (kN)
4.05
3.8
4.2
4.0
5.3 Figure 16. Impactor velocity curve (Al. plate + PU-NC3 – 7 m/s) Figure 16. Impactor velocity curve (Al. plate + PU-NC3 – 7 m/s) Fi
17 I
l
i
(Al
l
PU NC3
7
/ ) Figure 17. Impactor acceleration curve (Al. plate + PU-NC3 – 7 m/s) Figure 17. Impactor acceleration curve (Al. plate + PU-NC3 – 7 m/s) Figure 18. Deformation of plate central point (Al. plate + PU-NC3 – 7 m/s) Figure 18. Deformation of plate central point (Al. plate + PU-NC3 – 7 m/s) Table 3. Important parameters values at the impact from numerical study
Parameters
Aluminum
plate
Al. plate +
PU
Al. plate +
PU-NC2
Al. plate +
PU-NC3
Al. plate +
PU-NC4
Double
Al. plate
Velocity (m/s)
5.6
6
5.6
Residual Deform. (mm)
11.5
9.85
9.78
Maximum Deform. (mm)
12
10.2
10.1
Maximum Force (kN)
2.6
2.82
2.7
Velocity (m/s)
7.3
7.29
7.1
7
7.0
7.0
Residual Deform. (mm)
14.2
13.1
12.95
10
12.5
10.1
Maximum Deform. (mm)
14.7
13.5
13.2
12.3
12.85
10.6
Maximum Force (kN)
3.6
3.9
3.4
3.0
2.88
4.2
Velocity (m/s)
9.0
9.2
9.0
9.0
9
Residual Deform. (mm)
15.86
15.43
15.06
14.93
13.53
Maximum Deform. 3. Results and discussions Comparative evolution of maximum force – velocity curve Figure 13. Comparative evolution of maximum force – velocity curve y Figure 14. Comparative evolution of maximum force – deformation curve Regarding the numerical simulation, the constraints and contact chosen allow to observed both
loading and unloading phases. In Figure 15 is presented the Al. plate + PU-NC3 model for the maximum
plate deformation and a subsequent moment when the impact is no longer in contact with the plate. Regarding the numerical simulation, the constraints and contact chosen allow to observed both
loading and unloading phases. In Figure 15 is presented the Al. plate + PU-NC3 model for the maximum
plate deformation and a subsequent moment when the impact is no longer in contact with the plate. Regarding the numerical simulation, the constraints and contact chosen allow to observed both
loading and unloading phases. In Figure 15 is presented the Al. plate + PU-NC3 model for the maximum
plate deformation and a subsequent moment when the impact is no longer in contact with the plate. p
q
p
g
p
Based on the simulation results, the time-dependent variations of the velocity and acceleration of the
impactor were recorded, as well as the displacement of a node from the tested configuration for all
simulated scenarios. Typical curves evolutions are given in Figures 16-18. From these curves were
extracted the same parameters as those measured in the experimental study (Table 3). Based on the simulation results, the time-dependent variations of the velocity and acceleration of the
impactor were recorded, as well as the displacement of a node from the tested configuration for all
simulated scenarios. Typical curves evolutions are given in Figures 16-18. From these curves were
extracted the same parameters as those measured in the experimental study (Table 3). a) b)
Figure 15. Sequences for impact at 7 m/s velocity for Al. plate + PU-NC3:
a) maximum plate deformation, b) backward impactor movement b) )
Figure 15. Sequences for impact at 7 m/s velocity for Al. plate + PU-NC3:
a) maximum plate deformation, b) backward impactor movement Mater. Plast., 61 (1), 2024, 170-184 https://doi.org/10.37358/MP.24.1.5712 180 MATERIALE PLASTICE https://revmaterialeplastice.ro https://revmaterialeplastice.ro p
p
https://doi.org/10.37358/Mat.Plast.1964 rg/10.37358/Mat.Plast.1964
Figure 16. Impactor velocity curve (Al. plate + PU-NC3 – 7 m/s)
Figure 17. Impactor acceleration curve (Al. plate + PU-NC3 – 7 m/s)
Figure 18. Deformation of plate central point (Al. plate + PU-NC3 – 7 m/s) Figure 16. 84 181 https://doi.org/10.37358/MP.24.1.5712 3. Results and discussions (mm)
16.21
15.79
16.8
15.97
13.81
Maximum Force (kN)
4.05
3.8
4.2
4.0
5.3 Mater. Plast., 61 (1), 2024, 170-184 181 MATERIALE PLASTICE p
p
https://doi.org/10.37358/Mat.Plast.1964 Velocity (m/s)
10.5
11.0
11.0
10.66
10.66
Residual Deform. (mm)
17.89
17.54
18.96
17.72
14.5
Maximum Deform. (mm)
19.10
18.38
19.66
18.27
15.1
Maximum Force (kN)
4.1
4.3
5.3
4.8
6.8
Velocity (m/s)
10.7
11.7
11.63
Residual Deform. (mm)
18.37
19.64
16.4
Maximum Deform. (mm)
18.94
20.02
17.0
Maximum Force (kN)
4.75
5.03
7.2
Velocity (m/s)
13.12
Residual Deform. (mm)
17.6
Maximum Deform. (mm)
18.3
Maximum Force (kN)
7.8
*For the cases where the total perforation occurs, the numerical simulation was not performed. First of all, the simulation results in terms of plates deformations are in good agreement with the
experimental ones. Nevertheless, the differences between the maximum forces recorded and those
calculated are more significant, for example for a single aluminum plate impacted at 7 m/s, the
experimental result is value 2.6/2.9 kN and from numerical simulation is 3.6 kN. These differences may be explained by a possible misalignment between the moving part and the
target support where the sensor was mounted. This assumption is sustained also by the differences
between the recorded force profiles in time and calculated ones. As it can be seen in Figure 17, no
oscillations occur during the loading phase, as long as the simulations deals with an ideal axial
configuration. References 1. SAI S. SARVA, DESCHANEL, S., BOYCE, M.C., CHEN, W., Stress–strain behavior of a polyurea
and a polyurethane from low to high strain rates, Polymer, 48(8), 2007, 2208-2213. 1. SAI S. SARVA, DESCHANEL, S., BOYCE, M.C., CHEN, W., Stress strain behavior of a polyurea
and a polyurethane from low to high strain rates, Polymer, 48(8), 2007, 2208-2213. 2. RAMAN, S.N., NGO, T., LU, J., MENDIS, P., Experimental investigation on the tensile behavior of
polyurea at high strain rates. Mater. Des., 50, 2013,124-129. 3. AMINI, M.R., ISAACS, J., NEMAT-NASSER, S., Investigation of effect of polyurea on response of
steel plates to impulsive loads in direct pressure-pulse experiments. Mech. Mater., 42, 2010, 628–639. 4. TOADER, G., DIACON, A., AXINTE, S.M., MOCANU, A., RUSEN, E., State-of-the-Art Polyurea
Coatings: Synthesis Aspects, Structure–Properties Relationship, and Nanocomposites for Ballistic
Protection Applications Polymers 16 2014 454 2. RAMAN, S.N., NGO, T., LU, J., MENDIS, P., Experimental investigation on the tensile behavior of
polyurea at high strain rates. Mater. Des., 50, 2013,124-129. 3. AMINI, M.R., ISAACS, J., NEMAT-NASSER, S., Investigation of effect of polyurea on response of
steel plates to impulsive loads in direct pressure-pulse experiments. Mech. Mater., 42, 2010, 628–639. 4. TOADER, G., DIACON, A., AXINTE, S.M., MOCANU, A., RUSEN, E., State-of-the-Art Polyurea
Coatings: Synthesis Aspects, Structure–Properties Relationship, and Nanocomposites for Ballistic
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y
5. ***Raport stiintifico și tehnic, Contract nr. 278/2014, Sistem de Protectie Antiexplozie pentru
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2003, 187–192. Á 7. FINDIK, F., TARIM, N., Ballistic impact efficiency of polymer composites. Compos. Struct., 61,
2003, 187–192. Á 8. ÁVILA, A.F., NETO, A.S., NASCIMENTO, H., JR., Hybrid nanocomposites for mid-range ballistic
protection. Int. J. Impact Eng., 38, 2011, 669-676. 9. IFTIMIE, B., LUPOAE, M., ORBAN, O., Experimental Investigations Regarding the Behavior of
Composite Panels Based on Polyurea and Kevlar or Dyneema Layers Under Blast and Fragments, Mater. Plast., 56(3), 2019, 538-542. 10. ROTARIU, A., BUCUR F., TOADER, G., LUPOAE M., SAVA, A., ȘOMOIAG, P., CIRMACI-
MATEI, M.V., Experimental Study on Polyurea Coating Effects on Deformation of Metallic Plates
Subjected to Air Blast Loads, Mater. 4. Conclusions Laboratory impact tests at low velocity in a dynamic regime were performed on aluminum plates
coated with Hybrid Polyurea-Polyurethane-MWCNTs Nanocomposites. These samples have as the main layer an aluminum plate on which a layer of polyurea-polyurethane
with different compositions was deposited: simple polyurea-polyurethane and polyurea-polyurethane
containing carbon nanotubes in different concentrations. The experimental study investigation was based on the force sensor recordings, high-speed camera
images and 3D scans. While 3D Scans processing highlights the residual deformation, the video images
processing allows us to estimate the maximum deformation, a process parameter that cannot be measured
with post-test tools. The maximum force obtained by image processing is in good accordance with the values measured
by force sensor as long as no fracture occur in aluminum plates. The addition of hyperelastic coatings ensures a better resistance of the aluminum plates, the impact
velocity at which cracks appear increases, the best results being obtained for the composition with 0,2%
carbon nanotubes. The same trend is also observed in the case of the maximum forces recorded during
the tests. However, the presence of the hyperelastic layer does not significantly change the maximum
deformation supported by the aluminum plates before the appearance of cracks. Nevertheless, an
important change is observed regarding the position of the point where the fractures initiate. When
hyperelastic coatings are present, the fracture initiation point moves away from the central point with
the maximum deformation. The fracture propagation mode also changes. If in the case of a simple plate
the initial fracture tends to bifurcate quickly and lead to the formation of petals, when there are
hyperelastic coatings the crack propagates in a circular pattern that leads to the formation of a pseudo-
plug attached to the plate. These differences can be explained by the influence of the hyperelastic layer
on the necking phenomena occurrence and stress triaxiality. An LS-DYNA numerical study was performed for further investigations. The used material models
are proven to be accurate enough to obtain results similar to the experimental ones, in terms of plates
deformations. However, in term of maximum force, the results are no longer similar, a mismatch that
may be attributed to the lack of collinearity between the impactor and the target's support. Mater. 4. Conclusions Plast., 61 (1), 2024, 170-184 https://doi.org/10.37358/MP.24.1.5712 182 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 https://revmaterialeplastice.ro https://revmaterialeplastice.ro
https://doi.org/10.37358/Mat.Plast.1964 The negative residual velocity, observed both in the experimental and numerical studies, is caused by
the elastic component of the plate deformation that disappears during the unloading phase. The concordance between the results obtained based on image processing and the results obtained
through classical methods of measurement and study (sensors, 3D scanning and numerical simulation)
shows that the approach proposed in the present work is a viable one. Acknowledgments: This work is supported by the National Authority for Scientific Research from the
Ministry of Education, Research and Youth of Romania through the National Project contract number
278/2014. Manuscript received: 05.03.2024 References Plast., 53(4), 2016, 670-674. j
( )
11. BUCUR, F., TRANĂ, E., ROTARIU, A., Numerical and experimental study on the locally blast
loaded polyurea coated steel plates, Mater. Plast., 56(3), 2019, 492-499. 12. S. N. RAMAN, T. NGO, P. MENDIS, T. PHAM, Elastomeric Polymers for Retrofitting of Reinforced
Concrete Structures against the Explosive Effects of Blast, Adv. in Mater. Science and Eng., 2012, 754142, 2012. 13. HE, C., LIU, Y., YAO, Y., CHEN, Q., Experimental and Numerical Investigation of Ballistic
Resistance of Polyurea-Coated Aluminum Plates under Projectile Impacts, Crystals, 13, 2023, 1039. 14. HUANG, X.L., ZHANG, W., DENG, Y.F., JIANG, X.W., Experimental investigation on the
ballistic resistance of polymer-aluminum laminated plates, Int. J. Impact Eng. 2018. 15. MOHOTTI, D., NGO, T., RAMAN, S.N., ALI, M., MENDIS, P., Plastic deformation of polyurea
coated composite aluminium plates subjected to low velocity impact. Mater. Des. 2014, 56, 696-713. p
p
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y
p
16. MOHOTTI, D., NGO, T., MENDIS, P., RAMAN, S.N., Polyurea coated composite aluminium
plates subjected to high velocity projectile impact. Int. J. Mater. Des., 52, 2013, 1-16. 16. MOHOTTI, D., NGO, T., MENDIS, P., RAMAN, S.N., Polyurea coated composite aluminium
plates subjected to high velocity projectile impact. Int. J. Mater. Des., 52, 2013, 1-16. p
j
g
y p
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17. MOHOTTI, D., NGO, T., RAMAN, S.N., MENDIS, P., Analytical and numerical investigation of
polyurea layered aluminium plates subjected to high velocity projectile impact, Mater. Design., 82, 2015, 1-17. 17. MOHOTTI, D., NGO, T., RAMAN, S.N., MENDIS, P., Analytical and numerical investigation of
polyurea layered aluminium plates subjected to high velocity projectile impact, Mater. Design., 82, 2015, 1-17. Mater. Plast., 61 (1), 2024, 170-184 183 https://doi.org/10.37358/MP.24.1.5712 MATERIALE PLASTICE
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https://doi.org/10.37358/Mat.Plast.1964 18. WANG, Y., DING, L., LIN, J., QIU, X., WU, C., LIU, C., TIAN, Y., ZHANG, R., HUANG, W.,
MA, M., Recent Developments in Polyurea Research for Enhanced Impact Penetration Resistance and
Blast Mitigation, Polymers, 16, 2024, 440. 19. TOADER, G., MOLDOVAN, A.E., DIACON, A., F.M. DIRLOMAN, E. RUSEN, A. PODARU, T. ROTARIU, GINGHINA, R.E., HOZA, O.E., Effect of Aromatic Chain Extenders on Polyurea and
Polyurethane Coatings Designed for Defense Applications, Polymers, 15(3), 2023, 756. y
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,
20. TOADER, G., DIACON, A., RUSEN, E., RIZEA, F., TEODORESCU, M., STĂNESCU, P.O.,
DAMIAN, C., ROTARIU, A., TRANĂ, E., BUCUR, F., A Facile Synthesis Route of Hybrid Polyurea-
Polyurethane-MWCNTs Nanocomposite Coatings for Ballistic Protection and Experimental Testing in
Dynamic Regime, Polymers, 13, 2021, 1618. 21. NANTASETPHONG, W., AMIRKHIZI, A.V., JIA, Z., NEMAT-NASSER, S., Polyurea-Based
Composites: Ultrasonic Testing and Dynamic Mechanical Properties Modeling, Conference Proceedings
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22. BUCUR, F., TRANĂ, E., ROTARIU, A., GAVRUS A., BARBU, C., GUINES, D., Experimental
and numerical analysis concerning the behaviour of OL50 steel grade specimens coated with polyurea
layer under dynamics loadings, EPJ Web of Conferences, 94, 2015, 04044. 23.ROTARIU, A., TRANĂ, E., MATACHE, L., CIRMACI-MATEI, M., SANDU, S.,
MOLDOVEANU, C., BUCUR, F., Experimental Study on the Dynamic Response of Polyurethane/fly
Ash Ceramic Foam, Mater. Plast., 58(1), 2021, 106-112. 24. BACIU, C., LUPOAE, M., CONSTANTIN, D., Experimental analysis on reduced-scale protective
doors, WIT Transactions on the Built Environment, 180, 2018, 101-111. 25. BUCUR, F., ROTARIU, A., MATACHE, L., BACIU, F., JIGA, G., TRANĂ, E. Experimental and
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and numerical simulations, Mechanics of Materials, 43(7), 2011, 349-360. and numerical simulations, Mechanics of Materials, 43(7), 2011, 349-360. 30. MATERIALE PLASTICE
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https://doi.org/10.37358/Mat.Plast.1964 LIU, Q, WANG, S.P., LIN, X., CUI, P., ZHANG, S., Numerical simulation on the anti-penetration
performance of polyurea-core Weldox 460 E steel sandwich plates. Compos. Struct. 236, 2020, 111852. 31. MOHOTTI, D.; FERNANDO, P.L.N.; WEERASINGHE, D.; REMENNIKOV, A. Evaluation of effectiveness
of polymer coatings in reducing blast-induced deformation of steel plates. Def. Technol., 17, 2021, 1895–1904. 32. ILIESCU, N., ATANASIU, C., HADǍR, A., The simulation of the mechanical behaviour of engineering
structures on models made of plastic materials with special properties, Mater. Plast., 42(1), 2005, 72-76. 33.***https://www.creaform3d.com/en/news/creaform-adds-academia-20-3d-scanner-its-educational-solution- 30. LIU, Q, WANG, S.P., LIN, X., CUI, P., ZHANG, S., Numerical simulation on the anti-penetration
performance of polyurea-core Weldox 460 E steel sandwich plates. Compos. Struct. 236, 2020, 111852. performance of polyurea core Weldox 460 E steel sandwich plates. Compos. Struct. 236, 2020, 111852. 31. MOHOTTI, D.; FERNANDO, P.L.N.; WEERASINGHE, D.; REMENNIKOV, A. Evaluation of effectiveness
of polymer coatings in reducing blast-induced deformation of steel plates. Def. Technol., 17, 2021, 1895–1904. 32. ILIESCU, N., ATANASIU, C., HADǍR, A., The simulation of the mechanical behaviour of engineering
structures on models made of plastic materials with special properties, Mater. Plast., 42(1), 2005, 72-76. 33.***https://www.creaform3d.com/en/news/creaform-adds-academia-20-3d-scanner-its-educational-solution-
suite 31. MOHOTTI, D.; FERNANDO, P.L.N.; WEERASINGHE, D.; REMENNIKOV, A. Evaluation of effectiveness
of polymer coatings in reducing blast-induced deformation of steel plates. Def. Technol., 17, 2021, 1895–1904. 32. ILIESCU, N., ATANASIU, C., HADǍR, A., The simulation of the mechanical behaviour of engineering
structures on models made of plastic materials with special properties, Mater. Plast., 42(1), 2005, 72-76. 32. ILIESCU, N., ATANASIU, C., HADǍR, A., The simulation of the mechanical behaviour of engineering
structures on models made of plastic materials with special properties, Mater. Plast., 42(1), 2005, 72-76. 33.***https://www.creaform3d.com/en/news/creaform-adds-academia-20-3d-scanner-its-educational-solution-
suite 34. ***https://proto3000.com/product/goscan-3d/ 35.***LS-Dyna Keyword users’s manual 971 Mater. Plast., 61 (1), 2024, 170-184 184 https://doi.org/10.37358/MP.24.1.5712 https://doi.org/10.37358/MP.24.1.5712
|
https://openalex.org/W2586107431
|
https://www.nature.com/articles/ncomms14052.pdf
|
English
| null |
Nanoscale π–π stacked molecules are bound by collective charge fluctuations
|
Nature communications
| 2,017
|
cc-by
| 8,778
|
ARTICLE Received 23 Mar 2016 | Accepted 15 Nov 2016 | Published 7 Feb 2017 Received 23 Mar 2016 | Accepted 15 Nov 2016 | Published 7 Feb 2017 Results Binding energies of three supramolecular complexes. We start
by
analysing
a
set
of
three
already
synthesized20,21
and
theoretically investigated22–24 supramolecular p p complexes
(Fig. 1a), consisting of a C70 fullerene guest molecule hosted by
two different cycloparaphenylenes (CPPs; C1 and C2) and a
buckycatcher molecule (C3). The CPPs are the simplest structural
units of ‘armchair’ carbon nanotubes and their complexes are
precursors of fullerene peapods25,26. The buckycatcher complex,
on the other hand, represents a class of convex–concave p p
systems27. The experimental free energies of association of these
complexes are 7, 7 (ref. 28) and 5 kcal mol 1 (ref. 29),
respectively, making C1 and C2 degenerate in terms of stability. Reliable theoretical prediction of free energies of association is yet
an unsolved task burdened by numerous complications, especially
concerning the contribution of solvation effects30. Our focus here
is instead on the electronic origin of p p bonding and hence the
relevant quantities are the binding energies. As we need to
validate our vdW model as described below, we require reliable
reference results to begin with. To avoid the issues associated with
estimating binding energies from experimental free energies,
we have employed a theoretical reference in the form of DQMC
binding energies as an alternative. DQMC approximates the exact
solution of the electronic Schro¨dinger equation to an arbitrary
level of accuracy within the fixed-node approximation31 and has
been shown to yield agreement within 0.1 kcal mol 1 with the
quantum-chemical coupled-cluster method, which is considered
the
‘gold
standard’
for
mid-sized
molecular
complexes32. However, unlike the coupled-cluster method, DQMC scales
favourably with the system size and permits calculations of larger
systems33–35, including the ones of concern here. We note that
relative trends in the binding energies of the complexes (Fig. 2), In
this
work,
we
present
a
viewpoint
on
binding
in
supramolecular p p systems that is based on correlation of
collective quantum electron-density fluctuations, while being
backed up by a quantitative model that is able to provide highly
accurate predictions of binding energies. To this end, we employ
the many-body dispersion (MBD) method18,19, which captures the
anisotropy and collective nature of long-range correlation in such
systems, while maintaining a wavefunction that is transparent
enough for further analysis. Nanoscale p–p stacked molecules are bound by
collective charge fluctuations Jan Hermann1, Dario Alfe`2,3,4 & Alexandre Tkatchenko1,5 Non-covalent p p interactions are central to chemical and biological processes, yet the full
understanding of their origin that would unite the simplicity of empirical approaches with the
accuracy of quantum calculations is still missing. Here we employ a quantum-mechanical
Hamiltonian model for van der Waals interactions, to demonstrate that intermolecular
electron correlation in large supramolecular complexes at equilibrium distances is
appropriately described by collective charge fluctuations. We visualize these fluctuations and
provide connections both to orbital-based approaches to electron correlation, as well as to
the simple London pairwise picture. The reported binding energies of ten supramolecular
complexes obtained from the quantum-mechanical fluctuation model joined with density
functional calculations are within 5% of the reference energies calculated with the diffusion
quantum Monte-Carlo method. Our analysis suggests that p p stacking in supramolecular
complexes can be characterized by strong contributions to the binding energy from
delocalized, collective charge fluctuations—in contrast to complexes with other types of
bonding. 1 Fritz-Haber-Institut der Max-Planck-Gesellschaft, Faradayweg 4–6, 14195 Berlin, Germany. 2 Department of Earth Sciences, University College London,
London WC1E 6BT, UK. 3 Department of Physics and Astronomy, University College London, London WC1E 6BT, UK. 4 London Centre for Nanotechnology and
Thomas Young Centre@UCL, University College London, London WC1E 6BT, UK. 5 Physics and Materials Science Research Unit, University of Luxembourg,
162a Avenue de la Faiencerie, Luxembourg L-1511, Luxembourg. Correspondence and requests for materials should be addressed to A.T. (email: alexandre.tkatchenko@uni.lu). 1 NATURE COMMUNICATIONS | 8:14052 | DOI: 10.1038/ncomms14052 | www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14052 T T
he non-covalent p p interactions between conjugated
aromatic rings play a key role in a wide range of chemical
and biological processes. These interactions contribute
significantly to nucleobase stacking in RNA and DNA1, protein
folding2, molecular recognition3, template-directed synthesis4
and assembly of van der Waals (vdW) heterostructures5. Despite
intense
experimental
and
theoretical
studies,
the
conceptual
understanding
of
p p
interactions
in
these
complex systems is still largely based on small model systems
such as the prototypical benzene dimer. Hunter and Sanders6
coined a classical perspective on p p interactions, in which they
emphasized the electrostatic quadrupole interactions between
p orbitals. With later advances in electronic structure theory,
it became clear that the reality is in fact an intricate interplay
between electrostatic interactions, Pauli repulsion and London
dispersion. Nanoscale p–p stacked molecules are bound by
collective charge fluctuations Countless efforts have been put into the correct
prediction of the most stable conformer of the benzene dimer,
resulting
in
the
excellent
accuracy
of
B0.1 kcal mol 1
(refs
7–12). However,
due
to
the
complex
mathematical
structure
of
high-level
quantum
chemistry
methods,
the
complete understanding of the nature of the binding remains a
challenge even in such relatively small systems. This situation
even led to recent suggestions that there is really nothing special
about p p stacking in terms of intermolecular interactions, and
that the term should be abandoned altogether13,14. On the other
hand, Dobson and others15 have emphasized that collective
plasmon fluctuations in zero-gap one-dimensional and two-
dimensional systems, including conjugated graphene sheets, can
lead to unusual power laws for the binding energies at separations
beyond 10 nm15,16. The transition from the zero to finite gap
between the highest occupied and lowest unoccupied molecular
orbital (HOMO-LUMO) was further investigated by Misquitta
et al.17 who related the difference to the inherently non-local
response of the zero-gap systems. The plasmon-based approaches
are
effective
for
revealing
asymptotic
behaviour
of
vdW
interactions
between
prototypical
low-dimensional
systems. However,
conceptual
understanding
of
interactions
for
equilibrium
molecular
geometries
and
their
quantitative
description remains an open problem comparing with the binding energies of ten supramolecular
complexes obtained by high-level diffusion quantum Monte-
Carlo (DQMC) calculations. We then proceed to show how the
correlation mechanism in MBD corresponds to that of virtual
electronic excitations to unoccupied p-like orbitals in correlated
quantum chemistry methods. This enables us to interpret the
fluctuations in this model directly as correlated charge oscillations
of the electronic clouds. We provide visual representation of these
collective fluctuations and show how they build on, extend and
generalize the atomic pairwise picture of London dispersion from
small model molecules to large realistic systems. Furthermore, we
decompose the total binding energies of various types of
supramolecular complexes into the individual fluctuation modes
and thus provide evidence that the collective nature of these
fluctuations is characteristic of the conjugated systems. NATURE COMMUNICATIONS | 8:14052 | DOI: 10.1038/ncomms14052 | www.nature.com/naturecommunications ARTICLE ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14052 including the degeneracy of C1 and C2, match with the trends in
the free energies of association, further supporting our focus on
the binding energies. the latter improves the bare pairwise results (see Fig. 2), the
deviations from DQMC are still as much as 7 kcal mol 1. Without the three-body correction, the deviations would be as
much as 12 kcal mol 1, suggesting already at this level the
importance of the higher-order contributions. An explanation for
why the higher-order contributions destabilize the complexes
within the framework of MBD is given below. Although DQMC provides highly accurate predictions of the
binding energies, its wavefunction is not directly accessible,
hindering any conceptual
insight into the nature of the
binding and requiring calculations at the edge of current high-
performance computing. To overcome this limitation, we employ
the MBD model, which is built on a quantum-mechanical
Hamiltonian that can be solved exactly and at a manageable
computational
cost18,19,
and
show
that
the
resulting
wavefunctions can be interpreted in a straightforward manner. In particular, the MBD model represents each atom with a
pseudoelectron in a harmonic potential, which is constructed in
such a way as to reproduce the long-range dynamic response of
valence electrons of an atom within a 3% accuracy36. The
quantum charge fluctuations in such pseudoatoms are then
correlated within the dipole approximation to all orders of the
interaction potential, to obtain the long-range electron correlation
energy37,38. The MBD method has been successfully used to
model vdW interactions in a broad range of systems, ranging
from small gas-phase complexes to molecular crystals39, to hybrid
interfaces40, to complex nanostructured materials34. Changes in molecular polarizabilities. Having established the
accuracy of MBD for the systems in question, we proceed with
analysis of the mechanism of the binding by inspecting the
correlated wavefunctions of the MBD Hamiltonian. The many-
body correlation effects can be roughly divided into intra- and
intermolecular terms. Within an isolated molecule or material,
they manifest themselves in the non-trivial dependence of
the total polarizability on the system size41,42. The long-range
electrodynamic screening can lead to both increased or decreased
polarizability with respect to sum of atomic polarizabilities,
depending on the geometry and dimensionality of the system. For
example, fullerenes with their relatively bulky shape are typically
depolarized by these effects18, whereas linear and planar systems
often exhibit enhanced polarizability. ARTICLE The blue bar indicates the statistical sampling
errors in the energy, which are inherent to the method. MBD is the
MBD method19 calculated on top of the PBE exchange-correlation density
functional58. D3 is the DFT-D3 approach59 with the optional three-body
correction on top of the PW6B95 density functional; the values were taken
from refs 23,24. Numerical values are presented in Supplementary Table 1. [10]CPP
Fig
po
ne
ato
co quantum chemistry picture, the correlation of the fragment
wavefunctions in conjugated complexes occurs largely via p-p*
DQMC (reference)
MBD
D3
D3 (3-body)
–30
–40
–50
Interaction energy (kcal mol–1)
Figure 2 | Interaction energies of complexes C1–C3 (kcal mol 1). The energies are evaluated with respect to relaxed fragments (see
Supplementary Data 1). DQMC is the reference diffusion quantum
Monte-Carlo method31. The blue bar indicates the statistical sampling
errors in the energy, which are inherent to the method. MBD is the
MBD method19 calculated on top of the PBE exchange-correlation density
functional58. D3 is the DFT-D3 approach59 with the optional three-body
correction on top of the PW6B95 density functional; the values were taken
from refs 23,24. Numerical values are presented in Supplementary Table 1. [10]CPP
C70
Figure 3 | Heat map of the xy component of the interfragment non-local
polarizability axy
AB in [10]CPP–C70. Red and blue represent positive and
negative values, respectively. Rows and columns correspond to carbon
atoms of [10]CPP and C70, respectively, with four select carbon atoms
color-coded to the marked atoms in the structure in the inset. NATURE COMMUNICATIONS | 8:14052 | DOI: 10.1038/ncomms14052 | www.nature.com/naturecommunications
3 Interaction energy (kcal mol–1) [10]CPP
C70
Figure 3 | Heat map of the xy component of the interfragment non-local
polarizability axy
AB in [10]CPP–C70. Red and blue represent positive and
negative values, respectively. Rows and columns correspond to carbon
atoms of [10]CPP and C70, respectively, with four select carbon atoms
color-coded to the marked atoms in the structure in the inset. [10]CPP Figure 2 | Interaction energies of complexes C1–C3 (kcal mol 1). The energies are evaluated with respect to relaxed fragments (see
Supplementary Data 1). DQMC is the reference diffusion quantum
Monte-Carlo method31. The blue bar indicates the statistical sampling
errors in the energy, which are inherent to the method. MBD is the
MBD method19 calculated on top of the PBE exchange-correlation density
functional58. ARTICLE The latter can lead to
increased stabilization of some p p stacked systems as observed
by Grimme and colleagues13,43 for linear condensed acenes. In
the complexes studied here, the fullerene molecule shows 25%
depolarization with respect to the sum of atomic polarizabilities,
whereas the CPP rings in C1 and C2 show 31% and 35% increase,
respectively. Having said that, this intramolecular portion of the
electron correlation, while heavily influencing the magnitude of
the intermolecular binding, is not its cause. In particular, the
intramolecular correlation is captured in the diagonal blocks of
the non-local polarizability a(r, r0), the blocks being defined by
the interacting components (see Fig. 3 and Supplementary Figs 1
and 2 for plots of a). The intermolecular correlation, on the other
hand, is encoded in the much finer structure of the off-diagonal
blocks, which is propagated into differences in the wavefunction of
the whole complex with respect to those of the isolated fragments. Correct description of binding energetics in supramolecular
complexes is challenging due to the delicate balance between
different types of intermolecular interactions. Therefore, any
approximate model requires a systematic verifications of its
accuracy for a given class of supramolecular systems. In this
regard, Fig. 2 demonstrates that MBD is fully capable of
describing all three complexes C1–C3. The only deviation from
the reference occurs for C3 and amounts to an underbinding of
1.5 kcal mol 1 (4%) outside the statistical range given by DQMC,
which can be attributed to possible inaccuracies in the coupling of
MBD to the underlying density functional of Perdew, Burke, and
Ernzerhof (PBE) and to neglected higher-multipole coupling. To
put the predictions of MBD in context, they can be compared
with those of the D3 dispersion model as calculated by Antony
et al.24 D3 employs a pairwise approximation to London
dispersion with an optional three-body correction and, although Charge polarization induced by vdW interactions. In the
quantum chemistry picture, the correlation of the fragment
wavefunctions in conjugated complexes occurs largely via p-p* DQMC (reference)
MBD
D3
D3 (3-body)
–30
–40
–50
Interaction energy (kcal mol–1) qu
wa
DQMC (reference)
MBD
D3
D3 (3-body)
–30
–40
–50
Interaction energy (kcal mol–1)
Figure 2 | Interaction energies of complexes C1–C3 (kcal mol 1). The energies are evaluated with respect to relaxed fragments (see
Supplementary Data 1). DQMC is the reference diffusion quantum
Monte-Carlo method31. Results We establish validity of this model by C1
3.44
3.44
3.44
3.44
3.44
3.62
3.67
3.61
3.40
3.40
3.74
3.59
C3
C2
a
b
3.77
4.91
Figure 1 | Equilibrium structures of studied p p stacked complexes. Marked distances are in ångstro¨ms. Geometries of all structures are available
as XYZ files in Supplementary Data 1. (a) Three supramolecular complexes comprising the fullerene C70 molecule (grey) as a guest in three different
hosts (black and white): [10]-and [11]CPP, and a buckycatcher molecule, labelled C1, C2 and C3, respectively. (b) Benzene dimer in its two nearly
degenerate conformations: stacked parallel-displaced (top) and T-shaped (bottom). b a a C2 Figure 1 | Equilibrium structures of studied p p stacked complexes. Marked distances are in ångstro¨ms. Geometries of all structures are available
as XYZ files in Supplementary Data 1. (a) Three supramolecular complexes comprising the fullerene C70 molecule (grey) as a guest in three different
hosts (black and white): [10]-and [11]CPP, and a buckycatcher molecule, labelled C1, C2 and C3, respectively. (b) Benzene dimer in its two nearly
degenerate conformations: stacked parallel-displaced (top) and T-shaped (bottom). 2 NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14052 ^hMBD
i
fi xi
¼oifi xi
ð1Þ ð1Þ where oi are frequencies (energies) of the coupled fluctuations,
which have the form of linear combinations of the atomic
fluctuations,
xi¼ P
A CiAxA. The
binding
combinations
(in
analogy to bonding orbitals) have lower energies, which leads
to an increased spatial extent of the fluctuations as shown by the
charge density differences in Fig. 4b. The charge densities r(r) are
calculated explicitly from the MBD coupled wavefunctions
cMBD
j
i¼ Q
i fi xi
via the charge density operator ^r (see
Methods for details), rMBD rð Þ¼ cMBD ^r
j jcMBD
h
i
ð2Þ ð2Þ 0.3
0.4
0.5
0.6
–500
0
500
1,000
d
Density of modes i (a.u.)
Frequency i (a.u.) 0.3
0.4
0.5
0.6
–500
0
500
1,000
Fragments
Complex
Difference (×20)
b
a
d
e
f
Density of modes i (a.u.)
Frequency i (a.u.)
0.3
0.4
0.5
0.6
–5,000
0
5,000
Density of modes i (a.u.)
Frequency i (a.u.)
c
Figure 4 | Analysis of correlated charge fluctuations in supramolecular complexes and benzene dimer. (a–c) Electron density differences (charge
polarization) between a complex and its isolated fragments induced by vdW interactions. Yellow and blue colour denote accumulation and depletion of
charge density, respectively. Magnitude of the density difference is mapped to saturation of the colour with 50% saturation corresponding to the density of
2 10 5 and 4 10 5 a.u. for the benzene dimer and the complex C1, respectively. Charge polarizations were obtained either from a DFTcalculation with
a self-consistent TS functional in the parallel-displaced benzene dimer (see Fig. 1) (a) or directly from the correlated MBD wavefunctions (see main text for
details) for the benzene dimer (b) and the complex C1 (c). In the latter case, the relevant charge densities are calculated as expectation values of the charge
density operator, cMBD ^r
j jcMBD
h
i. (d) Energy densities of the oscillation states for parallel-displaced benzene dimer (top) and complex C1 (bottom). Gaussian smoothing with half-width of 0.06 eV was applied. (e,f) Select collective fluctuation modes. The arrows represent in-phase dipole fluctuations of
the electron density on the individual atoms. For benzene dimer (e), the lowest-energy dipole–dipole corresponds to the in-plane fluctuations on both
monomers in the parallel-displaced conformation (top), but not in the T-shaped conformation (bottom). NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14052 NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14052 excitations to the unoccupied p-like orbitals. As these orbitals
have a larger spatial extent, this correlation process leads to shift
of the electrons from the atoms to the outer regions. In the
density functional theory (DFT) picture, the correlation is
induced by a change in the local exchange-correlation potential,
which becomes decaying slightly slower with the distance from
the nuclei, allowing the electrons to shift outwards. Figure 4a
shows such a shift in a benzene dimer as obtained from a DFT
calculation with the self-consistent Tkatchenko–Scheffler (TS)
functional44. Finally, consider the MBD picture, where the
correlation arises via coupling of the charge fluctuations within
the individual fragments into collective fluctuations that may
span the whole complex. These are obtained directly as single-
particle solutions fi of the underlying many-body Hamiltonian
^HMBD (see equation (5) in Methods), The total displaced charge (integral over charge-accumulating
regions) amounts to 0.0101 and 0.0097 electrons, as obtained
from the TS density functional and the MBD wavefunctions,
respectively. Given the much different basis of these two
approaches and the absence of any explicit parametrization of
MBD with respect to spatial representation of the electron
density, we find the close match between the two approaches
reassuring of the solid physical foundation of the underlying
charge fluctuations in MBD. g
The orbital-based approaches of quantum chemistry can be
linked to MBD by considering the solution of the MBD
Hamiltonian as would be provided by standard quantum
chemistry methods. In such an approach, the ground s-states
of the harmonic oscillators in MBD would be correlated via
virtual excitations to their first excited states, which have the
symmetry of p-orbitals. As atomic p-orbitals form the basis of
the molecular p-orbitals, this may explain why MBD is
particularly fitting for description of p p systems. (We note
that this analogy is only partial though—the final states in the
transitions are corresponding, but the initial states have a
different symmetry.) Figure 4c illustrates that in a large
supramolecular
p p
complex,
the
charge
polarization
induced by vdW interactions fills the whole intermolecular
region. In this case, the total displaced charge amounts to 0.11
electrons, an order of magnitude increase from the benzene
dimer, which corresponds to the same increase in the vdW
interaction energy. ARTICLE ARTICLE NATURE COMMUNICATIONS | 8:14052 | DOI: 10.1038/ncomms14052 | www.nature.com/naturecommunications ARTICLE D3 is the DFT-D3 approach59 with the optional three-body
correction on top of the PW6B95 density functional; the values were taken
from refs 23,24. Numerical values are presented in Supplementary Table 1. C70 Figure 3 | Heat map of the xy component of the interfragment non-local
polarizability axy
AB in [10]CPP–C70. Red and blue represent positive and
negative values, respectively. Rows and columns correspond to carbon
atoms of [10]CPP and C70, respectively, with four select carbon atoms
color-coded to the marked atoms in the structure in the inset. 3 3 ARTICLE This can be related
to the standard London picture of pairwise dispersion, where
fluctuating dipoles on individual atoms are being correlated. Figure 4f demonstrates that this view can be generalized to the
case of large complexes not via sum over the atom pairs,
but rather by considering collective fluctuating dipoles and
higher multipoles of the whole molecules, akin to wavelike dipole
fluctuations or plasmons in nanomaterials45. In this framework,
the destabilization by the many-body correlation effects with
respect to the second-order (pairwise) approximation can be
understood by considering the degrees of freedom of the
correlation. In the pairwise picture, the oscillations within each
atom pair are correlated independently, essentially resulting in an Trends across structural motifs. We have demonstrated how
collective charge fluctuations are responsible for binding in the
three studied supramolecular complexes, and that quantitative
models,
which
take
this
into
account,
provide
accurate
predictions of the binding energies. The last part of our work
deals with two questions: (i) are these findings transferable to
other conjugated systems? (ii) Are they characteristic of con-
jugated systems? The magnitude of the binding and especially the
contribution of the higher-order many-body correlation effects is
a combined result of several factors including the degree of
symmetry of a system, its topology, compactness, and mutual
orientation of the host and the guest. To investigate whether our
model can account for these different factors, we have calculated
the binding energies of a set of conformations of the C70 fullerene
hosted in [N]cycloparaphenyleneacetylene rings, with N ranging
from 6 to 8 (see Fig. 5). The geometries were selected to cover a
spectrum of interaction motifs, ranging from tightly stacked to
open structures, and containing both symmetric as well as
distorted cases. The case of N ¼ 6 was previously studied by Yuan
et al.46 but their focus was mostly on equilibrium structures. The
MBD binding energies are compared with the results of
DQMC as well as the TS method36, which is a reliable model
for vdW interactions in smaller systems and simple solids, but
neglects the higher-order correlation effects. As in the case of the
CPP-based complexes, MBD provides binding energies in
excellent agreement with DQMC, with the largest difference of
1.7 kcal mol 1 (5%) and the mean absolute difference of
0.9 kcal mol 1. ARTICLE ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14052 ‘overcorrelation’. In contrast, the collective fluctuation model
correlates all atomic dipole oscillations at once, allowing only for
a moderate amount of correlation. As the differences in the
degrees of freedom grow with the system size, the many-body
effects are more pronounced in the supramolecular complexes,
where they reduce the binding energies by as much as 16%,
or 6 kcal mol 1, in the case of the buckyball catcher. Formation of collective charge fluctuations. The formation of
collective fluctuations in the complex is associated with a
broadening of the oscillation frequencies with respect to those in
the isolated fragments, which can be directly observed on the
energy densities of the oscillation states. Figure 4d shows that
although this broadening has a more complex shape in the
complex C1 than in the benzene dimer, the overall character is
similar in both cases, reflecting the fact that the mechanisms of
the binding are in fact the same. We note that in contrast to
the
splitting
of
atomic
orbital
energies
on
formation
of
molecular orbitals, which is symmetric and facilitates the bonding
via partial occupancy of the orbital space, here all states are singly
occupied (the fluctuations are bosonic) and the binding arises as a
result of an asymmetry in the splitting. Further insight can be
obtained by analysing individual oscillation modes. Many of the
collective modes have a distinct character that corresponds to
global
dipole,
quadrupole
or
higher-multipole
oscillations
extending over the whole complex. In general, the energy
ordering of the modes coincides with an increasing angular
moment, with collective dipole-like fluctuations having typically
the lowest energy. However, this is not always the case as can be
observed on the two nearly degenerate conformations of the
benzene dimer. Figure 4e shows that in the parallel-displaced
conformer
(top),
the
lowest-energy
collective
fluctuation
corresponds to two aligned in-plane dipole fluctuations in the
monomers, whereas in the T-shaped conformer (bottom) the
corresponding collective mode comprises one in-plane and
one out-of-plane monomer fluctuation, the latter having a sub-
stantially lower polarizability. These observations are manifested
in the vdW interaction energies as well, which are reduced
by the higher-order many-body effects by 8% and 3% in the
parallel-displaced and T-shaped conformation, respectively. In
the supramolecular complex C1 (Fig. 4f), the fluctuations most
contributing to the binding correspond to dipole and quadrupole
oscillations spanning the whole complex. ARTICLE Furthermore, there is no clear pattern in the
remaining small differences, suggesting that MBD treats the
various types of geometries included in the sample on an equal
footing. In contrast, the pairwise TS method in general overbinds,
with differences ranging from 2 to 13 kcal/mol. Part of this
overbinding can be attributed to the lack of short-range screening
in TS; however, most of the specific differences on individual
systems stem from the ‘overcorrelation’ introduced above. We
observe that these differences are largest in the tightly stacked
structures, resulting in the largest overbinding and a seeming
stability of these systems. With MBD and DQMC, on the other DQMC (reference)
MBD
TS
1 – 8 Rings
2 – 8 Rings
3 – 8 Rings
4 – 8 Rings
5 – 6 Rings
6 – 6 Rings
7 – 7 Rings
–20
–40
Binding energy (kcal mol–1)
Δ = 1.7
Δ = 6.3
Δ = 6.2
Δ = 8.9
Δ = 13.2
Δ = 12.5
Δ = 9.6
Figure 5 | Interaction energies of C70 in [N]cycloparaphenyleneacetylene (6rNr8). The sample was selected to cover a broad spectrum of
geometrical motifs and hence the individual geometries are not necessarily energy minima. However, all geometries are stationary points of the potential
energy surface. The energies are calculated with respect to relaxed fragments (see Supplementary Data 1). DQMC is the diffusion quantum Monte-Carlo
method (not given for system 7), whereas MBD and TS are the MBD model and TS method calculated on top of the PBE functional, respectively. D denotes
the energy difference between MBD and TS in kcal mol 1. Numerical values are presented in Supplementary Table 2. DQMC (reference)
MBD
TS
1 – 8 Rings
2 – 8 Rings
3 – 8 Rings
4 – 8 Rings
5 – 6 Rings
6 – 6 Rings
7 – 7 Rings
–20
–40
Binding energy (kcal mol–1)
Δ = 1.7
Δ = 6.3
Δ = 6.2
Δ = 8.9
Δ = 13.2
Δ = 12.5
Δ = 9.6 Figure 5 | Interaction energies of C70 in [N]cycloparaphenyleneacetylene (6rNr8). The sample was selected to cover a broad spectrum of
geometrical motifs and hence the individual geometries are not necessarily energy minima. However, all geometries are stationary points of the potential
energy surface. The energies are calculated with respect to relaxed fragments (see Supplementary Data 1). NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14052 For complex C1 (f), the dipole (left) and
quadrupole (right) in-plane oscillations are amongst the ones most contributing to the total binding energy. c a b d c Fragments
Complex
Difference (×20)
0.3
0.4
0.5
0.6
–5,000
0
5,000
Density of modes i (a.u.)
Frequency i (a.u.) Density of modes i (a.u.) e f Figure 4 | Analysis of correlated charge fluctuations in supramolecular complexes and benzene dimer. (a–c) Electron density differences (charge
polarization) between a complex and its isolated fragments induced by vdW interactions. Yellow and blue colour denote accumulation and depletion of
charge density, respectively. Magnitude of the density difference is mapped to saturation of the colour with 50% saturation corresponding to the density of
2 10 5 and 4 10 5 a.u. for the benzene dimer and the complex C1, respectively. Charge polarizations were obtained either from a DFTcalculation with
a self-consistent TS functional in the parallel-displaced benzene dimer (see Fig. 1) (a) or directly from the correlated MBD wavefunctions (see main text for
details) for the benzene dimer (b) and the complex C1 (c). In the latter case, the relevant charge densities are calculated as expectation values of the charge
density operator, cMBD ^r
j jcMBD
h
i. (d) Energy densities of the oscillation states for parallel-displaced benzene dimer (top) and complex C1 (bottom). Gaussian smoothing with half-width of 0.06 eV was applied. (e,f) Select collective fluctuation modes. The arrows represent in-phase dipole fluctuations of
the electron density on the individual atoms. For benzene dimer (e), the lowest-energy dipole–dipole corresponds to the in-plane fluctuations on both
monomers in the parallel-displaced conformation (top), but not in the T-shaped conformation (bottom). For complex C1 (f), the dipole (left) and
quadrupole (right) in-plane oscillations are amongst the ones most contributing to the total binding energy. 4 4 NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14052 NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14052 accuracy with respect to high-level reference data, (ii) obtain both
quantitatively and spatially correct charge polarization due to
long-range electron correlation with respect to density-functional
calculations, and (iii) analyse and visualize the individual
fluctuations and their contributions to the total binding energy. Two different models (TS and D3), which do not account for the
collective charge oscillations, are not capable of predicting binding
energies of a comparable accuracy. Our analysis also suggests that
such fluctuations are especially important in conjugated systems in
comparison with complexes with other types of bonding. One of
the main characteristics of aromatic systems is their relatively
narrow HOMO-LUMO gap, making them an interesting point on
the zero-gap to large-gap spectrum17,48. An essential property of
the dipole oscillators used in our model is that they are not point
dipoles, but rather have a natural width. This is crucial for
predicting accurate molecular polarizabilities, but also seems to
effectively provide the amount of delocalization in the molecular
response
that
is
required
to
describe
long-range
electron
correlation in finite-gap systems such as those studied in this
work. The fully delocalized electrons that can be found in metallic
systems and that can lead to type-C non-additivity as classified by
Dobson49 are not explicitly accounted for by our model and this
challenge is currently the topic of our work. hand, these structures are much closer in binding energy to the
more extended conformations. hand, these structures are much closer in binding energy to the
more extended conformations. accuracy with respect to high-level reference data, (ii) obtain both
quantitatively and spatially correct charge polarization due to
long-range electron correlation with respect to density-functional
calculations, and (iii) analyse and visualize the individual
fluctuations and their contributions to the total binding energy. Analysis of individual fluctuation modes. vdW interactions in
all materials are caused by correlation of charge fluctuations and
we have shown above that these fluctuations are non-local and
collective in the case of conjugated p p systems. To investigate
whether such characteristics are specific to these systems, we have
analysed the fluctuation mode that contributes most to the total
binding energy in seven select complexes from the data sets
S12L23 and S8 (ref. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14052 24; see Supplementary Figs 3 and 4), covering
all distinct types of bonding therein, which range from p p
stacking (two ‘tweezer’ complexes, one ‘pincer’ complex and the
[10]CPP complex studied above) to hydrogen bonding, to
electrostatic interactions (a pseudorotaxane complex, an amide
macrocycle complex and a cucurbituril complex). The fluctuation
modes of a complex can be expanded in terms of the fluctuation
modes of the individual monomers with equation (10) (see
Supplementary Methods for details). A simple yet revealing
measure of the number of components of such an expansion is
the inverse of the largest expansion coefficient (as an example,
consider the expansion 1¼1
3 þ 1
3 þ 1
3, where this measure would
be 3). In the four conjugated supramolecular complexes, we find
this measure to be 2.3, 2.8, 3.0 and 4.4, whereas in the three
complexes with other types of bonding, we find 1.1, 1.1 and 1.7,
demonstrating
that
the
modes
in
the
former
group
are
significantly more collective. As an illustrative example, the
most contributing fluctuation mode in the p p stacked ‘tweezer’
complex in Fig. 6a corresponds to a collective dipole-like
fluctuation, whereas it is mostly localized and unordered on the
guest molecule in the electrostatically bound cucurbituril complex
in Fig. 6b. In the latter case, the mode is strongly binding because
of its large polarizability and unspecific correlation with the
modes of the host molecule. Although performed on a limited
number of systems, these preliminary results already suggest that
the collective charge fluctuations indeed are characteristic of the
conjugated complexes. Two different models (TS and D3), which do not account for the
collective charge oscillations, are not capable of predicting binding
energies of a comparable accuracy. Our analysis also suggests that
such fluctuations are especially important in conjugated systems in
comparison with complexes with other types of bonding. One of
the main characteristics of aromatic systems is their relatively
narrow HOMO-LUMO gap, making them an interesting point on
the zero-gap to large-gap spectrum17,48. An essential property of
the dipole oscillators used in our model is that they are not point
dipoles, but rather have a natural width. This is crucial for
predicting accurate molecular polarizabilities, but also seems to
effectively provide the amount of delocalization in the molecular
response
that
is
required
to
describe
long-range
electron
correlation in finite-gap systems such as those studied in this
work. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14052 The fully delocalized electrons that can be found in metallic
systems and that can lead to type-C non-additivity as classified by
Dobson49 are not explicitly accounted for by our model and this
challenge is currently the topic of our work. Discussion
I
i In previous works, collective charge fluctuations (molecular
plasmons47) have been discussed predominantly within the
context of solids and low-dimensional extended systems15–17. Our findings show that they serve as a natural description of
long-range electron correlation in molecular systems, not only at
the asymptotic limit, but at equilibrium distances as well, and
furthermore that the corresponding models can be made fully
quantitative. We have demonstrated that using a relatively simple
Hamiltonian model for these fluctuations, one can: (i) calculate
binding energies of supramolecular complexes within chemical where nA¼
ffiffiffiffiffiffiffi
mA
p
rA RA
ð
Þ is the mass-weighted displacement of the
pseudoelectron on atom A from its equilibrium position RA, and Tlr is the
long-range part of the dipole potential. The solution is obtained by direct
diagonalization which is possible due to the biquadratic form of the Hamiltonian
and leads to the set of coupled fluctuation frequencies oi and harmonic modes
ni¼ P CiAnA. The MBD energy is then given by a plasmon-pole formula where nA¼
ffiffiffiffiffiffiffi
mA
p
rA RA
ð
Þ is the mass-weighted displacement of the
pseudoelectron on atom A from its equilibrium position RA, and Tlr is the
long-range part of the dipole potential. The solution is obtained by direct
diagonalization which is possible due to the biquadratic form of the Hamiltonian
and leads to the set of coupled fluctuation frequencies oi and harmonic modes
ni¼ P CiAnA. The MBD energy is then given by a plasmon-pole formula EMBD ¼ 1
2
X
i
oi 3
2
X
A
oA
ð6Þ ð6Þ An alternative and fully equivalent formulation, which allows for expansion of the
energy in orders of the coupling, starting with the second order, is provided by the
random phase approximation, An alternative and fully equivalent formulation, which allows for expansion of the
energy in orders of the coupling, starting with the second order, is provided by the
random phase approximation, EMBD¼
X
1
n¼2
1
ð
Þn
n
Z 1
0
du
2p Tr ~aT
ð
Þn
½
ð7Þ a
b
Figure 6 | The most binding fluctuation mode in two supramolecular
complexes. Only atoms where the magnitude of the dipole oscillation is
larger than 5% of the largest oscillation are shown. (a) Conjugated
‘tweezer’ complex. (b) Electrostatically bound cucurbituril complex. Methods
M
b d Many-body dispersion. MBD18,19,50 models the electrons of each atom as a
harmonic oscillator with the static polarizability a0 and oscillation frequency
o ¼ 4C6=3a2
0 calculated from the C6 dispersion coefficient. These parameters are
obtained from the electron density by scaling the corresponding free-atom values
with a ratio of the Hirshfeld volumes of the atoms in the molecule and free atoms, a0 ¼ afree
0
V
Vfree
ð3Þ ð3Þ The scaled atomic polarizabilities are then screened via the Dyson screening
equation using the short-range part of the dipole potential. ð4Þ ~a ¼ a aTsr~a ~a ¼ a aTsr~a
ð4Þ The resulting fully non-local polarizability ~a (see Supplementary Fig. 1) is then
contracted back to individual atoms. The polarizabilities obtained in this way are
then used as an input into the MBD Hamiltonian, HMBD¼ 1
2
X
A
=2
xA þ 1
2
X
A
o2
Ax2
A þ 1
2
X
AB
oAoB
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
~a0;A~a0;B
p
nATlr
ABnB
ð5Þ ARTICLE ARTICLE ARTICLE DQMC is the diffusion quantum Monte-Carlo
method (not given for system 7), whereas MBD and TS are the MBD model and TS method calculated on top of the PBE functional, respectively. D denotes
the energy difference between MBD and TS in kcal mol 1. Numerical values are presented in Supplementary Table 2. Figure 5 | Interaction energies of C70 in [N]cycloparaphenyleneacetylene (6rNr8). The sample was selected to cover a broad spectrum of
geometrical motifs and hence the individual geometries are not necessarily energy minima. However, all geometries are stationary points of the potential
energy surface. The energies are calculated with respect to relaxed fragments (see Supplementary Data 1). DQMC is the diffusion quantum Monte-Carlo
method (not given for system 7), whereas MBD and TS are the MBD model and TS method calculated on top of the PBE functional, respectively. D denotes
the energy difference between MBD and TS in kcal mol 1. Numerical values are presented in Supplementary Table 2. NATURE COMMUNICATIONS | 8:14052 | DOI: 10.1038/ncomms14052 | www.nature.com/naturecommunications 5 References 31. Foulkes, W. M. C., Mitas, L., Needs, R. J. & Rajagopal, G. Quantum Monte
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interactions with subchemical accuracy. J. Chem. Theory Comput. 9, 4287–4292
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self-assembly. Sci. Technol. Adv. Mater. 9, 014109 (2008). 4. Ariga, K. et al. Challenges and breakthroughs in recent research on
lf
bl
S
T h
l Ad
(
) 34. Ambrosetti, A., Alfe`, D., DiStasio, R. A. Jr & Tkatchenko, A. Hard numbers for
large molecules: toward exact energetics for supramolecular systems. J. Phys. Chem. Lett. 5, 849–855 (2014). self-assembly. Sci. Technol. Adv. Mater. 9, 014109 (2008). 5. Geim, A. K. & Grigorieva, I. V. Van der Waals heterostructures. Nature 499,
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g
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41. Gobre, V. V. & Tkatchenko, A. Scaling laws for van der Waals interactions in
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of p–p interactions in benzene dimers. J. Phys. Chem. A 110, 10656–10668
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43. Ehrlich, S., Moellmann, J. & Grimme, S. Dispersion-corrected density functional
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self-consistent interatomic van der Waals density functional. Phys. Rev. Let
114, 176802 (2015). 14. Martinez, C. R. & Iverson, B. L. Rethinking the term ‘pi-stacking’. Chem. Sci. 3,
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analytic benchmarks for van der Waals energy functionals. Phys. Rev. Lett. 96,
073201 (2006). 45. Ambrosetti, A., Ferri, N., DiStasio, J., Robert, A. & Tkatchenko, A. Wavelike
charge density fluctuations and van der Waals interactions at the nanoscale. Science 351, 1171–1176 (2016). 16. CSJ ¼ ^D" ^D#eJ ð11Þ 21. Jasti, R., Bhattacharjee, J., Neaton, J. B. & Bertozzi, C. R. Synthesis,
characterization, and theory of [9]-, [12]-, and [18]cycloparaphenylene:
nanohoop structures. J. Am. Chem. Soc. 130, 17646–17647 (2008). characterization, and theory of [9]-, [12]-, and [18]cycloparaphenylene: carbon
nanohoop structures. J. Am. Chem. Soc. 130, 17646–17647 (2008). where ^D", ^D# are Slater determinants constructed from single-particle spin
orbitals representing the up and down electron-spin projections, respectively, and
eJ is the so-called Jastrow factor, which is an exponential of a sum of explicitly
correlated one- (electron–nucleus), two- (electron–electron) and three-body
(electron–electron–nucleus) terms. Single-particle orbitals were obtained from
local density approximation (LDA) plane-wave calculations performed with the
PWSCF program55 and expanded in terms of B-splines56. DQMC binding energies
have been computed with respect to the fragments separated by at least 10 Å, with
the residual binding energy of less than 0.6 kcal mol 1 (estimated with MBD). This
procedure avoids the size-consistency problems of DQMC (Zen, A. et al., in
preparation). 22. Grimme, S. Supramolecular binding thermodynamics by dispersion-corrected
density functional theory. Chem. Eur. J. 18, 9955–9964 (2012). 23. Risthaus, T. & Grimme, S. Benchmarking of London dispersion-accounting
density functional theory methods on very large molecular complexes. J. Chem. Theory Comput. 9, 1580–1591 (2013). y
p
24. Antony, J., Sure, R. & Grimme, S. Using dispersion-corrected density functional
theory to understand supramolecular binding thermodynamics. Chem. Commun. 51, 1764–1774 (2015). 25. Okada, S., Saito, S. & Oshiyama, A. Energetics and electronic structures of
encapsulated C60 in a carbon nanotube. Phys. Rev. Lett. 86, 3835–3838 (2001). p
y
,
(
)
26. Monthioux, M. Filling single-wall carbon nanotubes. Carbon 40, 1809–1823 (2002). 26. Monthioux, M. Filling single-wall carbon nanotubes. Carbon 40, 1809–1823 (2002). 26. Monthioux, M. Filling single-wall carbon nanotubes. Carbon 40, ux, M. Filling single-wall carbon nanotubes. Carbon 40, 1809–1823 (2 27. Kawase, T. & Kurata, H. Ball-, bowl-, and belt-shaped conjugated systems and
their complexing abilities: exploration of the concave–convex p–p interaction. Chem. Rev. 106, 5250–5273 (2006). DFT calculations. All DFT calculations were performed with the FHI-aims
program57 using the ‘tight’ settings for basis sets and real-space grids. FHI-aims is
an all-electron code with numerical basis sets. The Kohn–Sham self-consistent
cycle was converged to 10 6 eV in energy and 10 5 electrons in density. 28. Iwamoto, T. et al. Size- and orientation-selective encapsulation of C70 by
cycloparaphenylenes. Chem. Eur. J 19, 14061–14068 (2013). 29. CSJ ¼ ^D" ^D#eJ Mu¨ck-Lichtenfeld, C., Grimme, S., Kobryn, L. & Sygula, A. Inclusion complexes
of buckycatcher with C60 and C70. Phys. Chem. Chem. Phys. 12, 7091–7097
(2010). Data availability. The authors declare that the data necessary for reproducing the
results of this study are available within the article and its Supplementary
Information files. Data availability. The authors declare that the data necessary for reproducing the
results of this study are available within the article and its Supplementary
Information files. 30. Yang, L., Adam, C., Nichol, G. S. & Cockroft, S. L. How much do van der Waals
dispersion forces contribute to molecular recognition in solution? Nat. Chem. 5,
1006–1010 (2013). ARTICLE ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14052 Diffusion quantum Monte Carlo. All DQMC energies presented in this work
were calculated using the CASINO programme52, employing relativistic
pseudopotentials53 with the locality approximation54 and the Slater–Jastrow trial
wave functions Diffusion quantum Monte Carlo. All DQMC energies presented in this work
were calculated using the CASINO programme52, employing relativistic 19. Ambrosetti, A., Reilly, A. M., DiStasio, R. A. Jr & Tkatchenko, A. Long-range correlation energy calculated from coupled atomic response
functions. J. Chem. Phys. 140, 18A508 (2014). tions. J. Chem. Phys. 140, 18A508 (2014). 20. Sygula, A., Fronczek, F. R., Sygula, R., Rabideau, P. W. & Olmstead, M. M. A double concave hydrocarbon buckycatcher. J. Am. Chem. Soc. 129,
3842–3843 (2007). CSJ ¼ ^D" ^D#eJ Discussion
I
i a ð7Þ b b a The MBD charge densities are obtained from the correlated wavefunctions The MBD charge densities are obtained from the correlated wavefunctions cMBD
j
i
Y
i
e 1
2oix2
i
ð8Þ ð8Þ as an expectation value of the charge density operator r rð Þ ¼ cMBD
h
j
X
A
qAd r RA
ð
Þ cMBD
j
i
ð9Þ ð9Þ where the charges are set to 1 and the effective masses of the pseudoelectrons then
follow from a0 ¼ q/mo2. The collective fluctuation modes xi of the complex can be
expressed in terms of the modes x1;i, x2;i of its fragments via n ¼ C
C 1
1
n1
C 1
2
n2
ð10Þ Figure 6 | The most binding fluctuation mode in two supramolecular
complexes. Only atoms where the magnitude of the dipole oscillation is
larger than 5% of the largest oscillation are shown. (a) Conjugated
‘tweezer’ complex. (b) Electrostatically bound cucurbituril complex. ð10Þ All MBD calculations were performed with a standalone program available
in ref. 51. NATURE COMMUNICATIONS | 8:14052 | DOI: 10.1038/ncomms14052 | www.nature.com/naturecommunications 6 NATURE COMMUNICATIONS | 8:14052 | DOI: 10.1038/ncomms14052 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14052 NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14052 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/ Author contributions 49. Dobson, J. F. Beyond pairwise additivity in London dispersion interactions. Int. J. Quantum Chem. 114, 1157–1161 (2014). J.H. and A.T. conceived the research. J.H. carried out the MBD calculations and analysis. D.A. carried out the DQMC calculations. J.H. wrote the paper with contributions from all
authors. 50. DiStasio, R. A. Jr, Gobre, V. V. & Tkatchenko, A. Many-body van der Waals
interactions in molecules and condensed matter. J. Phys. Condens. Matter 26,
213202 (2014). 51. Hermann, J. Source code of program ‘MBD’. doi:10.5281/zenodo.47528. Additional information 52. Needs, R. J., Towler, M. D., Drummond, N. D. & Ros, P. L. Continuum
variational and diffusion quantum Monte Carlo calculations. J. Phys. Condens
Matter 22, 023201 (2010). Supplementary Information accompanies this paper at http://www.nature.com/
naturecommunications 53. Trail, J. R. & Needs, R. J. Norm-conserving Hartree–Fock pseudopotentials and
their asymptotic behavior. J. Chem. Phys. 122, 014112 (2005). Competing financial interests: The authors declare no competing financial
interests. References Bordag, M., Geyer, B., Klimchitskaya, G. L. & Mostepanenko, V. M. Lifshitz-type formulas for graphene and single-wall carbon nanotubes:
Van der Waals and Casimir interactions. Phys. Rev. B 74, 205431 (2006 46. Yuan, K. et al. Theoretical insights into the host–guest interactions between
[6]cycloparaphenyleneacetylene and its anthracene-containing derivative and
fullerene C70. J. Phys. Org. Chem. 27, 772–782 (2014). 17. Misquitta, A. J., Spencer, J., Stone, A. J. & Alavi, A. Dispersion interactions
between semiconducting wires. Phys. Rev. B 82, 075312 (2010). 47. Lauchner, A. et al. Molecular plasmonics. Nano Lett. 15, 6208–6214 (2015). 48. Misquitta, A. J., Maezono, R., Drummond, N. D., Stone, A. J. & Needs, R. J. Anomalous nonadditive dispersion interactions in systems of three
one-dimensional wires. Phys. Rev. B 89, 045140 (2014). 18. Tkatchenko, A., DiStasio, R. A. Jr, Car, R. & Scheffler, M. Accurate and efficient
method for many-body van der Waals interactions. Phys. Rev. Lett. 108, 236402
(2012). 7 NATURE COMMUNICATIONS | 8:14052 | DOI: 10.1038/ncomms14052 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | 8:14052 | DOI: 10.1038/ncomms14052 | www.nature.com/naturecommunications 7 Competing financial interests: The authors declare no competing financial
interests. y
p
y
54. Mita´sˇ, L., Shirley, E. L. & Ceperley, D. M. Nonlocal pseudopotentials and
diffusion Monte Carlo. J. Chem. Phys. 95, 3467–3475 (1991). r The Author(s) 2017 NATURE COMMUNICATIONS | 8:14052 | DOI: 10.1038/ncomms14052 | www.nature.com/naturecommunications Reprints and permission information is available online at http://npg.nature.com/
reprintsandpermissions/ Reprints and permission information is available online at http://npg.nature.com/
reprintsandpermissions/ 55. Baroni, S., Dal Corso, A., de Gironcoli, S. & Giannozzi, P. Program ‘pwscf’. URL https://www.quantum-espresso.org/. 56. Alfe`, D. & Gillan, M. J. Efficient localized basis set for quantum Monte Carlo
calculations on condensed matter. Phys. Rev. B 70, 161101 (2004). How to cite this article: Hermann, J. et al. Nanoscale p–p stacked molecules are bound
by collective charge fluctuations. Nat. Commun. 8, 14052 doi: 10.1038/ncomms14052
(2017). y
57. Blum, V. et al. Ab initio molecular simulations with numeric atom-centered
orbitals. Comput. Phys. Commun. 180, 2175–2196 (2009). Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. 58. Perdew, J. P., Burke, K. & Ernzerhof, M. Generalized gradient approximation
made simple. Phys. Rev. Lett. 77, 3865–3868 (1996). 58. Perdew, J. P., Burke, K. & Ernzerhof, M. Generalized gradient approximation
made simple. Phys. Rev. Lett. 77, 3865–3868 (1996). 59. Grimme, S., Antony, J., Ehrlich, S. & Krieg, H. A consistent and accurate ab
initio parametrization of density functional dispersion correction (DFT-D) for
the 94 elements H-Pu. J. Chem. Phys. 132, 154104 (2010). Acknowledgements We acknowledge use of the resources of the Oak Ridge Leadership Computing Facility at
the Oak Ridge National Laboratory, which is supported by the Office of Science of the
US Department of Energy (DOE) under Contract Number DEAC05-00OR22725. A.T. acknowledges financial support by the European Research Council (ERC-StG
VDW-CMAT) and the DFG SPP-1807 Research Network. r The Author(s) 2017 NATURE COMMUNICATIONS | 8:14052 | DOI: 10.1038/ncomms14052 | www.nature.com/naturecommunications 8
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https://openalex.org/W4327632684
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https://hal.science/hal-01813014/document
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English
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Non destructive testing using non linear vibroacoustic
|
HAL (Le Centre pour la Communication Scientifique Directe)
| 2,005
|
public-domain
| 2,587
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To cite this version: Bernard Rousselet, Gérard Vanderborck. Non destructive testing using non linear vibroacoustic. 7e
colloque national en calcul des structures, CSMA, May 2005, Giens, France. hal-01813014 HAL Id: hal-01813014
https://hal.science/hal-01813014v1
Submitted on 12 Jun 2018 L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d’enseignement et de
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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. Public Domain Very recent experiments – have been performed on a real bridge by G. Vanderborck with four prestressed cables:
two undamaged cables, a damaged cable and a safe one but damaged at the anchor; – See Vanderborck-Lagier(2004) [VAN ] for a presentation of the results of the experiment
with a new post processing graphic presentation of experimental results. Difficulties of the experiments: Difficulties of the experiments: – non linearities of the shakers (including piezoelectric actuators) – non linearities of the shakers (including piezoelectric actuators) – Natural non linearities: supports, links of complex multi structures as air planes, bridges
etc etc Non destructive testing
using non linear vibroacoustic B. Rousselet*, — G. Vanderborck**
* U.N.S.A. Laboratoire J.A. Dieudonné
U.M.R. C.N.R.S. 6621, Parc Valrose, F 06108 Nice, Cédex 2, email : br@math.unice.fr
** Thales Underwater Systems, Département acoustique
06903 Sophia-Antipolis CEDEX
and Laboratoire J.A. Dieudonné
U.M.R. C.N.R.S. 6621 B. Rousselet*, — G. Vanderborck**
* U.N.S.A. Laboratoire J.A. Dieudonné
U.M.R. C.N.R.S. 6621, Parc Valrose, F 06108 Nice, Cédex 2, email : br@math.unice.fr
** Thales Underwater Systems, Département acoustique
06903 Sophia-Antipolis CEDEX
and Laboratoire J.A. Dieudonné
U.M.R. C.N.R.S. 6621 * U.N.S.A. Laboratoire J.A. Dieudonné
U.M.R. C.N.R.S. 6621, Parc Valrose, F 06108 Nice, Cédex 2, email : br@math.unice.fr
** Thales Underwater Systems, Département acoustique
06903 S
hi
A i
li CEDEX ABSTRACT. Several recent experimental studies show that it is possible to detect defects in a
structure by considering its vibro-acoustic response to an external actuation. On this topic
there is a vast literature in applied physics. We recall some papers related to the use of the
frequency response for non destructive testing; in particular generation of higher harmonics,
cross-modulation of a high frequency by a low frequency: We intend to present simple spring
mass models, simple bar and beam models with damage and use asymptotic expansions and
numerical methods to try to get results which show some similarity with the experiments of
[P.D 03]. Asymptotic expansions have been used for at least a century and for example has been
used recently for numerical approximation of bifurcation of structures in PotierFerry-Cochelin
and coworkers (1993) [E.H 93]. The key idea is to look at the solution in the frequency
domain for the experiments and consequently for the numerics. RÉSUMÉ KEYWORDS: Non destructive testing, non linear vibroacoustic KEYWORDS: Non destructive testing, non linear vibroacoustic MOTS-CLÉS : Essais non destructifs, vibroacoustique non linà c
aire Non destructive testing
1 1. Some previous papers – In Ekimov-Didenkulov-Kasakov (1999), [A.E 99], – In Zaitsev-Sas (1999), [V.Z 99], – Other results may be found in Sedunov-Tsionsky-Donskoy(2002) [D.D 02],Sutin-
Donskoy (1998), [A.S 98], Moussatov-Castagnede-Gusev(2002), [GUS 02] ... – Other results may be found in Sedunov-Tsionsky-Donskoy(2002) [D.D 02],Sutin-
Donskoy (1998), [A.S 98], Moussatov-Castagnede-Gusev(2002), [GUS 02] ... – GDR 2501 (Etude de la propagation ultrasonore en milieux inhomogènes en vue du con-
trole non destructif) – In Vanderborck-Lagier-Groby (2003) [P.D 03], "a vibro-acoustic method, based on fre-
quency modulation, is developed in order to detect defects on aluminum and concrete beams. – In Vanderborck-Lagier-Groby (2003) [P.D 03], "a vibro-acoustic method, based on fre-
quency modulation, is developed in order to detect defects on aluminum and concrete beams. – Question: algorithm and software for detecting the secondary picks? – then find (by optimization) datas such that the secondary picks are important: criteria for
damage. General tendency: – The pick of
W
is much larger than the pick in
]
X
which are the most natural picks
in the experiments;
^ it is delicate to find datas such that the secondary picks at
]
X
3. Transverse vibrations: vibrating masses on streched cables in large
displacement Work performed with Theissen (doctoral student of U. Muenster); Erasmus students N. Goris and I. Altrogge worked on this topic during their stay in UNSA (2004-2005). We consider
n masses attached to horizontal springs (or cables) which are in tension
_
, at rest ; the tension
is positive when the cable is in traction which is assumed; at rest the mass
a` is submited to the
force
_
the masses are moving (vertically) transversely to the springs; we denote by uper case
letters quantities in the rest position and lower case in the current configuration. Other terms The term
is zero but the third term satisfies:
L
*
*
(2.5)
set
9
D
G
2HJ
H
)M
ONQP
>2R
'
so that:
*
*
(2.5)
G
2J
)
OQ
2
'
*
*
(2.5)
to simplify, we assume
(+*
& and set
9
D
EGF
>2HJI
? H
@K
,)M
ONQP
?@
>2R
?'@
so that:
S
$
"$) (2.5) to simplify, we assume
(
*
& and set
9
D
E
F
>
H
I
? H
@
K
,
M
N
P
? @
>
R
? @
so that:
S
$
"$)
*
M
! #
9
! #
#
(2.6)
A-TVU
M
W
!)#
W
TYX
M
AJTVU
M
9
#)
!$#
(2.7)
W
TJU
M
9
)
X
#Z!$#
W
TVU
M
9
)
X
[#Z!$#
(2.8)
W
TJU
M
9
)
X
#Z!$#
W
TVU
M
9
S )
X
#Z!)#
(2.9)
W
TBX
M
9
AJTYXY9
#) 7!$#
A-TVU'9
WY "!)#
(2.10)
he expansion, we get terms of pulsation
U
,
\
etc If we go on in the expansion, we get terms of pulsation
U
,
\
etc General tendency: Here we assume that the masses can move only vertically. Here we assume that the masses can move only vertically. 2. Simplest mechanical example in which we can exhibit intermodulations. We consider a 1 d.o.f example of a spring mass
system with a non linear spring.
"$
%
! #
with
(2.1)
with initial conditions
&'#
)(+*-,/. 0
)12*
(2.2)
56 (2.1) (2.2) We are going to solve this equation symbolically with an asymptotic expansion with respect
to
:
*
4
4
4 ; then numerically... The linear case
The linear case The linear case The first term is solution of:
*
*
! #
with
&
#
(
*
,
.
1
* which gives:
(2.3)
with
9
:
#
,
*
(
*$;6<
'
!$#
18*
9
#)
!$#
9
"!$#
(2.4)
=
(2.3)
with
9
#
,
*
(
*
;
<
! #
1
*
9
#
! #
9
! #
(2.4)
=
Remarque 1. If we set
(
*
&
,
1
*
& , then the term of pulsation
has magnitude
>
? @ times
the magnitude of the term of pulsation
; this is not a good choice for the non linear case in
which
>
?@
is of order
A
&B& ; it seems good choice
(+*
&
, 12*
C
>
?@
A
#
D
EGF
>2HJI
? H
@-K
#
9
#
9 2
Giens05. 2
Giens05. Other terms Other terms 3.1.1. A possible damage of a cable is breakage of several fibers, this will cause decrease of rigidity
* say for cable 1. Non destructive testing
3 0
4
8
12
16
20
24
28
32
36
0
200
400
600
800
1000
1200
absolute value of Fourier transform of non lin displacement with
alpha=62.831853, nualpha=10, F=1450, dt=0.01, y0=0, v0=−27.972687 ,k1=950 ,lambda=0.2
freqin Hz
F(u) 0
4
8
12
16
20
24
28
32
36
0
200
400
600
800
1000
1200
absolute value of Fourier transform of non lin displacement with
alpha=62.831853, nualpha=10, F=1450, dt=0.01, y0=0, v0=−27.972687 ,k1=950 ,lambda=0.2
freqin Hz
F(u) absolute value of Fourier transform of non lin displacement with
alpha=62.831853, nualpha=10, F=1450, dt=0.01, y0=0, v0=−27.972687 ,k1=950 ,lambda=0.2
F(u) – Let us start with undamaged cables of same rigidity
. If we note
b
, the common length
of the unstressed cables, and L their common stressed lenth, their tension is
_
b
b
# ;
d
f
) – now, after damage,
*
c
, cable 1 becomes longer and cable 2 shorter,
b
*
e
b
,
the tension goes down to
_
*
b
*
b
#
b
b
# ; – now, after damage,
*
c
, cable 1 becomes longer and cable 2 shorter,
b
*
e
b
,
the tension goes down to
_
*
b
*
b
#
b
b
# ; – note the limit case of cable 1 broken is
*
& so that the cable 2 gets lenth
b
but the
system is no longer working properly! – note the limit case of cable 1 broken is
*
& so that the cable 2 gets lenth
b
but the
system is no longer working properly! – Before such a breakdown, if the change of tension is substantial, this causes a substantial
change of the fundamental frequency; indeed, this is the routine monitoring of cable bridges! – The nonlinear vibroacoustic testing aims at monitoring the cables before such a substan-
tial change. 4. A non linear string model A model of non linear string has been introduced first by Kirchoff in 1877 and rederived by
Carrier in 1945. 'h"
+
-
m
g
g
_
i
j
k
#
k
k
l
(4.1) (4.1) (4.1) Several mathematical studies of this type of equations have been performed recently (Medeiros(1994),
Clark- Lima (1997). 4
Giens05. nananan
nananan
nananan
nananan
oaoaoao
oaoaoao
oaoaoao
papapapap
papapapap
papapapap
papapapap
qaqaqaqaq
qaqaqaqaq
qaqaqaqaq
rarararararararararararararararararararararararararararararararararararar
rarararararararararararararararararararararararararararararararararararar
rarararararararararararararararararararararararararararararararararararar
sasasasasasasasasasasasasasasasasasasasasasasasasasasasasasasasasasasasas
sasasasasasasasasasasasasasasasasasasasasasasasasasasasasasasasasasasasas
sasasasasasasasasasasasasasasasasasasasasasasasasasasasasasasasasasasasas
L 1
L
3
l 1
X
Y
2
L
m2
θ 3
m1
θ2
l2
l3
θ1
Two masses on stretched cables (cable 2 damaged) moving freely Giens05. Two masses on stretched cables (cable 2 damaged) moving freely 5. Masses free to move in a plane Equation of the dynamics Equation of the dynamics
^ u w
`
t
`
v
_
`
;
<
x
`
#
_
`
y
*
;
<
x
`
y
*
#
l
`
A
4
4
4
(5.1)
{` B`Sv
_
`
x
`
#
_
`|y
*
x
`zy
*
#
~}
`
A
444
(5.2) 6. Bar models with defects Bar models with longitudinal waves (dynamical traction and compression) are considered. Q
m
!
t
l
t
,
! #
(6.1) (6.1) With a non linear stress-strain law:
t
t
#
#
(6.2) (6.2) We could as well assume that the applied load is of order epsilon without any assumption
on the nonlinearity. Assuming
to be small an approximate solution is searched for with the
following "ansatz":
)
*
444 Theoretical justification of the expansions: the situation is complex in full generality: non
linear hyperbolic equations exhibit a singularity after a finite time! But: the experiments are
performed during a short time interval and the Fourier transforms are computed on these time 5 Non destructive testing Non destructive testing Non destructive testing intervals! Following a suggestion of Guy Metivier we are addressing the problem during a
small initial time interval in which the solution is smooth: plan to use an approximation of
the equation with a fixed point method proposed in Majda. In any case we should smooth the
characteristic function (the material is changing smoothly)! – But what is the relative level of secondary peaks for a given s
tigations: indeed it is also the difficulty of the real experiments – Need to include other behaviors: shocks, friction – Need of more precise models: non linear beams including tractional, flexural, torsional
effects – Mixture of local models for the defect and global models for the undamaged structure to
obtain precise results at low computational cost. 7. Conclusion – Some simple models governed by ODE pr PDE show intermodulations; – But what is the relative level of secondary peaks for a given
tigations: indeed it is also the difficulty of the real experiments 8. References [A.E 99] A.E.EKIMOV I.N.DIDENKULOV V., “Modulation of torsional waves in a rod with a
crack”, J.Acoust. Soc. AM., vol. 3, num. 106, 1999, p. 1289-1291. [A.S 98] A. SUTIN D. D., “Nonlinear vibro-acoustic nondestructive testing technique”, Non-
destructive characterisation of material, 7 Ed R.E. green, Plenum press, New York, 1998. [D.D 02] D. DONSKOY A. EKIMOV N. S. T., “Nonlinear seismo-acoustic land mine detection
and discrimination”, . Acoust. Soc. Am, vol. 111, 2002, p. 2705-2714. [E.H 93] E.H. BOUTYOUR B. COCHELIN N. D. M. P.-F., “Calculs non linéaires par des
méthodes asymptotiques-numériques: applications aux structures élastiques”,
Colloque
national en calcul de structures, 11-14 mai 1993, Hermes, 1993. [GUS 02] GUSEV A. M.-B. C.-V., “Frequency up-conversion and frequency down-
conversion of acoustic waves in damaged materials”, Physics letter A, vol. 301, 2002,
p. 281-290. [P.D 03] P. DUFOURCQ JP. GROBY M. L. P. T. E. G. V., “Détection vibro-acoustique non
linéaire d’ endomagements dans une structure poutre”, Communication au Congrès français
de mécanique, septembre 2003. [VAN ] VANDERBORCK GERARD L. M., “Application of non-linear ultrasonic spectroscopy
to health monitoring and damage detection in structures,. 38p.”, 75th Shock and Vibration
Symposium, Virginia Beach (VA) USA, du 18/10/2004 au 21/10/2004. [V.Z 99] V. ZAITSEV P. S., “Nonlinear vibro-acoustic response of a metal sample with a dis-
continuity like defect as related to damage detection problems”, Proceedings of DECT 99,
Las Vegas, Nevada, 1999.
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|
English
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Age-related change in task-evoked amygdala—prefrontal circuitry: a multiverse approach with an accelerated longitudinal cohort aged 4-22 years
|
bioRxiv (Cold Spring Harbor Laboratory)
| 2,021
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cc-by
| 44,247
|
Age-related change in task-evoked amygdala—prefrontal circuitry: a multiverse approach
with an accelerated longitudinal cohort aged 4-22 years Paul Alexander Bloom1*, Michelle VanTieghem2, Laurel Gabard-Durnam3, Dylan G. Gee4,
Jessica Flannery5, Christina Caldera6, Bonnie Goff6, Eva H. Telzer7, Kathryn L. Humphreys8,
Dominic S. Fareri9, Mor Shapiro10, Sameah Algharazi11, Niall Bolger1, Mariam Aly1, & Nim
Tottenham1 1Columbia University 2Virta Health 3Northeastern University 4Yale University 5Limbix Health
6University of California Los Angeles 7University of North Carolina at Chapel Hill 8Vanderbilt
University 9Adelphi University 10Kaiser Permanente 11City College of New York Please address correspondence to:
Paul Alexander Bloom
Columbia University Psychology, New York, NY
Email: paul.bloom@columbia.edu Keywords: Amygdala, prefrontal cortex, development, longitudinal, multiverse, robustness Data Availability Because participants and their parents did not consent to data sharing at the time of
participation, we cannot make data from the current study publicly available. .
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
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which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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bioRxiv preprint Funding This work was supported by funding from the National Institutes of Mental Health
(2R01MH091864) and the Dana Foundation for Nim Tottenham, and a National Science
Foundation Graduate Research Fellowship (DGE 1644869) for Paul Alexander Bloom. The
authors declare no competing interests. . CC-BY 4.0 International license
available under a
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The copyright holder for this preprint
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The copyright holder for this preprint
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bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Abstract CC-BY 4.0 International license
available under a
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bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Abstract The amygdala and its connections with medial prefrontal cortex (mPFC) play central roles in
the development of emotional processes. While several studies have suggested that this
circuitry exhibits functional changes across the first two decades of life, findings have been
mixed – perhaps resulting from differences in analytic choices across studies. Here we used
multiverse analyses to examine the robustness of task-based amygdala–mPFC function
findings to analytic choices within the context of an accelerated longitudinal design (4-22 years-
old; N=98; 183 scans; 1-3 scans/participant). Participants, recruited from the greater Los
Angeles area, completed an event-related emotional face (fear, neutral) task. Parallel analyses
varying in preprocessing and modeling choices found that age-related change estimates for
amygdala reactivity were more robust than task-evoked amygdala–mPFC functional
connectivity to varied analytical choices. Specification curves indicated evidence for age-
related decreases in amygdala reactivity to faces, though within-participant changes in
amygdala reactivity could not be differentiated from between-participant differences. In
contrast, amygdala—mPFC functional connectivity results varied across methods much more,
and evidence for age-related change in amygdala–mPFC connectivity was not consistent. Generalized psychophysiological interaction (gPPI) measurements of connectivity were
especially sensitive to whether a deconvolution step was applied. Our findings demonstrate
the importance of assessing the robustness of findings to analysis choices, although the age-
related changes in our current work cannot be overinterpreted given low test-retest reliability. Together, these findings highlight both the challenges in estimating developmental change in
longitudinal cohorts and the value of multiverse approaches in developmental neuroimaging
for assessing robustness of results. (Preprint: Generalized psychophysiological interaction (gPPI) measurements of connectivity were
especially sensitive to whether a deconvolution step was applied. Our findings demonstrate
the importance of assessing the robustness of findings to analysis choices, although the age-
related changes in our current work cannot be overinterpreted given low test-retest reliability. Together, these findings highlight both the challenges in estimating developmental change in
longitudinal cohorts and the value of multiverse approaches in developmental neuroimaging
for assessing robustness of results. (Preprint: https://www.biorxiv.org/content/10.1101/2021.10.08.463601v1). . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint . Key Points •
Multiverse analyses applied to fMRI data are valuable for determining the robustness of
findings to varied analytical choices •
In the current study, age-related change estimates for amygdala reactivity were
relatively robust to analytical decisions, though gPPI functional connectivity analyses
were much more sensitive, leading some estimates to flip sign •
Both test-retest reliability and robustness to analytical choices are important
considerations for developmental research Running head: AMYGDALA
MPFC AGE RELATED CHANGE
. CC-BY 4.0 International license
available under a
which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Rodent models and human neuroimaging have provided converging evidence for the
1
importance of the amygdala and medial prefrontal cortex (mPFC) in the development of threat
2
processing (Adolphs, 2008; Forbes et al., 2011), emotion regulation (Pozzi et al., 2020; Silvers
3
et al., 2015; Sullivan & Perry, 2015), and affective learning (Moriceau & Sullivan, 2006; Pattwell
4
et al., 2016). Characterizing growth trajectories of these regions may provide insight into
5
neural constructions underlying emotional development (Meyer & Lee, 2019). To probe
6
amygdala–mPFC circuitry across development, face stimuli are frequently employed because
7
they effectively engage this circuitry while being child-appropriate (Hariri et al., 2002). Though
8
a number of studies have examined age-related changes from childhood to young adulthood
9
in amygdala responses and amygdala–mPFC functional connectivity (FC) associated with
10
emotional face stimuli, findings have varied widely (likely due in part to differences in sample
11
composition and task design; see sTable 1 for details). Key Points 30
While the small sample sizes examined in many studies on amygdala–mPFC
31
development likely contribute to differences in findings (Marek et al., 2020), especially given
32
low reliability of many amygdala—mPFC measures (Elliott et al., 2020; Herting et al., 2017;
33
Sauder et al., 2013), important methodological differences also exist across studies. 34
Differences in age range or sample demographics, stimuli, task (e.g. passive viewing
35
vs. emotion labeling or matching (Lieberman et al., 2007), task design (blocked vs. event-
36
related; Sergerie et al., 2008), or contrast used (faces > shapes vs. faces > baseline) may also
37
contribute to discrepancies (see sTable 1). The brain regions under investigation also differ
38
across studies; for example, prefrontal clusters with which amygdala connectivity was
39
assessed. Interpreting discrepancies across studies without appreciation for these
40
methodological differences would be inappropriate, and in fact, incorrect. Yet, such
41
differences do not account for all discrepancies in findings across studies. Variation in
42
processing pipelines is another source of differences across studies, as varying analytic
43
decisions can produce qualitatively different findings, even between putatively identical
44
analyses of the same dataset (Botvinik-Nezer et al., 2020). Choices including software
45
package (Bowring et al., 2019), spatial smoothing (Jo et al., 2007), treatment of head motion
46 Running head: AMYGDALA—MPFC AGE-RELATED CHANGE related change (Zhang et al., 2019). While some investigations have found differing age-
25
related change for faces displaying different emotions (Killgore & Yurgelun-Todd, 2007; Swartz
26
et al., 2014; Vijayakumar et al., 2019), even investigations of fearful faces specifically have
27
varied in their developmental findings for both amygdala reactivity and amygdala—mPFC
28
functional connectivity (Forbes et al., 2011; Gee et al., 2013; Killgore et al., 2001; Wu et al.,
29
2016, 2016; Zhang et al., 2019). 30 While the small sample sizes examined in many studies on amygdala–mPFC
31
development likely contribute to differences in findings (Marek et al., 2020), especially given
32
low reliability of many amygdala—mPFC measures (Elliott et al., 2020; Herting et al., 2017;
33
Sauder et al., 2013), important methodological differences also exist across studies. 34
Differences in age range or sample demographics, stimuli, task (e.g. passive viewing
35
vs. emotion labeling or matching (Lieberman et al., 2007), task design (blocked vs. event-
36
related; Sergerie et al., 2008), or contrast used (faces > shapes vs. Key Points Several studies have found age-
12
related change in amygdala reactivity, including decreases as a function of age in response to
13
emotional faces (Gee et al., 2013; Guyer et al., 2008; Killgore et al., 2001; Passarotti et al.,
14
2009; Swartz et al., 2014; Telzer et al., 2015) as well as other images (Decety et al., 2012;
15
Silvers et al., 2017b; Vink et al., 2014), increases in amygdala reactivity with age (Joseph et al.,
16
2015; Todd et al., 2011), developmental sex differences (Xu et al., 2021) or peaks during
17
adolescence (Hare et al., 2008; Vijayakumar et al., 2019). Others have observed no age-
18
related changes (Kujawa et al., 2016; Pfeifer et al., 2011; Pine et al., 2001; Wu et al., 2016;
19
Yurgelun-Todd & Killgore, 2006; Zhang et al., 2019). 20
With task-evoked amygdala–mPFC FC, several studies have found age-related
21 With task-evoked amygdala–mPFC FC, several studies have found age-related
21
decreases from childhood to young adulthood (Gee et al., 2013; Kujawa et al., 2016; Silvers et
22
al., 2017a; Wu et al., 2016), while others have found increases (Decety et al., 2012; Perlman &
23
Pelphrey, 2011; Vink et al., 2014), developmental sex differences (Xu et al., 2021), or little age-
24 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE
related change (Zhang et al., 2019). While some investigations have found differing age-
25
related change for faces displaying different emotions (Killgore & Yurgelun-Todd, 2007; Swartz
26
et al., 2014; Vijayakumar et al., 2019), even investigations of fearful faces specifically have
27
varied in their developmental findings for both amygdala reactivity and amygdala—mPFC
28
functional connectivity (Forbes et al., 2011; Gee et al., 2013; Killgore et al., 2001; Wu et al.,
29
2016, 2016; Zhang et al., 2019). Key Points faces > baseline) may also
37
contribute to discrepancies (see sTable 1). The brain regions under investigation also differ
38
across studies; for example, prefrontal clusters with which amygdala connectivity was
39
assessed. Interpreting discrepancies across studies without appreciation for these
40
methodological differences would be inappropriate, and in fact, incorrect. Yet, such
41
differences do not account for all discrepancies in findings across studies. Variation in
42
processing pipelines is another source of differences across studies, as varying analytic
43
decisions can produce qualitatively different findings, even between putatively identical
44
analyses of the same dataset (Botvinik-Nezer et al., 2020). Choices including software
45
package (Bowring et al., 2019), spatial smoothing (Jo et al., 2007), treatment of head motion
46
(Achterberg & van der Meulen, 2019), parcellation (Bryce et al., 2021), and functional
47
connectivity approach (Di et al., 2020) can also impact results and qualitatively change
48
findings (Cisler et al., 2014). Additionally, the majority of developmental investigations of
49 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Running head: AMYGDALA—MPFC AGE-RELATED CHANGE u
g
ead
G
C
G
C
G
analytical choices, highlighting the importance of considering both robustness and reliability in
75
developmental research. 76
____________________________
77
Insert Figure 1 about here
78
____________________________
79
80
____________________________
81
Insert Table 1 about here
82
____________________________
83
84
85
Methods
86
Before completing analyses, we preregistered methods for the current study through the
87
Open Science Framework at https://osf.io/8nyj7/. Only analyses for age-related changes in
88
amygdala reactivity and amygdala–mPFC gPPI were preregistered in detail (see Table 1 Aims
89
1a & 2a), and we did not preregister multiverse analyses. Methods detailed below include both
90
information included in the preregistration and additional information and analyses not
91
preregistered. Analysis code & documentation can be found at
92
https://github.com/pab2163/amygdala_mpfc_multiverse. 93
94
Participants
95
Participants were recruited as part of a larger study examining brain development as a
96
f
ti
f
l lif
i i
i
Th
t
l
(N 98 55 f
l
43
l )
97 u
g
ead
G
C
G
C
G
analytical choices, highlighting the importance of considering both robustness and reliability in
75
developmental research. 76
____________________________
77
Insert Figure 1 about here
78
____________________________
79
80
____________________________
81
Insert Table 1 about here
82
____________________________
83
84
85
Methods
86
Before completing analyses, we preregistered methods for the current study through the
87
Open Science Framework at https://osf.io/8nyj7/. Only analyses for age-related changes in
88
amygdala reactivity and amygdala–mPFC gPPI were preregistered in detail (see Table 1 Aims
89
1a & 2a), and we did not preregister multiverse analyses. Methods detailed below include both
90
information included in the preregistration and additional information and analyses not
91
preregistered. Analysis code & documentation can be found at
92
https://github.com/pab2163/amygdala_mpfc_multiverse. 93
94
Participants
95
Participants were recruited as part of a larger study examining brain development as a
96
f
f
f
(
f
)
9 analytical choices, highlighting the importance of considering both robustness and reliability in
75
developmental research. 76 Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Running head: AMYGDALA—MPFC AGE-RELATED CHANGE
amygdala–mPFC function have studied cross-sectional samples. Because cross-sectional
50
studies are susceptible to cohort effects and cannot measure within-participant change,
51
longitudinal work has been recommended for better charting of developmental trajectories
52
(Crone & Elzinga, 2015; Madhyastha et al., 2017). 53
Here, we used multiverse analyses to examine the robustness of developmental
54
changes to varied analytical decisions. We focused on task-related amygdala–mPFC
55
functional development in an accelerated longitudinal sample ranging from ages 4-22 years. 56
We selected a task that was designed to be appropriate for young ages to characterize
57
developmental change in amygdala–mPFC responses to fear and neutral faces across
58
childhood and adolescence, and we asked whether findings were robust to analytical choices. 59
This accelerated longitudinal design is an extension of the sample reported on by Gee et al. 60
(2013). We preregistered two hypotheses (https://osf.io/8nyj7/) predicting that both amygdala
61
reactivity (1) and amygdala–mPFC connectivity (2) as measured with generalized
62
psychophysiological interaction models (gPPI), would decrease as a function of age during
63
viewing of fearful faces relative to baseline (see Table 1 Aims 1a & 2a). 64 Although we did not preregister further hypotheses, we also investigated age-related
65
changes in within-scan differences in amygdala responses across trials and FC using beta
66
series correlations. As previous work identified associations between amygdala–mPFC FC
67
and separation anxiety (Carpenter et al., 2015; Gee et al., 2013), we asked whether any
68
amygdala–mPFC measures were associated longitudinally with separation anxiety behaviors
69
(see Table 1 Aim 3). We used ‘multiverse’ analyses and specification curves to examine the
70
impact of analytical decisions on results. We also investigated test-retest reliability of all brain
71
measurements across longitudinal study visits, given the importance of such reliability for
72
interpreting individual differences or developmental change (Herting et al., 2017). Our
73
multiverse approach allows us to thoroughly explore the robustness of different findings to
74 . CC-BY 4.0 International license
available under a
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The copyright holder for this preprint
this version posted February 22, 2022. ;
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doi:
bioRxiv preprint Methods . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Running head: AMYGDALA—MPFC AGE-RELATED CHANGE
years-old (M = 11.9 years old) who enrolled to participate in a study on emotional development. 99
All participants were reported to be physically and psychiatrically healthy (no medical or
100
psychiatric disorders), as indicated by a telephone screening before participation, and free of
101
MRI contraindications. All except 4 participants fell below clinical cutoffs (see sFigure 2) on the
102
Child Behavior Checklist (CBCL) Total Problems, Internalizing Problems, and Externalizing
103
Problems scales (Achenbach, 1991). The larger study also included youths with a history of
104
institutional and/or foster care outside of the United States, who are not included here. 105
Participants from the greater Los Angeles area were recruited through flyers, state birth records,
106
community events, online advertising, lab website and newsletters, psychologists’ offices,
107
psychology courses at a local university (participants ages 18-22 years old only), and word-of-
108
mouth. Each participant completed up to 3 MRI scans spaced at an average interval of 18
109
months between visits. Parents provided written consent, children 7+ years old gave written
110
assent, and children under 7 years old gave verbal assent. Study protocols were approved by
111
the local university institutional review board. These data were collected between 2009 and
112
2015. 113 An accelerated longitudinal design was used such that participants’ starting ages at scan
114
1 comprised the entire range of sample ages (4-22 years old), and coverage was approximately
115
balanced across the entire age range (see Figure 1B). The design was structured into 3 study
116
‘waves’ corresponding with recruitment efforts and visit protocols. Participants were
117
overenrolled at wave 1 to account for anticipated attrition (e.g., braces, relocation, etc) to
118
achieve the desired sample size across the three waves. Methods 137
Separation Anxiety
138
For each participant (except for 10 adults 18-22 years), a parent completed both the
139
Revised Children’s Anxiety and Depression Scale (RCADS-P) and the Screen for Child Anxiety
140
Related Emotional Disorders (SCARED-P) to assess the frequency of symptoms of anxiety and
141
low mood (Birmaher et al., 1999; Chorpita et al., 2000). Following prior work suggesting
142
associations between task-evoked amygdala–mPFC functional connectivity and separation
143
anxiety (Carpenter et al., 2015; Gee et al., 2013), we used the separation anxiety subscales
144
from both the SCARED-P and RCADS-P as measures of anxiety-related behaviors in asking
145
. CC BY 4.0 International license
available under a Running head: AMYGDALA—MPFC AGE-RELATED CHANGE
Of the 191 participants participating in the longitudinal study, 140 completed at least one
123
MRI scan. After exclusions for incomplete task runs (including falling asleep), computer errors
124
resulting in missing stimulus timing files, high head motion, and failed visual QA
125
(scanner/motion artifacts), a final sample of 98 participants (N = 183 total scans) was included
126
for analysis (see Figure 1). Exclusion criteria were preregistered after conducting preliminary
127
preprocessing, but before construction of group-level models and multiverse analysis plans. 128
This sample included 40 participants with 1 scan, 31 with 2 scans, and 27 with 3 scans (one
129
more participant than preregistered due to an initial coding error). Wave 1 data from forty-two of
130
these participants were reported on by Gee et al. (2013). 131 This sample included 40 participants with 1 scan, 31 with 2 scans, and 27 with 3 scans (one
129
more participant than preregistered due to an initial coding error). Wave 1 data from forty-two of
130
these participants were reported on by Gee et al. (2013). 131
The median annual household income for participating families was $85,001-$100,000
132
(for reference, median annual household income in Los Angeles County from 2015-2019 was
133
$68,044; US Census Bureau, 2021). Epidemiological methods were not used to recruit a
134
sample representative of the Los Angeles or United States populations (Heeringa et al., 2004),
135
and Hispanic or Latinx participants were particularly underrepresented. Further sample
136
demographics can be found in the supplementary materials (see sTables 2-3, sFigures 1-2). Methods While most participants were recruited
119
such that their first scan occurred at wave 1, a smaller set of participants were recruited at wave
120
2, such that some participants completed their first scan at wave 2 (see Figure 1). For these
121
participants, only 2 scans were planned. 122 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE
Of the 191 participants participating in the longitudinal study, 140 completed at least on
123
MRI scan. After exclusions for incomplete task runs (including falling asleep), computer errors
124
resulting in missing stimulus timing files, high head motion, and failed visual QA
125
(scanner/motion artifacts), a final sample of 98 participants (N = 183 total scans) was included
126
for analysis (see Figure 1). Exclusion criteria were preregistered after conducting preliminary
127
preprocessing, but before construction of group-level models and multiverse analysis plans. 128
This sample included 40 participants with 1 scan, 31 with 2 scans, and 27 with 3 scans (one
129
more participant than preregistered due to an initial coding error). Wave 1 data from forty-two o
130
these participants were reported on by Gee et al. (2013). 131
The median annual household income for participating families was $85,001-$100,000
132
(for reference, median annual household income in Los Angeles County from 2015-2019 was
133
$68,044; US Census Bureau, 2021). Epidemiological methods were not used to recruit a
134
sample representative of the Los Angeles or United States populations (Heeringa et al., 2004),
135
and Hispanic or Latinx participants were particularly underrepresented. Further sample
136
demographics can be found in the supplementary materials (see sTables 2-3, sFigures 1-2). Methods As expected, raw separation anxiety scores on both measures
150
decreased as a function of age, while standardized scores (which are normed based on gender
151
and grade level) were consistent across development with few children at or near clinical
152
threshold (see Figure 6). 153
154
Emotion Discrimination Task
155
Participants completed either two (at wave 3) or three (at waves 1 and 2) runs of a
156
modified ‘go/no-go’ task with emotional faces during fMRI scanning. Runs varied by emotional
157
expression (fear, happy, sad), and within each run participants viewed emotional faces
158
interspersed with neutral faces. To ensure that participants were paying attention, they were
159
asked to press a button whenever they saw a neutral face (no response was required for any
160
other face expression). The order of the runs was counterbalanced across participants; the
161
stimuli within each run were pseudorandomized (Wager & Nichols, 2003) to allow for event-
162
related estimates of the hemodynamic response, and fixed across participants. For the present
163
analysis, only the fear run of the task was used. The other two runs, which used happy and sad
164
faces in place of fear, are not included in the present analysis as these conditions were not
165
present at all waves of data collection. As 50% of trials were ‘go’ trials under this paradigm, we
166
refer to the task as an emotion discrimination task, rather than a true ‘go/no-go’ paradigm since
167
there was no strong prepotent motor response. Stimuli within each run were presented with a
168
jittered ITI (3-10s, Median = 4.93s]) according to a genetic algorithm with a fixation cross on the
169
. CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint . Methods 137
Separation Anxiety
138
For each participant (except for 10 adults 18-22 years), a parent completed both the
139
Revised Children’s Anxiety and Depression Scale (RCADS-P) and the Screen for Child Anxiety
140
Related Emotional Disorders (SCARED-P) to assess the frequency of symptoms of anxiety and
141
low mood (Birmaher et al., 1999; Chorpita et al., 2000). Following prior work suggesting
142
associations between task-evoked amygdala–mPFC functional connectivity and separation
143
anxiety (Carpenter et al., 2015; Gee et al., 2013), we used the separation anxiety subscales
144
from both the SCARED-P and RCADS-P as measures of anxiety-related behaviors in asking
145
whether such functional connectivity may be linked to anxiety levels during childhood and
146 The median annual household income for participating families was $85,001-$100,000
132
(for reference, median annual household income in Los Angeles County from 2015-2019 was
133
$68,044; US Census Bureau, 2021). Epidemiological methods were not used to recruit a
134
sample representative of the Los Angeles or United States populations (Heeringa et al., 2004),
135
and Hispanic or Latinx participants were particularly underrepresented. Further sample
136
demographics can be found in the supplementary materials (see sTables 2-3, sFigures 1-2). 137 Separation Anxiety
138
For each participant (except for 10 adults 18-22 years), a parent completed both the
139
Revised Children’s Anxiety and Depression Scale (RCADS-P) and the Screen for Child Anxiety
140
Related Emotional Disorders (SCARED-P) to assess the frequency of symptoms of anxiety and
141
low mood (Birmaher et al., 1999; Chorpita et al., 2000). Following prior work suggesting
142
associations between task-evoked amygdala–mPFC functional connectivity and separation
143
anxiety (Carpenter et al., 2015; Gee et al., 2013), we used the separation anxiety subscales
144
from both the SCARED-P and RCADS-P as measures of anxiety-related behaviors in asking
145
whether such functional connectivity may be linked to anxiety levels during childhood and
146 Running head: AMYGDALA—MPFC AGE-RELATED CHANGE
adolescence. For 11 participants who had missing items on the SCARED-P, indicating parents
147
had skipped or forgotten to answer a question, we imputed responses using 5-Nearest Neighbor
148
imputation using only the other items included in the SCARED-P separation anxiety subscale
149
(Beretta & Santaniello, 2016). Methods CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Running head: AMYGDALA—MPFC AGE-RELATED CHANGE
Karolinska Directed Emotional Faces database (Calvo & Lundqvist, 2008), and the same face
171
stimuli were used across longitudinal study visits (Vijayakumar et al., 2019). Each run (130 TRs,
172
duration of 4:20) consisted of 48 trials (24 neutral faces, 24 fearful faces), each presented for
173
350ms. All fMRI analyses of this task used event-related designs. 174
175
MRI Acquisition
176
Participants under 18-years-old completed a mock scanning session before the MRI
177
scan to acclimate to the scanner environment and practice lying still for data collection. Waves 1
178
and 2 were collected on a Siemens 3T TIM Trio MRI scanner using a standard radiofrequency
179
head coil. A 2D spin echo image (TR, 4000 ms; TE, 40 ms; matrix size, 256 x 256; 4 mm thick;
180
0mm gap) was acquired in the oblique plane to guide slice configuration in both structural and
181
functional scans. A whole-brain high-resolution T1-weighted anatomical scan (MPRAGE; 256 x
182
256 in-plane resolution; 256mm FOV; 192 x 1 mm sagittal slices) was acquired for each
183
participant for registration of functional data. The task was presented through MR-compatible
184
goggles during scanning. T2*-weighted echoplanar images (interleaved slice acquisition) were
185
collected at an oblique angle of ~30 degrees (130 volumes/run; TR=2000ms; TE=30 ms; flip
186
angle=90°; matrix size=64 x 64; FOV=192 mm; 34 slices; 4 mm slice thickness; skip=0 mm). 187
Wave 3 was collected on a Siemens 3T TIM Trio MRI scanner at a different location using
188
identical acquisition parameters. 189
190
Behavioral Analyses
191 Karolinska Directed Emotional Faces database (Calvo & Lundqvist, 2008), and the same face
171
stimuli were used across longitudinal study visits (Vijayakumar et al., 2019). Methods CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE adolescence. For 11 participants who had missing items on the SCARED-P, indicating parents
147
had skipped or forgotten to answer a question, we imputed responses using 5-Nearest Neighbor
148
imputation using only the other items included in the SCARED-P separation anxiety subscale
149
(Beretta & Santaniello, 2016). As expected, raw separation anxiety scores on both measures
150
decreased as a function of age, while standardized scores (which are normed based on gender
151
and grade level) were consistent across development with few children at or near clinical
152
threshold (see Figure 6). 153 Participants completed either two (at wave 3) or three (at waves 1 and 2) runs of a
156
modified ‘go/no-go’ task with emotional faces during fMRI scanning. Runs varied by emotional
157
expression (fear, happy, sad), and within each run participants viewed emotional faces
158
interspersed with neutral faces. To ensure that participants were paying attention, they were
159
asked to press a button whenever they saw a neutral face (no response was required for any
160
other face expression). The order of the runs was counterbalanced across participants; the
161
stimuli within each run were pseudorandomized (Wager & Nichols, 2003) to allow for event-
162
related estimates of the hemodynamic response, and fixed across participants. For the present
163
analysis, only the fear run of the task was used. The other two runs, which used happy and sad
164
faces in place of fear, are not included in the present analysis as these conditions were not
165
present at all waves of data collection. As 50% of trials were ‘go’ trials under this paradigm, we
166
refer to the task as an emotion discrimination task, rather than a true ‘go/no-go’ paradigm since
167
there was no strong prepotent motor response. Stimuli within each run were presented with a
168
jittered ITI (3-10s, Median = 4.93s]) according to a genetic algorithm with a fixation cross on the
169
screen (Wager & Nichols, 2003). Face images were adult White female faces from the
170 . Methods If there were outstanding issues with a particular scan run (areas of
206
brain tissue cut off, or significant areas of skull left in, 30/195 scans), FSL’s brain extraction tool
207
(BET; Jenkinson et al., 2012) was used instead. We used robust brain center estimation, and
208
modified the fractional intensity values between 0.5-0.7 to optimize quality. Slice-time correction
209
was not used. Timeseries of the 6 motion parameters were calculated and subsequent spatial
210
realignment of BOLD volumes was completed using MCFLIRT in FSL (Jenkinson et al., 2002). 211
Scans over a threshold of >40 volumes with > .9mm framewise displacement (framewise
212
displacement calculated as the sum of absolute frame-to-frame differences between head
213
realignment estimates; Power et al., 2012) were excluded from analysis (12 out of an initial 195,
214
or 6.2%). After this exclusion, an average of 96.7% (range = [70.1-100%]) of stimulus-coincident
215
volumes in each scan were below the 0.9mm framewise displacement threshold. The mean age
216
of participants with excluded scans was 7.16 and 8/12 were male. Registration matrices were
217
calculated for registration of functional images to high-resolution structural T1 images using
218
FSL’s FLIRT with boundary-based registration. Registration matrices for standard MNI space
219
. CC-BY 4.0 International license
available under a Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Running head: AMYGDALA—MPFC AGE-RELATED CHANGE 3dskullstrip from the Analysis of Functional NeuroImages (AFNI, v20.1.16) software
204
package (Cox, 1996) was first run on all MPRAGE scans. Next, experimenters checked the
205
quality of the skull stripping. If there were outstanding issues with a particular scan run (areas of
206
brain tissue cut off, or significant areas of skull left in, 30/195 scans), FSL’s brain extraction tool
207
(BET; Jenkinson et al., 2012) was used instead. We used robust brain center estimation, and
208
modified the fractional intensity values between 0.5-0.7 to optimize quality. Slice-time correction
209
was not used. Timeseries of the 6 motion parameters were calculated and subsequent spatial
210
realignment of BOLD volumes was completed using MCFLIRT in FSL (Jenkinson et al., 2002). 211
Scans over a threshold of >40 volumes with > .9mm framewise displacement (framewise
212
displacement calculated as the sum of absolute frame-to-frame differences between head
213
realignment estimates; Power et al., 2012) were excluded from analysis (12 out of an initial 195,
214
or 6.2%). Methods Each run (130 TRs,
172
duration of 4:20) consisted of 48 trials (24 neutral faces, 24 fearful faces), each presented for
173
350ms. All fMRI analyses of this task used event-related designs. 174 Participants under 18-years-old completed a mock scanning session before the MRI
177
scan to acclimate to the scanner environment and practice lying still for data collection. Waves 1
178
and 2 were collected on a Siemens 3T TIM Trio MRI scanner using a standard radiofrequency
179
head coil. A 2D spin echo image (TR, 4000 ms; TE, 40 ms; matrix size, 256 x 256; 4 mm thick;
180
0mm gap) was acquired in the oblique plane to guide slice configuration in both structural and
181
functional scans. A whole-brain high-resolution T1-weighted anatomical scan (MPRAGE; 256 x
182
256 in-plane resolution; 256mm FOV; 192 x 1 mm sagittal slices) was acquired for each
183 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE
alarm rate (on fear face trials) as the respective outcomes, and included nested random effects
195
for task sessions within participants (models were not nested for d’ as this analysis used only 1
196
metric per session rather than trial-wise outcomes, but still included random effects for
197
participants). Additionally, to model age-related change in reaction times during correct hit trials,
198
we fit linear, quadratic, cubic, and inverse age (1/age; Luna et al., 2004, 2021) regressions with
199
identical random effects structures. Model equations and results for all behavioral analyses can
200
be found in the supplement (see supplemental methods p.12-14, sFigures 3-4). 201
202
Preregistered fMRI Pipeline
203
3dskullstrip from the Analysis of Functional NeuroImages (AFNI, v20.1.16) software
204
package (Cox, 1996) was first run on all MPRAGE scans. Next, experimenters checked the
205
quality of the skull stripping. Methods After this exclusion, an average of 96.7% (range = [70.1-100%]) of stimulus-coincident
215
volumes in each scan were below the 0.9mm framewise displacement threshold. The mean age
216
of participants with excluded scans was 7.16 and 8/12 were male. Registration matrices were
217
calculated for registration of functional images to high-resolution structural T1 images using
218
FSL’s FLIRT with boundary-based registration. Registration matrices for standard MNI space
219 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE
were also calculated using both FLIRT (linear registration) and FNIRT (nonlinear registration)
220
with 12 DOF and a warp resolution of 10mm. Data were high-pass filtered at .01Hz and
221
smoothed with an isotropic Gaussian kernel with FWHM of 6mm before running general linear
222
models (GLMs), and 4d volumes were grand mean scaled such that the average intensity value
223
was 10000. 224
Following preprocessing, we ran scan-level GLMs using FSL’s FEAT (v6.00). We
225
included event-related regressors for fear and neutral faces (convolved with a double-gamma
226
HRF), their temporal derivatives (Pernet, 2014), and 24 head motion nuisance regressors (the 6
227
head realignment parameters, their temporal derivatives, and their squares (Power et al., 2012)
228
Volumes with FD > .9mm were downweighed to 0 in the GLM. Pre-whitening was used to
229
estimate and remove temporal autocorrelation (Woolrich et al., 2001). For each scan, we
230
calculated fear > baseline, neutral > baseline, and fear > neutral contrasts. We used native-
231
space bilateral amygdala masks generated using Freesurfer (v6.0; Fischl, 2012) by
232
VanTieghem et al. (2021). 233
234
Multiverse Analyses and Specification Curves
235
In addition to the preregistered pipelines, we conducted multiverse analyses to address
236
all aims in Table 1 and constructed sets of separate specification curves for each aim (see
237
Table 2). Methods In general, multiverse analyses aim to probe the consistency of results across all
238
‘reasonable’ possible combinations of analysis decisions (i.e. simultaneously taking all possible
239
‘forking paths’)(Steegen et al., 2016). Because analyzing fMRI data using all reasonable
240
specifications was infeasible (i.e., possibilities are virtually infinite), we took the approach of
241
‘sampling’ from the many reasonable or commonly-used analysis choices for each multiverse. 242
Despite not being completely comprehensive, this approach still allowed for thorough
243
investigation into the robustness of results. For all multiverse analyses, we constructed
244 Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Running head: AMYGDALA—MPFC AGE-RELATED CHANGE
were also calculated using both FLIRT (linear registration) and FNIRT (nonlinear registration)
220
with 12 DOF and a warp resolution of 10mm. Data were high-pass filtered at .01Hz and
221
smoothed with an isotropic Gaussian kernel with FWHM of 6mm before running general linear
222
models (GLMs), and 4d volumes were grand mean scaled such that the average intensity value
223
was 10000. 224
Following preprocessing, we ran scan-level GLMs using FSL’s FEAT (v6.00). We
225
included event-related regressors for fear and neutral faces (convolved with a double-gamma
226
HRF), their temporal derivatives (Pernet, 2014), and 24 head motion nuisance regressors (the 6
227
head realignment parameters, their temporal derivatives, and their squares (Power et al., 2012). 228
Volumes with FD > .9mm were downweighed to 0 in the GLM. Pre-whitening was used to
229
estimate and remove temporal autocorrelation (Woolrich et al., 2001). For each scan, we
230
calculated fear > baseline, neutral > baseline, and fear > neutral contrasts. We used native-
231
space bilateral amygdala masks generated using Freesurfer (v6.0; Fischl, 2012) by
232
VanTieghem et al. (2021). 233 Running head: AMYGDALA—MPFC AGE-RELATED CHANGE
were also calculated using both FLIRT (linear registration) and FNIRT (nonlinear registration)
220
with 12 DOF and a warp resolution of 10mm. Data were high-pass filtered at .01Hz and
221
smoothed with an isotropic Gaussian kernel with FWHM of 6mm before running general linear
222
models (GLMs), and 4d volumes were grand mean scaled such that the average intensity value
223
was 10000. 224
Following preprocessing, we ran scan-level GLMs using FSL’s FEAT (v6.00). Methods We
225
included event-related regressors for fear and neutral faces (convolved with a double-gamma
226
HRF), their temporal derivatives (Pernet, 2014), and 24 head motion nuisance regressors (the 6
227
head realignment parameters, their temporal derivatives, and their squares (Power et al., 2012). 228
Volumes with FD > .9mm were downweighed to 0 in the GLM. Pre-whitening was used to
229
estimate and remove temporal autocorrelation (Woolrich et al., 2001). For each scan, we
230
calculated fear > baseline, neutral > baseline, and fear > neutral contrasts. We used native-
231
space bilateral amygdala masks generated using Freesurfer (v6.0; Fischl, 2012) by
232
VanTieghem et al. (2021). 233
234 were also calculated using both FLIRT (linear registration) and FNIRT (nonlinear registration)
220
with 12 DOF and a warp resolution of 10mm. Data were high-pass filtered at .01Hz and
221
smoothed with an isotropic Gaussian kernel with FWHM of 6mm before running general linear
222
models (GLMs), and 4d volumes were grand mean scaled such that the average intensity value
223
was 10000. 224 Following preprocessing, we ran scan-level GLMs using FSL’s FEAT (v6.00). We
225
included event-related regressors for fear and neutral faces (convolved with a double-gamma
226
HRF), their temporal derivatives (Pernet, 2014), and 24 head motion nuisance regressors (the 6
227
head realignment parameters, their temporal derivatives, and their squares (Power et al., 2012). 228
Volumes with FD > .9mm were downweighed to 0 in the GLM. Pre-whitening was used to
229
estimate and remove temporal autocorrelation (Woolrich et al., 2001). For each scan, we
230
calculated fear > baseline, neutral > baseline, and fear > neutral contrasts. We used native-
231
space bilateral amygdala masks generated using Freesurfer (v6.0; Fischl, 2012) by
232
VanTieghem et al. (2021). 233 Multiverse Analyses and Specification Curves
235
In addition to the preregistered pipelines, we conducted multiverse analyses to address
236
all aims in Table 1 and constructed sets of separate specification curves for each aim (see
237
Table 2). In general, multiverse analyses aim to probe the consistency of results across all
238
‘reasonable’ possible combinations of analysis decisions (i.e. simultaneously taking all possible
239
‘forking paths’)(Steegen et al., 2016). Because analyzing fMRI data using all reasonable
240
specifications was infeasible (i.e., possibilities are virtually infinite), we took the approach of
241
‘sampling’ from the many reasonable or commonly-used analysis choices for each multiverse. Methods We varied analysis choices of GLM software,
265
hemodynamic response function, nuisance regressors, first-level GLM estimates, amygdala
266
ROI, exclusion criteria (exclude vs. include scans analyzed by Gee et al., 2013), group-level
267
model outlier treatment, and group-level model covariates (see Table 2 & supplemental
268
methods p.14-17). Multiverse analyses of amygdala reactivity included a total of 2808 model
269
. CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made Running head: AMYGDALA—MPFC AGE-RELATED
specification curves by ranking models by their beta e
245
interest for interpretation and visualization (Cosme &
246
& Przybylski, 2019; Simonsohn et al., 2015, 2020). B
247
preregistered, we did not conduct formal null hypothe
248
as continuous measures of evidence, we report the p
249
estimate of the same sign, as well as the proportion o
250
intervals excluding 0 in the same direction. In addition
251
specific choices, we submitted point estimates for pa
252
specifications to multiple regression models. From th
253
effects of each analysis decision point on the parame
254
p.30-31, sFigures 11-13, 41-43, & 55-57). 255
256
Multiverse amygdala reactivity analyses
257
For amygdala reactivity analyses, we examine
258
estimates to a variety of analytical decisions. In addit
259
pipeline, we preprocessed data using C-PAC softwar
260
C-PAC to take advantage of features supporting runn
261
example multiple nuisance regression forks using the
262
was used in C-PAC pipelines (see supplemental met
263
preprocessing, we examined the impact of different s
264
on age-related change in amygdala reactivity. We va
265
hemodynamic response function, nuisance regressor
266
ROI, exclusion criteria (exclude vs. include scans ana
267
model outlier treatment, and group-level model covar
268
methods p.14-17). Multiverse analyses of amygdala r
269
CC BY 4.0 Inte
available under a Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Running head: AMYGDALA—MPFC AGE-RELATED CHANGE specification curves by ranking models by their beta estimates (ascending) for parameters of
245
interest for interpretation and visualization (Cosme & Lopez, 2020; Klapwijk et al., 2019; Orben
246
& Przybylski, 2019; Simonsohn et al., 2015, 2020). Because specification choices were not
247
preregistered, we did not conduct formal null hypothesis testing of specification curves. Methods 242
Despite not being completely comprehensive, this approach still allowed for thorough
243
investigation into the robustness of results. For all multiverse analyses, we constructed
244 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE
specification curves by ranking models by their beta estimates (ascending) for parameters of
245
interest for interpretation and visualization (Cosme & Lopez, 2020; Klapwijk et al., 2019; Orben
246
& Przybylski, 2019; Simonsohn et al., 2015, 2020). Because specification choices were not
247
preregistered, we did not conduct formal null hypothesis testing of specification curves. Instead,
248
as continuous measures of evidence, we report the proportion of specifications resulting in an
249
estimate of the same sign, as well as the proportion of specifications resulting in 95% posterior
250
intervals excluding 0 in the same direction. In addition, to analyze in more detail the impact of
251
specific choices, we submitted point estimates for parameters of interest across all
252
specifications to multiple regression models. From these models, we examined the conditional
253
effects of each analysis decision point on the parameter of interest (see supplemental methods
254
p.30-31, sFigures 11-13, 41-43, & 55-57). 255
256
Multiverse amygdala reactivity analyses
257
For amygdala reactivity analyses, we examined the robustness of age-related change
258
estimates to a variety of analytical decisions. In addition to the preregistered FSL-based
259
pipeline, we preprocessed data using C-PAC software (v1.4.1; Craddock et al., 2013). We used
260
C-PAC to take advantage of features supporting running multiple pipeline ‘forks’ in parallel (for
261
example multiple nuisance regression forks using the same registration). No spatial smoothing
262
was used in C-PAC pipelines (see supplemental methods p.14). Following C-PAC and FSL
263
preprocessing, we examined the impact of different sets of commonly-used analysis methods
264
on age-related change in amygdala reactivity. Methods In addition, we
271
examined nonlinear age-related changes using quadratic and inverse age models (see sFigures
272
14-17) and ran a smaller set of analyses (sFigure 19) to ask whether we could differentiate
273
within-participant change over time from between-participant differences through alternative
274
model parametrization (see supplemental methods p.19). 275
For all specifications, individual-level amygdala reactivity estimates were submitted to a
276
group-level multilevel regression model for estimation of age-related changes. All models
277
allowed intercepts to vary by participant, and some specifications also allowed for varying
278
slopes (see supplemental methods p.15 for model syntax). All models also included a scan-level
279
covariate for head motion (mean framewise displacement [FD]; Power et al., 2012;
280
Satterthwaite et al., 2012, 2013). Consistent with prior work, head motion was higher on
281
average in younger children, and decreased with age (see sFigure 5), though head motion was
282
not associated with amygdala reactivity estimates for most specifications (see sFigure 26). Age-
283
related change findings examined for the preregistered pipeline also remained consistent under
284
more stringent exclusion thresholds based on mean framewise displacement (see sFigure 27). 285
Across preprocessing specifications, we also examined within-scan similarity of amygdala and
286
whole-brain voxelwise reactivity patterns (see sFigures 20-21) and between-scan correlations of
287
average amygdala reactivity estimates (sFigures 22-24). 288
289
Change in amygdala reactivity across trials
290
T
b
h
h
d l
i i
hibi
d
i hi
h
i
d
d
291 specifications (156 ways of defining participant-level amygdala reactivity x 18 group-level model
270
specifications) for each contrast. We analyzed all specifications in parallel. In addition, we
271
examined nonlinear age-related changes using quadratic and inverse age models (see sFigures
272
14-17) and ran a smaller set of analyses (sFigure 19) to ask whether we could differentiate
273
within-participant change over time from between-participant differences through alternative
274
model parametrization (see supplemental methods p.19). 275 specifications (156 ways of defining participant-level amygdala reactivity x 18 group-level model
270
specifications) for each contrast. We analyzed all specifications in parallel. In addition, we
271
examined nonlinear age-related changes using quadratic and inverse age models (see sFigures
272
14-17) and ran a smaller set of analyses (sFigure 19) to ask whether we could differentiate
273
within-participant change over time from between-participant differences through alternative
274
model parametrization (see supplemental methods p.19). Methods Instead,
248
as continuous measures of evidence, we report the proportion of specifications resulting in an
249
estimate of the same sign, as well as the proportion of specifications resulting in 95% posterior
250
intervals excluding 0 in the same direction. In addition, to analyze in more detail the impact of
251
specific choices, we submitted point estimates for parameters of interest across all
252
specifications to multiple regression models. From these models, we examined the conditional
253
effects of each analysis decision point on the parameter of interest (see supplemental methods
254
p.30-31, sFigures 11-13, 41-43, & 55-57). 255 For amygdala reactivity analyses, we examined the robustness of age-related change
258
estimates to a variety of analytical decisions. In addition to the preregistered FSL-based
259
pipeline, we preprocessed data using C-PAC software (v1.4.1; Craddock et al., 2013). We used
260
C-PAC to take advantage of features supporting running multiple pipeline ‘forks’ in parallel (for
261
example multiple nuisance regression forks using the same registration). No spatial smoothing
262
was used in C-PAC pipelines (see supplemental methods p.14). Following C-PAC and FSL
263
preprocessing, we examined the impact of different sets of commonly-used analysis methods
264
on age-related change in amygdala reactivity. We varied analysis choices of GLM software,
265
hemodynamic response function, nuisance regressors, first-level GLM estimates, amygdala
266
ROI, exclusion criteria (exclude vs. include scans analyzed by Gee et al., 2013), group-level
267
model outlier treatment, and group-level model covariates (see Table 2 & supplemental
268
methods p.14-17). Multiverse analyses of amygdala reactivity included a total of 2808 model
269 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE
specifications (156 ways of defining participant-level amygdala reactivity x 18 group-level model
270
specifications) for each contrast. We analyzed all specifications in parallel. Methods 275 For all specifications, individual-level amygdala reactivity estimates were submitted to a
276
group-level multilevel regression model for estimation of age-related changes. All models
277
allowed intercepts to vary by participant, and some specifications also allowed for varying
278
slopes (see supplemental methods p.15 for model syntax). All models also included a scan-level
279
covariate for head motion (mean framewise displacement [FD]; Power et al., 2012;
280
Satterthwaite et al., 2012, 2013). Consistent with prior work, head motion was higher on
281
average in younger children, and decreased with age (see sFigure 5), though head motion was
282
not associated with amygdala reactivity estimates for most specifications (see sFigure 26). Age-
283
related change findings examined for the preregistered pipeline also remained consistent under
284
more stringent exclusion thresholds based on mean framewise displacement (see sFigure 27). 285
Across preprocessing specifications, we also examined within-scan similarity of amygdala and
286
whole-brain voxelwise reactivity patterns (see sFigures 20-21) and between-scan correlations of
287
average amygdala reactivity estimates (sFigures 22-24). 288 To probe whether amygdala reactivity exhibited within-scan change in an age-dependent
291
manner, we modeled reactivity to each face trial using a Least Squares Separate method (LSS;
292
Abdulrahman & Henson, 2016). After preprocessing, we used FEAT to fit 48 separate GLMs
293
corresponding to each trial in each scan. A given trial was modeled with its own regressor and
294 . CC-BY 4.0 International license
available under a
which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE
the remaining 47 trials were modeled with a single regressor. Each GLM also included 24 head
295
motion nuisance regressors and had TRs with framewise displacement > .9mm downweighted
296
to 0. BOLD data were high-pass filtered at .01Hz before the GLM. Methods From each of the 48 GLMs,
297
we extracted the mean amygdala beta estimates corresponding to a contrast for each single
298
trial > baseline. 299
We constructed separate multiverse analyses using three different methods for
300
measuring change in amygdala reactivity across trials. For method 1 (slopes), we measured
301
rank-order correlations between trial number and trial-wise amygdala betas. For method 2 (trial
302
halves), we split trials into the first half (trials 1-12) and second half (trials 13-24), and modeled
303
age-related change in each half. For method 3 (single-trial models), we constructed larger
304
multilevel models with individual trials as the unit of observation. We conducted several analysis
305
specifications for each method (see Table 2 & supplemental methods p.21-23), and generated
306
corresponding specification curves. 307
308
Multiverse amygdala–mPFC functional connectivity (FC) analyses
309
We applied multiverse analysis techniques towards examining age-related changes in
310
amygdala–mPFC FC using gPPI and beta-series correlation (BSC) methods. Briefly, gPPI
311
estimates functional connectivity by constructing an interaction term between the timecourse in
312
a seed region of interest and a stimulus (task) regressor. Voxels whose activity is well fit by this
313
interaction term (a psychological-physiological interaction, or PPI) are assumed to be
314
“functionally coupled” with the seed region in a way that depends on the behavioral task
315
(McLaren et al., 2012; O’Reilly et al., 2012). BSC offers a different way of estimating functioning
316
connectivity, by constructing ‘timeseries’ of beta values (i.e., a beta series) in a condition of
317
interest for two regions of interest, and calculating the product-moment correlation between
318
those beta series. 319 Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Running head: AMYGDALA—MPFC AGE-RELATED CHANGE
the remaining 47 trials were modeled with a single regressor. Each GLM also included 24 head
295
motion nuisance regressors and had TRs with framewise displacement > .9mm downweighted
296
to 0. BOLD data were high-pass filtered at .01Hz before the GLM. From each of the 48 GLMs,
297
we extracted the mean amygdala beta estimates corresponding to a contrast for each single
298
trial > baseline. 299
We constructed separate multiverse analyses using three different methods for
300
measuring change in amygdala reactivity across trials. For method 1 (slopes), we measured
301
rank-order correlations between trial number and trial-wise amygdala betas. Methods For method 2 (trial
302
halves), we split trials into the first half (trials 1-12) and second half (trials 13-24), and modeled
303
age-related change in each half. For method 3 (single-trial models), we constructed larger
304
multilevel models with individual trials as the unit of observation. We conducted several analysis
305
specifications for each method (see Table 2 & supplemental methods p.21-23), and generated
306
corresponding specification curves. 307
308
Multiverse amygdala–mPFC functional connectivity (FC) analyses
309
We applied multiverse analysis techniques towards examining age-related changes in
310
amygdala–mPFC FC using gPPI and beta-series correlation (BSC) methods. Briefly, gPPI
311 the remaining 47 trials were modeled with a single regressor. Each GLM also included 24 head
295
motion nuisance regressors and had TRs with framewise displacement > .9mm downweighted
296
to 0. BOLD data were high-pass filtered at .01Hz before the GLM. From each of the 48 GLMs,
297
we extracted the mean amygdala beta estimates corresponding to a contrast for each single
298
trial > baseline. 299 We constructed separate multiverse analyses using three different methods for
300
measuring change in amygdala reactivity across trials. For method 1 (slopes), we measured
301
rank-order correlations between trial number and trial-wise amygdala betas. For method 2 (trial
302
halves), we split trials into the first half (trials 1-12) and second half (trials 13-24), and modeled
303
age-related change in each half. For method 3 (single-trial models), we constructed larger
304
multilevel models with individual trials as the unit of observation. We conducted several analysis
305
specifications for each method (see Table 2 & supplemental methods p.21-23), and generated
306
corresponding specification curves. 307 Multiverse amygdala–mPFC functional connectivity (FC) analyses
309 We applied multiverse analysis techniques towards examining age-related changes in
310
amygdala–mPFC FC using gPPI and beta-series correlation (BSC) methods. Briefly, gPPI
311
estimates functional connectivity by constructing an interaction term between the timecourse in
312
a seed region of interest and a stimulus (task) regressor. Voxels whose activity is well fit by this
313
interaction term (a psychological-physiological interaction, or PPI) are assumed to be
314
“functionally coupled” with the seed region in a way that depends on the behavioral task
315
(McLaren et al., 2012; O’Reilly et al., 2012). BSC offers a different way of estimating functioning
316
connectivity, by constructing ‘timeseries’ of beta values (i.e., a beta series) in a condition of
317
interest for two regions of interest, and calculating the product-moment correlation between
318
those beta series. 319 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Running head: AMYGDALA—MPFC AGE-RELATED CHANGE g
We constructed separate specification curves for age-related change in gPPI and BSC
320
for each contrast. Across gPPI specifications, we varied whether to use a deconvolution step in
321
creating interaction regressors (Di & Biswal, 2017; Gitelman et al., 2003), as well as several
322
other analysis decision points (see Table 2 & supplemental methods p.24-25). The
323
deconvolution step applies to the preprocessed BOLD data from the seed timecourse: these
324
data are first deconvolved to estimate the “underlying neural activity” that produced the BOLD
325
signal (Gitelman et al., 2003), then these deconvolved signals are multiplied with the task
326
regressor (e.g., for fear faces). Finally, this new interaction term is convolved with a
327
hemodynamic response to produce the BOLD functional connectivity regressor of interest. Multiverse amygdala–mPFC functional connectivity (FC) analyses
309 353 Age-related changes in gPPI and BSC were estimated using multilevel regression
354
models as described for the amygdala reactivity analyses. We focused primarily on linear age-
355
related change, but also examined quadratic and inverse age associations (see sFigures 45-48
356
& 58-61). We separately examined group mean gPPI and BSC for each contrast (see sFigures
357
38 & 52), as well as associations between mean framewise displacement and both FC
358
measures across specifications (see sFigures 49 & 62). Additionally, we examined mean
359
estimates and age-related change in ‘task-independent’ FC as measured by beta weight of the
360
‘physio’ term from the seed amygdala timeseries within the gPPI model (representing baseline
361
amygdala–mPFC functional connectivity controlling for task-induced variance; sFigures 50-51). 362
363
Multiverse analyses of associations between amygdala–mPFC circuitry and separation anxiety
364
behaviors. 365
We used further multiverse analyses to ask whether amygdala reactivity, change in
366
amygdala reactivity over the course of the task, or amygdala–mPFC FC were associated with
367
separation anxiety behaviors. Separate specification curves were created for each brain
368 Age-related changes in gPPI and BSC were estimated using multilevel regression
354
models as described for the amygdala reactivity analyses. We focused primarily on linear age-
355
related change, but also examined quadratic and inverse age associations (see sFigures 45-48
356
& 58-61). We separately examined group mean gPPI and BSC for each contrast (see sFigures
357
38 & 52), as well as associations between mean framewise displacement and both FC
358
measures across specifications (see sFigures 49 & 62). Additionally, we examined mean
359
estimates and age-related change in ‘task-independent’ FC as measured by beta weight of the
360
‘physio’ term from the seed amygdala timeseries within the gPPI model (representing baseline
361
amygdala–mPFC functional connectivity controlling for task-induced variance; sFigures 50-51). 362
363
Multiverse analyses of associations between amygdala–mPFC circuitry and separation anxiety
364
behaviors. 365
We used further multiverse analyses to ask whether amygdala reactivity, change in
366
amygdala reactivity over the course of the task, or amygdala–mPFC FC were associated with
367
separation anxiety behaviors. Separate specification curves were created for each brain
368 Age-related changes in gPPI and BSC were estimated using multilevel regression
354
models as described for the amygdala reactivity analyses. We focused primarily on linear age-
355
related change, but also examined quadratic and inverse age associations (see sFigures 45-48
356
& 58-61). Multiverse amygdala–mPFC functional connectivity (FC) analyses
309 Across BSC specifications we varied
347
analyses across several decision points (see Table 2 & supplemental methods p.26), including
348
whether to include a correction for global signal (post-hoc distribution centering(Fox et al.,
349
2009)). We extracted mean beta estimates for amygdala and mPFC ROIs for each trial, then
350
calculated product-moment correlations between the timeseries of betas across trials (neutral
351
and fear separately) for both regions (Di et al., 2020). These correlation coefficients were
352
transformed to z-scores, then submitted to group-level models. 353
Age-related changes in gPPI and BSC were estimated using multilevel regression
354
models as described for the amygdala reactivity analyses. We focused primarily on linear age-
355
related change, but also examined quadratic and inverse age associations (see sFigures 45-4
356
& 58-61). We separately examined group mean gPPI and BSC for each contrast (see sFigures
357
38 & 52), as well as associations between mean framewise displacement and both FC
358
measures across specifications (see sFigures 49 & 62). Additionally, we examined mean
359
estimates and age-related change in ‘task-independent’ FC as measured by beta weight of the
360
‘physio’ term from the seed amygdala timeseries within the gPPI model (representing baseline
361
amygdala–mPFC functional connectivity controlling for task-induced variance; sFigures 50-51)
362
363
Multiverse analyses of associations between amygdala–mPFC circuitry and separation anxiety
364
behaviors. 365
We used further multiverse analyses to ask whether amygdala reactivity, change in
366
amygdala reactivity over the course of the task, or amygdala–mPFC FC were associated with
367
separation anxiety behaviors Separate specification curves were created for each brain
368 areas within the ventromedial prefrontal cortex derived from Mackey & Petrides (Mackey &
344
Petrides, 2014). 345
For BSC analyses, we used beta estimates from the LSS GLMs described above for
346
analyses of within-scan change in amygdala reactivity. Across BSC specifications we varied
347
analyses across several decision points (see Table 2 & supplemental methods p.26), including
348
whether to include a correction for global signal (post-hoc distribution centering(Fox et al.,
349
2009)). We extracted mean beta estimates for amygdala and mPFC ROIs for each trial, then
350
calculated product-moment correlations between the timeseries of betas across trials (neutral
351
and fear separately) for both regions (Di et al., 2020). These correlation coefficients were
352
transformed to z-scores, then submitted to group-level models. Multiverse amygdala–mPFC functional connectivity (FC) analyses
309 328
Given recent work indicating that centering the task regressor before creation of the interaction
329
term can mitigate spurious effects (Di et al., 2017), we also compared pipelines in which we
330
centered the task regressor before deconvolution (pipelines including deconvolution in main
331
analyses did not include this step; see sFigure 44). 332 We preregistered constructing an mPFC ROI containing 120 voxels centered at the peak
333
coordinates reported by Gee at al. (2013) for age-related change in fear > baseline gPPI
334
(Talairach 2,32,8; or MNI 3,35,8). However, after preregistration we discovered that these peak
335
coordinates were not at the center of the ROI reported by Gee at al. (2013), and were quite
336
close to the corpus callosum. The 120-voxel ROI we created that was centered at this peak
337
coordinate would have contained a high proportion of white matter voxels relative to cortical
338
voxels (though this was not true for the mPFC ROI identified by Gee et al. (2013). To address
339
this issue, we instead constructed three spherical ROIs with 5mm radii; the first centered at the
340
above peak coordinates, the second shifted slightly anterior, and the third shifted slightly ventral
341
relative to the second (see Figure 4). Lastly, to examine amygdala functional connectivity with a
342
more broadly-defined mPFC, we also used a ‘large vmPFC’ mask encompassing many of the
343 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
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doi:
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available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
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doi:
bioRxiv preprint Running head: AMYGDALA
MPFC AGE RELATED CHANGE
areas within the ventromedial prefrontal cortex derived from Mackey & Petrides (Mackey &
344
Petrides, 2014). 345
For BSC analyses, we used beta estimates from the LSS GLMs described above for
346
analyses of within-scan change in amygdala reactivity. Multiverse amygdala–mPFC functional connectivity (FC) analyses
309 We separately examined group mean gPPI and BSC for each contrast (see sFigures
357
38 & 52), as well as associations between mean framewise displacement and both FC
358
measures across specifications (see sFigures 49 & 62). Additionally, we examined mean
359
estimates and age-related change in ‘task-independent’ FC as measured by beta weight of the
360
‘physio’ term from the seed amygdala timeseries within the gPPI model (representing baseline
361
amygdala–mPFC functional connectivity controlling for task-induced variance; sFigures 50-51). 362
363
M lti
l
f
i ti
b t
d l
PFC i
it
d
ti
i t
364 We used further multiverse analyses to ask whether amygdala reactivity, change in
366
amygdala reactivity over the course of the task, or amygdala–mPFC FC were associated with
367
separation anxiety behaviors. Separate specification curves were created for each brain
368 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
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doi:
bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Running head: AMYGDALA—MPFC AGE-RELATED CHANGE
measure type (amygdala reactivity, amygdala reactivity change across trials, amygdala–mPFC
369
FC). All analyses used multilevel regression models with covariates for age, and specification
370
curves included both RCADS-P and SCARED-P separation anxiety subscales as outcomes
371
(see Table 2 & supplemental methods p.29-30). Because we did not have parent-reported
372
RCADS-P or SCARED-P scores for 10 adult participants, these analyses had an N=173. Multiverse amygdala–mPFC functional connectivity (FC) analyses
309 373
374
Reliability analyses
375
To better understand the proportion of variance in each measure explained by the
376
grouping of observations within repeated measurements of the same participants over time, we
377
computed Bayesian intraclass correlation (ICC) estimates through variance decomposition of
378
the posterior predictive distributions of the multilevel regression models previously described. 379
We implemented these through the ‘performance’ R package (Lüdecke et al., 2021; Nakagawa
380
et al., 2017). Negative ICC estimates under this method are possible, and indicate that the
381
posterior predictive distribution has higher variance when not conditioning on random effects
382
than when conditioning on them (likely indicating the posterior predictive variance is large, and
383
random effects explain very little of this variance). 384
385
Model-fitting
386
All statistical models fit at the group level were run in the R (v 3.6.1) computing
387 measure type (amygdala reactivity, amygdala reactivity change across trials, amygdala–mPFC
369
FC). All analyses used multilevel regression models with covariates for age, and specification
370
curves included both RCADS-P and SCARED-P separation anxiety subscales as outcomes
371
(see Table 2 & supplemental methods p.29-30). Because we did not have parent-reported
372
RCADS-P or SCARED-P scores for 10 adult participants, these analyses had an N=173. 373 Reliability analyses
375
To better understand the proportion of variance in each measure explained by the
376
grouping of observations within repeated measurements of the same participants over time, we
377
computed Bayesian intraclass correlation (ICC) estimates through variance decomposition of
378
the posterior predictive distributions of the multilevel regression models previously described. 379
We implemented these through the ‘performance’ R package (Lüdecke et al., 2021; Nakagawa
380
et al., 2017). Negative ICC estimates under this method are possible, and indicate that the
381
posterior predictive distribution has higher variance when not conditioning on random effects
382
than when conditioning on them (likely indicating the posterior predictive variance is large, and
383
random effects explain very little of this variance). 384
385
Model-fitting
386
All statistical models fit at the group level were run in the R (v 3.6.1) computing
387
environment. In order to most accurately model age-related changes in each of our measures,
388
we attempted to take into account both between-participants information and repeated
389
measurements within participants over time. Multiverse amygdala–mPFC functional connectivity (FC) analyses
309 Unless otherwise indicated, models were estimated
390 To better understand the proportion of variance in each measure explained by the
376
grouping of observations within repeated measurements of the same participants over time, we
377
computed Bayesian intraclass correlation (ICC) estimates through variance decomposition of
378
the posterior predictive distributions of the multilevel regression models previously described. 379
We implemented these through the ‘performance’ R package (Lüdecke et al., 2021; Nakagawa
380
et al., 2017). Negative ICC estimates under this method are possible, and indicate that the
381
posterior predictive distribution has higher variance when not conditioning on random effects
382
than when conditioning on them (likely indicating the posterior predictive variance is large, and
383
random effects explain very little of this variance). 384 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE using Hamiltonian Markov chain Monte Carlo sampling as implemented in the Stan
391
programming language through the brms package in R (Bürkner, 2019; Gelman et al., 2015). 392
Unless otherwise indicated, all models used package default weakly-informative priors (student-
393
t distributions with mean 0, scale parameter of 10 standardized units, and 3 degrees of freedom
394
for all fixed effects), and were run with 4 chains of 2000 sampling iterations (1000 warmup) each
395
(see supplemental methods p.18-19 and p.30 for syntax). 396
397
Interactive visualizations
398
Because static plots visualizing the model predictions for all models in each multiverse
399
would require far more page space than available, we created web-based interactive
400
visualization tools for exploring different model specifications and viewing the corresponding raw
401
(participant-level) data and fitted model predictions using R and Shiny (Beeley, 2013). Multiverse amygdala–mPFC functional connectivity (FC) analyses
309 These
402
visualizations can be found at https://pbloom.shinyapps.io/amygdala_mpfc_multiverse/
403
404
Deviations from preregistration
405
Although we largely completed the preregistered analyses, the current study includes
406
many analyses beyond those proposed in the initial preregistration. Because the additional
407
analyses (i.e., all multiverses) conducted here give us substantial analytical flexibility over that
408
initially indicated by preregistration, we consider all results here to be at least in part exploratory
409
(rather than completely confirmatory), despite the preregistered hypotheses. Additionally, we
410
note that BSC analyses, analyses of change in amygdala reactivity across trials, and analyses
411
of associations between all brain measures and separation anxiety were exploratory, and
412
conducted after we had seen the results of the preregistered reactivity and gPPI analyses. In
413
addition, to avoid possible selection bias introduced by the analytical flexibility inherent in
414
running many parallel analyses, we consider all analysis specifications simultaneously,
415 using Hamiltonian Markov chain Monte Carlo sampling as implemented in the Stan Thus, over half
430
of models indicated that on average, increases in age were associated with decreases in
431
amygdala reactivity to fear faces > baseline. Because the timepoint 1 data in the current study
432
included the 42 scans used by Gee et al. (2013) to age-related changes in amygdala—mPFC
433
circuitry for the fear > baseline contrast, results including these scans may have been more
434
likely to find similar change (particularly for fear > baseline, see sFigures 11-13, & 25). 435
Estimated age-related change was on average weaker, though still largely negative (98.1%
436
negative, 25.3% of posterior intervals excluding 0) when 42 previously analyzed scans (ages 4-
437
17 years) were excluded to provide stricter independence from previously analyzed data (see
438
. CC-BY 4.0 International license
available under a emphasizing that without further methodological work, we consider all such choices in tandem
416
as providing equal evidential value. While reliability analyses were not preregistered, they too
417
provide key information for interpreting the current analyses. 418
419
Results
420
421
Age-related change in amygdala reactivity
422
We used multilevel regression models and specification curve analyses to examine age-
423
related changes in amygdala reactivity to faces in an accelerated longitudinal sample ranging
424
from ages 4-22 years (Figure 2). Across specifications, we found relatively consistent evidence
425
for negative age-related change in anatomically-defined (Harvard-Oxford atlas and Freesurfer-
426
defined) amygdala reactivity to fear faces > baseline, such that the vast majority of analysis
427
specifications (99.6%) estimated linear slopes at the group level that were negative in sign, and
428
the majority (60.0%) of 95% posterior intervals about these slopes excluded 0 (Figure 2A;
429
interactive version at https://pbloom.shinyapps.io/amygdala_mpfc_multiverse/). Thus, over half
430
of models indicated that on average, increases in age were associated with decreases in
431
amygdala reactivity to fear faces > baseline. Because the timepoint 1 data in the current study
432
included the 42 scans used by Gee et al. (2013) to age-related changes in amygdala—mPFC
433
circuitry for the fear > baseline contrast, results including these scans may have been more
434
likely to find similar change (particularly for fear > baseline, see sFigures 11-13, & 25). using Hamiltonian Markov chain Monte Carlo sampling as implemented in the Stan Although we largely completed the preregistered analyses, the current study includes
406
many analyses beyond those proposed in the initial preregistration. Because the additional
407
analyses (i.e., all multiverses) conducted here give us substantial analytical flexibility over that
408
initially indicated by preregistration, we consider all results here to be at least in part exploratory
409
(rather than completely confirmatory), despite the preregistered hypotheses. Additionally, we
410
note that BSC analyses, analyses of change in amygdala reactivity across trials, and analyses
411
of associations between all brain measures and separation anxiety were exploratory, and
412
conducted after we had seen the results of the preregistered reactivity and gPPI analyses. In
413
addition, to avoid possible selection bias introduced by the analytical flexibility inherent in
414
running many parallel analyses, we consider all analysis specifications simultaneously,
415 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE
emphasizing that without further methodological work, we consider all such choices in tandem
416
as providing equal evidential value. While reliability analyses were not preregistered, they too
417
provide key information for interpreting the current analyses. 418
419
Results
420
421
Age-related change in amygdala reactivity
422
We used multilevel regression models and specification curve analyses to examine age-
423
related changes in amygdala reactivity to faces in an accelerated longitudinal sample ranging
424
from ages 4-22 years (Figure 2). Across specifications, we found relatively consistent evidence
425
for negative age-related change in anatomically-defined (Harvard-Oxford atlas and Freesurfer-
426
defined) amygdala reactivity to fear faces > baseline, such that the vast majority of analysis
427
specifications (99.6%) estimated linear slopes at the group level that were negative in sign, and
428
the majority (60.0%) of 95% posterior intervals about these slopes excluded 0 (Figure 2A;
429
interactive version at https://pbloom.shinyapps.io/amygdala_mpfc_multiverse/). using Hamiltonian Markov chain Monte Carlo sampling as implemented in the Stan 442
Parallel multiverse analyses found similarly consistent age-related decreases in neutral
443
faces > baseline amygdala reactivity (see Figure 2C for an example pipeline & sFigure 9 for
444
specification curve), but no consistent evidence for age-related change for the fear > neutral
445
contrast (see sFigure 10). However, there was consistent evidence for higher reactivity for fear
446
faces > neutral on average as well as each emotion compared to baseline (sFigures 6-8),
447
indicating that while the amygdala responses were robust and generally stronger for fear faces
448
compared to neutral, such fear > neutral differences did not change with age. Across contrasts,
449
varying the inclusion of block order or scanner covariates, inclusion of random intercepts, and
450
use of robust regression models had little impact on age-related change estimates (see Figure
451
2B sFigures 6-8). 452
While group-level estimates of average age-related change were relatively consistent
453
across specifications, the estimated age terms in these models could be influenced by both
454
within-participant change and between-participant differences (King et al., 2018; Madhyastha et
455
al., 2018). A smaller separate specification curve indicated that when models were parametrized
456
to differentiate within-participant change and between-participant differences, average within-
457
participant change was not consistent across specifications and could not be estimated with
458
precision (Figure 2D). In contrast, estimates of between-participant differences largely indicated
459
negative age-related change in concurrence with our initial model parametrization. At the same
460
time, within-participant versus between-participant terms were not reliably different from one
461
another, indicating that models could not distinguish them despite higher precision for
462
estimating between-participant differences (see sFigure 19). We did not find consistent
463
evidence for quadratic age-related changes in amygdala reactivity (see sFigures 14-17). Inverse
464
age models (i.e. amygdala reactivity modeled as a function of 1/age) indicated results similar to
465 amygdala, and when using t-stats extracted from scan-level GLMs rather than beta estimates
441
for group-level models (see sFigure 11). 442 indicating that while the amygdala responses were robust and generally stronger for fear faces
448
compared to neutral, such fear > neutral differences did not change with age. Across contrasts,
449
varying the inclusion of block order or scanner covariates, inclusion of random intercepts, and
450
use of robust regression models had little impact on age-related change estimates (see Figure
451
2B sFigures 6-8). using Hamiltonian Markov chain Monte Carlo sampling as implemented in the Stan 435
Estimated age-related change was on average weaker, though still largely negative (98.1%
436
negative, 25.3% of posterior intervals excluding 0) when 42 previously analyzed scans (ages 4-
437
17 years) were excluded to provide stricter independence from previously analyzed data (see
438 Age-related change in amygdala reactivity
422
We used multilevel regression models and specification curve analyses to examine age-
423
related changes in amygdala reactivity to faces in an accelerated longitudinal sample ranging
424
from ages 4-22 years (Figure 2). Across specifications, we found relatively consistent evidence
425
for negative age-related change in anatomically-defined (Harvard-Oxford atlas and Freesurfer-
426
defined) amygdala reactivity to fear faces > baseline, such that the vast majority of analysis
427
specifications (99.6%) estimated linear slopes at the group level that were negative in sign, and
428
the majority (60.0%) of 95% posterior intervals about these slopes excluded 0 (Figure 2A;
429
interactive version at https://pbloom.shinyapps.io/amygdala_mpfc_multiverse/). Thus, over half
430
of models indicated that on average, increases in age were associated with decreases in
431
amygdala reactivity to fear faces > baseline. Because the timepoint 1 data in the current study
432
included the 42 scans used by Gee et al. (2013) to age-related changes in amygdala—mPFC
433
circuitry for the fear > baseline contrast, results including these scans may have been more
434
likely to find similar change (particularly for fear > baseline, see sFigures 11-13, & 25). 435 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Running head: AMYGDALA—MPFC AGE-RELATED CHANGE
amygdala, and when using t-stats extracted from scan-level GLMs rather than beta estimates
441
for group-level models (see sFigure 11). using Hamiltonian Markov chain Monte Carlo sampling as implemented in the Stan 452 While group-level estimates of average age-related change were relatively consistent
453
across specifications, the estimated age terms in these models could be influenced by both
454
within-participant change and between-participant differences (King et al., 2018; Madhyastha et
455
al., 2018). A smaller separate specification curve indicated that when models were parametrized
456
to differentiate within-participant change and between-participant differences, average within-
457
participant change was not consistent across specifications and could not be estimated with
458
precision (Figure 2D). In contrast, estimates of between-participant differences largely indicated
459
negative age-related change in concurrence with our initial model parametrization. At the same
460
time, within-participant versus between-participant terms were not reliably different from one
461
another, indicating that models could not distinguish them despite higher precision for
462
estimating between-participant differences (see sFigure 19). We did not find consistent
463
evidence for quadratic age-related changes in amygdala reactivity (see sFigures 14-17). Inverse
464
age models (i.e. amygdala reactivity modeled as a function of 1/age) indicated results similar to
465 Running head: AMYGDALA—MPFC AGE-RELATED CHANGE
those of linear and quadratic models with most specifications for the fear > baseline and neutral
466
> baseline (though less consistent) contrasts indicating age-related decreases (see sFigure 18). 467
468
469
____________________________
470
Insert Figure 2 about here
471
____________________________
472
473
Age-related differences in within-scan amygdala reactivity change
474
To ask whether age-related changes in amygdala reactivity could be due to
475
developmental changes in patterns of amygdala reactivity across face trials (within a run), we
476
examined whether within-scan change in amygdala reactivity varied with age (see Table 1 Aim
477
1b). Analyses included 42 specifications (3 amygdala regions of interest [ROIs] x 2 global signal
478
correction options x 7 group-level models). Across both fear and neutral trials, linear slopes of
479
amygdala reactivity were negative on average, indicating higher amygdala reactivity at the
480
beginning of the run (Figure 3A, sFigure 30). Across specifications, for both fear (100% of
481
estimates had the same sign, 95.2% of posterior intervals excluding 0 in the same direction) and
482
neutral trials (100% of estimates in the same direction, 38.1% of posterior intervals excluding 0),
483
there was evidence that these within-scan slopes were steeper (i.e., more negative) at younger
484
ages, though evidence was relatively weaker for neutral trials (Figure 3D-E). using Hamiltonian Markov chain Monte Carlo sampling as implemented in the Stan Specifications with
485
a global signal subtraction step also tended to find stronger age-related change. 486
Similarly, when splitting trials into the first half (trials 1-12) versus second half (trials 13-
487
24), there was consistent evidence (100% of estimates had the same sign, 69.2% with posterior
488
interval excluding 0) for an interaction between age and trial half, such that average reactivity to
489
. CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE
those of linear and quadratic models with most specifications for the fear > bas
466
> baseline (though less consistent) contrasts indicating age-related decreases
467
468
469
____________________________
470
Insert Figure 2 about here
471
____________________________
472
473
Age-related differences in within-scan amygdala reactivity change
474
To ask whether age-related changes in amygdala reactivity could be du
475
developmental changes in patterns of amygdala reactivity across face trials (w
476
examined whether within-scan change in amygdala reactivity varied with age (
477
1b). Analyses included 42 specifications (3 amygdala regions of interest [ROIs
478
correction options x 7 group-level models). Across both fear and neutral trials,
479
amygdala reactivity were negative on average, indicating higher amygdala rea
480
beginning of the run (Figure 3A, sFigure 30). using Hamiltonian Markov chain Monte Carlo sampling as implemented in the Stan It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE
average reactivity during the second half of trials (see Figure 3B, sFigure 32). This interaction
491
was in the same direction for neutral trials across most specifications (88.5% of estimates), but
492
was typically not as strong (3.8% of posterior intervals excluding 0). Single-trial models
493
indicated similar age-related change in within-scan amygdala dynamics (see Figure 3C,
494
sFigures 33-34). Mean group-level amygdala reactivity was higher for the first half of trials for
495
fear faces > baseline across several specifications, though there were not consistent differences
496
between trial halves for mean amygdala reactivity to neutral faces (sFigure 31). 497
498
____________________________
499
Insert Figure 3 about here
500
____________________________
501
502
Age-related change in task-evoked amygdala–mPFC functional connectivity
503
We used multilevel regression modelling and specification curve analyses to examine
504
age-related change in task-evoked amygdala–mPFC functional connectivity within the
505
accelerated longitudinal cohort (see Table 1 Aims 2a-b). For the fear > baseline contrast, a
506
specification curve with 288 total specifications (4 definitions of participant-level gPPI estimates
507
x 4 mPFC ROIs x 18 group-level models) of amygdala–mPFC gPPI did not find consistent
508
evidence of age-related change: while 59.0% of models found point estimates in the positive
509
direction, only 23% of posterior intervals excluded 0 (Figure 4C-D, interactive version at
510
https://pbloom.shinyapps.io/amygdala_mpfc_multiverse/). Specification curve analyses found
511
that the sign of the estimated age-related change depended almost entirely on deconvolution,
512
such that most specifications including a deconvolution step resulted in negative age-related
513
change estimates never distinguishable from 0 (78.5% of point estimates negative, 0% of
514
posterior intervals excluding 0), and most specifications not including a deconvolution step
515
. using Hamiltonian Markov chain Monte Carlo sampling as implemented in the Stan Across specifications, for both fea
481
estimates had the same sign, 95.2% of posterior intervals excluding 0 in the sa
482
neutral trials (100% of estimates in the same direction, 38.1% of posterior inter
483
there was evidence that these within-scan slopes were steeper (i.e., more neg
484
ages, though evidence was relatively weaker for neutral trials (Figure 3D-E). S
485
a global signal subtraction step also tended to find stronger age-related chang
486
Similarly, when splitting trials into the first half (trials 1-12) versus secon
487
24), there was consistent evidence (100% of estimates had the same sign, 69. 488 Age-related differences in within-scan amygdala reactivity change
474
To ask whether age-related changes in amygdala reactivity could be due to
475
developmental changes in patterns of amygdala reactivity across face trials (within a run), we
476
examined whether within-scan change in amygdala reactivity varied with age (see Table 1 Aim
477
1b). Analyses included 42 specifications (3 amygdala regions of interest [ROIs] x 2 global signal
478
correction options x 7 group-level models). Across both fear and neutral trials, linear slopes of
479
amygdala reactivity were negative on average, indicating higher amygdala reactivity at the
480
beginning of the run (Figure 3A, sFigure 30). Across specifications, for both fear (100% of
481
estimates had the same sign, 95.2% of posterior intervals excluding 0 in the same direction) and
482
neutral trials (100% of estimates in the same direction, 38.1% of posterior intervals excluding 0),
483
there was evidence that these within-scan slopes were steeper (i.e., more negative) at younger
484
ages, though evidence was relatively weaker for neutral trials (Figure 3D-E). Specifications with
485
a global signal subtraction step also tended to find stronger age-related change. 486 Similarly, when splitting trials into the first half (trials 1-12) versus second half (trials 13-
487
24), there was consistent evidence (100% of estimates had the same sign, 69.2% with posterior
488
interval excluding 0) for an interaction between age and trial half, such that average reactivity to
489
fear faces > baseline in the first half of trials decreased as a function of age more so than did
490 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. using Hamiltonian Markov chain Monte Carlo sampling as implemented in the Stan CC BY 4.0 International license
available under a Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Insert Figure 3 about here Age-related change in task-evoked amygdala–mPFC functional connectivity
503
We used multilevel regression modelling and specification curve analyses to examine
504
age-related change in task-evoked amygdala–mPFC functional connectivity within the
505
accelerated longitudinal cohort (see Table 1 Aims 2a-b). For the fear > baseline contrast, a
506
specification curve with 288 total specifications (4 definitions of participant-level gPPI estimates
507
x 4 mPFC ROIs x 18 group-level models) of amygdala–mPFC gPPI did not find consistent
508
evidence of age-related change: while 59.0% of models found point estimates in the positive
509
direction, only 23% of posterior intervals excluded 0 (Figure 4C-D, interactive version at
510
https://pbloom.shinyapps.io/amygdala_mpfc_multiverse/). Specification curve analyses found
511
that the sign of the estimated age-related change depended almost entirely on deconvolution,
512
such that most specifications including a deconvolution step resulted in negative age-related
513
change estimates never distinguishable from 0 (78.5% of point estimates negative, 0% of
514
posterior intervals excluding 0), and most specifications not including a deconvolution step
515 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
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bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Running head: AMYGDALA—MPFC AGE-RELATED CHANGE resulted in positive age-related change estimates (96.5% of point estimates positive, 47.9% of
516
posterior intervals excluding 0). A visualization of the effects of the deconvolution step on
517
amygdala FC with each of four mPFC ROIs is presented in Figure 4B. While mPFC ROI
518
definition and other analysis decision points also influenced estimates of age-related change in
519
gPPI (Figure 4D), follow-up regression models indicated that the effect of including the
520
deconvolution step was several times larger for the fear > baseline contrast (see sFigures 41-
521
43). using Hamiltonian Markov chain Monte Carlo sampling as implemented in the Stan (Gee et al., 2013), exclusion of these scans had little
529
impact on age-related change results (see Figure 4D). 530 Insert Figure 4 about here using Hamiltonian Markov chain Monte Carlo sampling as implemented in the Stan 522
Through equivalent multiverse analyses we also found no evidence of consistent linear
523
age-related change in amygdala–mPFC gPPI for the neutral > baseline and fear > neutral
524
contrasts (see sFigures 39-40), or nonlinear change for any contrast (see sFigures 45-48). In
525
addition, we did not see consistent evidence for group average amygdala–mPFC gPPI for any
526
contrast, though such results often differed as a function of whether a deconvolution step was
527
included (see sFigure 38). Though we included gPPI analysis specifications excluding the 42
528
scans at timepoint 1 studied by Gee et al. (Gee et al., 2013), exclusion of these scans had little
529
impact on age-related change results (see Figure 4D). 530
531
____________________________
532
Insert Figure 4 about here
533
____________________________
534
535
In addition to gPPI analyses, we used beta series correlation (BSC) analyses to examine
536
age-related changes in task-evoked amygdala–mPFC connectivity (see Table 1 Aim 2b). As
537
with gPPI, multiverse analyses of amygdala–mPFC BSC (168 total specifications; 3 amygdala
538
ROI definitions x 4 mPFC ROI definitions x 2 global signal options x 7 group-level models) for
539
fear trials (vs baseline) did not yield strong evidence of age-related change across pipelines
540 resulted in positive age-related change estimates (96.5% of point estimates positive, 47.9% of
516
posterior intervals excluding 0). A visualization of the effects of the deconvolution step on
517
amygdala FC with each of four mPFC ROIs is presented in Figure 4B. While mPFC ROI
518
definition and other analysis decision points also influenced estimates of age-related change in
519
gPPI (Figure 4D), follow-up regression models indicated that the effect of including the
520
deconvolution step was several times larger for the fear > baseline contrast (see sFigures 41-
521
43). 522 Through equivalent multiverse analyses we also found no evidence of consistent linear
523
age-related change in amygdala–mPFC gPPI for the neutral > baseline and fear > neutral
524
contrasts (see sFigures 39-40), or nonlinear change for any contrast (see sFigures 45-48). In
525
addition, we did not see consistent evidence for group average amygdala–mPFC gPPI for any
526
contrast, though such results often differed as a function of whether a deconvolution step was
527
included (see sFigure 38). Though we included gPPI analysis specifications excluding the 42
528
scans at timepoint 1 studied by Gee et al. Insert Figure 5 about here
5 Insert Figure 4 about here In addition to gPPI analyses, we used beta series correlation (BSC) analyses to examine
536
age-related changes in task-evoked amygdala–mPFC connectivity (see Table 1 Aim 2b). As
537
with gPPI, multiverse analyses of amygdala–mPFC BSC (168 total specifications; 3 amygdala
538
ROI definitions x 4 mPFC ROI definitions x 2 global signal options x 7 group-level models) for
539
fear trials (vs baseline) did not yield strong evidence of age-related change across pipelines
540 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE (84.5% of point estimates in the same direction, 24.4% of posterior intervals excluding 0; Figure
541
5A, interactive version at https://pbloom.shinyapps.io/amygdala_mpfc_multiverse). Unlike gPPI
542
analyses, however, choice of mPFC ROI (as well as amygdala ROI, though this was not
543
examined for gPPI) most impacted age-related change in BSC estimates, rather than
544
preprocessing or modeling analytical choices (Figure 5B, sFigures 55-57). Accordingly, while
545
global signal subtraction resulted in weaker amygdala–mPFC BSC on average (see sFigure
546
52), inclusion of this step did not consistently affect age-related change estimates (Figure 4C). 547
We did not find consistent evidence for age-related change in amygdala–mPFC BSC for neutral
548
trials (vs baseline), or for fear > neutral trials (sFigures 53-54). We did not find consistent
549
evidence for nonlinear age-related change for any contrast (sFigures 58-61). 550 examined for gPPI) most impacted age-related change in BSC estimates, rather than
544
preprocessing or modeling analytical choices (Figure 5B, sFigures 55-57). Accordingly, while
545
global signal subtraction resulted in weaker amygdala–mPFC BSC on average (see sFigure
546
52), inclusion of this step did not consistently affect age-related change estimates (Figure 4C). 547
We did not find consistent evidence for age-related change in amygdala–mPFC BSC for neutral
548
trials (vs baseline), or for fear > neutral trials (sFigures 53-54). Insert Figure 4 about here CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
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doi:
bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE
____________________________
566
567
568
Amygdala–mPFC Measures & Separation Anxiety
569
We conducted multiverse analyses of associations between several amygdala–mPFC
570
measures (amygdala reactivity, amygdala–mPFC FC, within-scan changes in amygdala
571
reactivity) and separation anxiety behaviors (see Table 1 Aim 3). Separation anxiety behaviors
572
on average decreased with age, as indicated by the RCADS-P and SCARED-P raw scores
573
(Figure 6A-C). Neither specification curves for amygdala reactivity (18 total specifications, 56%
574
of point estimates in the same direction as median estimate, 0% of posterior intervals excluding
575
0), amygdala–mPFC gPPI FC (90 total specifications, 72% of point estimates in the same
576
direction as median estimate, 1% of posterior intervals excluding 0), amygdala–mPFC BSC FC
577
(18 total specifications, 83% of point estimates in the same direction as median estimate, 0% of
578
posterior intervals excluding 0), nor slope of amygdala responses across trials (12 total
579
specifications, 75% of point estimates in the same direction as median estimate, 17% of
580
posterior intervals excluding 0), found consistent evidence for associations between brain
581
measures and separation anxiety. Similar specification curves found little consistent evidence
582
for associations between brain measures and generalized anxiety, social anxiety, or total
583
anxiety behaviors (see sFigure 67). All specifications controlled for age (see supplemental
584
methods p.30). 585
T
ifi
ll f ll
i
k
ti
i ti
b t
586 Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Amygdala–mPFC Measures & Separation Anxiety
569
We conducted multiverse analyses of associations between several amygdala–mPFC
570
measures (amygdala reactivity, amygdala–mPFC FC, within-scan changes in amygdala
571
reactivity) and separation anxiety behaviors (see Table 1 Aim 3). Separation anxiety behaviors
572
on average decreased with age, as indicated by the RCADS-P and SCARED-P raw scores
573
(Figure 6A-C). .
CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022.
;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint Insert Figure 4 about here We did not find consistent
549
evidence for nonlinear age-related change for any contrast (sFigures 58-61). 550
Additionally, we constructed a correlation matrix using rank-order correlations of scan-
551
level BSC and gPPI estimates for the fear (vs baseline) condition. Across scans, there was little
552
evidence of correspondence between BSC and gPPI metrics for amygdala–mPFC connectivity
553
(Figure 5D, sFigures 63-66). Further, FC estimates tended to be positively correlated within a
554
method type (BSC, gPPI) across mPFC ROIs, though less strongly for gPPI estimates with
555
versus without a deconvolution step. 556
In addition to gPPI and BSC methods for functional connectivity, we also explored
557
between-scan associations between amygdala reactivity and mPFC reactivity (sFigures 28-29). 558
Multilevel models indicated that amygdala reactivity for fear faces > baseline was positively
559
associated with mPFC reactivity for fear faces > baseline for all mPFC ROIs, though we did not
560
find consistent evidence for age-related changes in associations between amygdala and mPFC
561
reactivity to fear faces > baseline (see sFigure 29). 562
563
____________________________
564
Insert Figure 5 about here
565 Additionally, we constructed a correlation matrix using rank-order correlations of scan-
551
level BSC and gPPI estimates for the fear (vs baseline) condition. Across scans, there was little
552
evidence of correspondence between BSC and gPPI metrics for amygdala–mPFC connectivity
553
(Figure 5D, sFigures 63-66). Further, FC estimates tended to be positively correlated within a
554
method type (BSC, gPPI) across mPFC ROIs, though less strongly for gPPI estimates with
555
versus without a deconvolution step. 556 In addition to gPPI and BSC methods for functional connectivity, we also explored
557
between-scan associations between amygdala reactivity and mPFC reactivity (sFigures 28-29). 558
Multilevel models indicated that amygdala reactivity for fear faces > baseline was positively
559
associated with mPFC reactivity for fear faces > baseline for all mPFC ROIs, though we did not
560
find consistent evidence for age-related changes in associations between amygdala and mPFC
561
reactivity to fear faces > baseline (see sFigure 29). 562 Insert Figure 5 about here
565 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint . Insert Figure 4 about here Neither specification curves for amygdala reactivity (18 total specifications, 56%
574
of point estimates in the same direction as median estimate, 0% of posterior intervals excluding
575
0), amygdala–mPFC gPPI FC (90 total specifications, 72% of point estimates in the same
576
direction as median estimate, 1% of posterior intervals excluding 0), amygdala–mPFC BSC FC
577
(18 total specifications, 83% of point estimates in the same direction as median estimate, 0% of
578
posterior intervals excluding 0), nor slope of amygdala responses across trials (12 total
579
specifications, 75% of point estimates in the same direction as median estimate, 17% of
580
posterior intervals excluding 0), found consistent evidence for associations between brain
581
measures and separation anxiety. Similar specification curves found little consistent evidence
582
for associations between brain measures and generalized anxiety, social anxiety, or total
583
anxiety behaviors (see sFigure 67). All specifications controlled for age (see supplemental
584
methods p.30). 585 To more specifically follow up on previous work reporting associations between
586
separation anxiety behaviors and amygdala–mPFC gPPI for fear > baseline specifically (Gee et
587
al., 2013), we plotted model predictions for such models from the above multiverse analysis for
588
each of the four mPFC ROIs, across all three separation anxiety outcome measures, and both
589
with and without a deconvolution step (Figure 6E). We did not find consistent evidence for
590 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
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doi:
bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE associations with separation anxiety, and results showed high sensitivity to the deconvolution
591
step, mPFC ROI, and outcome measure used. 592
____________________________
593
Insert Figure 6 about here
594
____________________________
595
Reliability:
596
597
To examine test-retest reliability estimates of amygdala—mPFC measures across
598
longitudinal visits, we computed Bayesian ICC estimates using a variance decomposition
599
method (Lüdecke et al., 2021). Because such models can accommodate missing data, all
600
observations (98 participants, 183 total scans) were used, including participants with only 1 visit. 601
All amygdala reactivity (Figure 7A) and amygdala—mPFC functional connectivity (Figure 7C)
602
measures, as well as slopes of amygdala reactivity estimates across trials (Figure 7B),
603
demonstrated poor reliability (ICC < 0.4; Cicchetti & Sparrow, 1981; Elliott et al., 2020). 604
Separation anxiety measures demonstrated somewhat higher, though still largely poor reliability
605
(point estimates ~0.4, 95% CIs included values below 0.4; Figure 7D). Head motion in the
606
scanner (mean framewise displacement) showed the highest reliability (ICC = 0.52, 95% CI
607
[0.29, 0.68]),
608
609 associations with separation anxiety, and results showed high sensitivity to the deconvolution
591
step, mPFC ROI, and outcome measure used. 592 Insert Figure 6 about here Insert Figure 6 about here Reliability:
596
597
To examine test-retest reliability estimates of amygdala—mPFC measures across
598
longitudinal visits, we computed Bayesian ICC estimates using a variance decomposition
599
method (Lüdecke et al., 2021). Because such models can accommodate missing data, all
600
observations (98 participants, 183 total scans) were used, including participants with only 1 visit. 601
All amygdala reactivity (Figure 7A) and amygdala—mPFC functional connectivity (Figure 7C)
602
measures, as well as slopes of amygdala reactivity estimates across trials (Figure 7B),
603
demonstrated poor reliability (ICC < 0.4; Cicchetti & Sparrow, 1981; Elliott et al., 2020). 604
Separation anxiety measures demonstrated somewhat higher, though still largely poor reliability
605
(point estimates ~0.4, 95% CIs included values below 0.4; Figure 7D). Head motion in the
606
scanner (mean framewise displacement) showed the highest reliability (ICC = 0.52, 95% CI
607
[0.29, 0.68]),
608 Discussion
613
Measures that are both robust to researcher decisions and reliable across
614
measurement instances are critical for studies of the human brain (Botvinik-Nezer et al., 2020;
615
Bowring et al., 2019; Elliott et al., 2020; T. Xu et al., 2022). The accelerated longitudinal design
616 Discussion
613
Measures that are both robust to researcher decisions and reliable across
614
measurement instances are critical for studies of the human brain (Botvinik-Nezer et al., 2020;
615
Bowring et al., 2019; Elliott et al., 2020; T. Xu et al., 2022). The accelerated longitudinal design
616 Discussion
613
Measures that are both robust to researcher decisions and reliable across
614 measurement instances are critical for studies of the human brain (Botvinik-Nezer et al., 2020;
615
Bowring et al., 2019; Elliott et al., 2020; T. Xu et al., 2022). The accelerated longitudinal design
616 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. Insert Figure 6 about here ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Running head: AMYGDALA—MPFC AGE-RELATED CHANGE and multiverse analysis approach used in the current study allowed a rare opportunity to
617
examine both reliability and robustness of amygdala—mPFC measures using a rapid event-
618
related face task from early childhood through young adulthood. Overall, estimates for age-
619
related change in amygdala reactivity were relatively robust to a variety of analytical decision
620
points, while age-related change estimates for amygdala—mPFC connectivity were more
621
sensitive to researcher choices. gPPI analyses were particularly sensitive to whether a
622
deconvolution step was applied. Yet, in concurrence with previous work (Elliott et al., 2020;
623
Haller et al., 2022; Herting et al., 2017; Infantolino et al., 2018; Kennedy et al., 2021; Nord et al.,
624
2017; Sauder et al., 2013), amygdala—mPFC measures displayed consistently poor test-
625
retest reliability across many analytical specifications. While low reliability estimates in the
626
present study may be due in part to the long (~18 months) test-retest interval (Elliott et al.,
627
2020) and potential true developmental change (Herting et al., 2017), low reliability
628
nevertheless imposes a major caveat towards interpretation of the current developmental
629
findings. 630 The present findings are valuable from a methodological standpoint in evaluating the
631
robustness of analytical tools used. A measurement can have high test-retest reliability yet
632
low robustness (high sensitivity) to analytical decisions, or vice versa (Li et al., 2021). Because
633
neither robustness nor reliability guarantee the other, current findings on the impacts of
634
analytic choices will likely be informative in guiding future studies. Thus, we discuss each of
635
the main analyses below, with particular emphasis on how findings are impacted by analytic
636
choices. 637
638
Amygdala reactivity
639
While there were differences across model specifications, the majority of pipelines
640
supported our hypothesis that amygdala reactivity to fearful faces decreases with age from
641 The present findings are valuable from a methodological standpoint in evaluating the
631
robustness of analytical tools used. A measurement can have high test-retest reliability yet
632
low robustness (high sensitivity) to analytical decisions, or vice versa (Li et al., 2021). Because
633
neither robustness nor reliability guarantee the other, current findings on the impacts of
634
analytic choices will likely be informative in guiding future studies. Insert Figure 6 about here Although estimating nonlinear
657
age-related change was not a primary goal of the present study, future work should use model
658
comparisons for better differentiating nonlinear patterns (Curran et al., 2010; Luna et al.,
659
2021). 660 early childhood through early adulthood (see Table 1 Aim 1a). Across specifications, we found
642
relatively robust evidence for age-related decreases in amygdala reactivity to both fearful and
643
neutral faces (Figure 2A). Yet, findings also varied considerably across specifications. For
644
example, only 60% of pipelines produced results that would be individually labeled as
645
‘significant’ (under a = .05), indicating that multiple investigations of this dataset could likely
646
lead to qualitatively different conclusions. While over half of analyses found evidence
647
consistent with studies indicating greater amygdala reactivity to fear faces > baseline in
648
younger children (Forbes et al., 2011; Gee et al., 2013; Guyer et al., 2008; Swartz et al., 2014),
649
the other 40% of specifications would have been consistent with investigations that found little
650
age-related change (NB: there were also differences in samples, age ranges, task
651
parameters, and behavioral demands across these studies; Kujawa et al., 2016; Wu et al.,
652
2016; Zhang et al., 2019). We also found that different specifications resulted in somewhat
653
different nonlinear trajectories (see sFigures 14-18). Not only did inverse age and quadratic
654
age models find different trajectories (as would be expected), but quadratic trajectories
655
themselves also displayed considerable analytic variability, with some specifications finding
656
“convex” and others finding “concave” fits (see sFigure 17). Although estimating nonlinear
657
age-related change was not a primary goal of the present study, future work should use model
658
comparisons for better differentiating nonlinear patterns (Curran et al., 2010; Luna et al.,
659
2021). 660 Models also found evidence for between-participant differences, but could neither
661
identify within-participant change (Figure 2D) nor differentiate between-participant from within-
662
participant estimates. As such, interpretation of the age-related change reported here is
663
subject to many of the same limitations that apply to cross-sectional designs (Glenn, 2003),
664
where age-related changes may not necessarily indicate ‘true’ developmental growth. High
665 Models also found evidence for between-participant differences, but could neither
661
identify within-participant change (Figure 2D) nor differentiate between-participant from within-
662
participant estimates. Insert Figure 6 about here Thus, we discuss each of
635
the main analyses below, with particular emphasis on how findings are impacted by analytic
636
choices. 637
638
Amygdala reactivity
639
While there were differences across model specifications, the majority of pipelines
640
supported our hypothesis that amygdala reactivity to fearful faces decreases with age from
641 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Running head: AMYGDALA—MPFC AGE-RELATED CHANGE early childhood through early adulthood (see Table 1 Aim 1a). Across specifications, we found
642
relatively robust evidence for age-related decreases in amygdala reactivity to both fearful and
643
neutral faces (Figure 2A). Yet, findings also varied considerably across specifications. For
644
example, only 60% of pipelines produced results that would be individually labeled as
645
‘significant’ (under a = .05), indicating that multiple investigations of this dataset could likely
646
lead to qualitatively different conclusions. While over half of analyses found evidence
647
consistent with studies indicating greater amygdala reactivity to fear faces > baseline in
648
younger children (Forbes et al., 2011; Gee et al., 2013; Guyer et al., 2008; Swartz et al., 2014),
649
the other 40% of specifications would have been consistent with investigations that found little
650
age-related change (NB: there were also differences in samples, age ranges, task
651
parameters, and behavioral demands across these studies; Kujawa et al., 2016; Wu et al.,
652
2016; Zhang et al., 2019). We also found that different specifications resulted in somewhat
653
different nonlinear trajectories (see sFigures 14-18). Not only did inverse age and quadratic
654
age models find different trajectories (as would be expected), but quadratic trajectories
655
themselves also displayed considerable analytic variability, with some specifications finding
656
“convex” and others finding “concave” fits (see sFigure 17). Insert Figure 6 about here As such, interpretation of the age-related change reported here is
663
subject to many of the same limitations that apply to cross-sectional designs (Glenn, 2003),
664
where age-related changes may not necessarily indicate ‘true’ developmental growth. High
665 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE
uncertainty in estimating average within-participant change could be driven by several factors,
666
including true heterogeneity in individual trajectories, low measurement reliability, scanner
667
differences across longitudinal timepoints, or unmodeled variables impacting amygdala
668
reactivity. Additionally, the within-participants terms represent a smaller age range (a
669
maximum of 4 years for any given participant), relative to the broader age range assessed by
670
the between-participants terms (18 years), which may have placed additional limits on
671
identifying reliable within-participant change. 672
Age-related change in amygdala responses to fear faces over baseline seemed largely
673
the result of earlier trials in the task (see sFigures 32-34). While differences in task design and
674
contrast across studies have been highlighted as potential sources of discrepant findings on the
675
development of amygdala function (Killgore & Yurgelun-Todd, 2007; Lieberman et al., 2007;
676
Swartz et al., 2014), this result indicates that attention to trial structure and task duration may
677
also be necessary in comparing studies. Because the paradigm used in the current study
678
involved a task requiring participants to press for one face (‘neutral’) and not press for ‘fear’
679
faces, findings specific to fear faces over baseline under the current paradigm may also be
680
driven by behavioral task demands. Insert Figure 6 about here 681
682
Amygdala–mPFC Functional Connectivity
683
We did not find evidence for our second hypothesis as neither gPPI nor BSC analyses
684 uncertainty in estimating average within-participant change could be driven by several factors,
666
including true heterogeneity in individual trajectories, low measurement reliability, scanner
667
differences across longitudinal timepoints, or unmodeled variables impacting amygdala
668
reactivity. Additionally, the within-participants terms represent a smaller age range (a
669
maximum of 4 years for any given participant), relative to the broader age range assessed by
670
the between-participants terms (18 years), which may have placed additional limits on
671
identifying reliable within-participant change. 672 uncertainty in estimating average within-participant change could be driven by several factors,
666
including true heterogeneity in individual trajectories, low measurement reliability, scanner
667
differences across longitudinal timepoints, or unmodeled variables impacting amygdala
668
reactivity. Additionally, the within-participants terms represent a smaller age range (a
669
maximum of 4 years for any given participant), relative to the broader age range assessed by
670
the between-participants terms (18 years), which may have placed additional limits on
671
identifying reliable within-participant change. 672 differences across longitudinal timepoints, or unmodeled variables impacting amygdala
668
reactivity. Additionally, the within-participants terms represent a smaller age range (a
669
maximum of 4 years for any given participant), relative to the broader age range assessed by
670
the between-participants terms (18 years), which may have placed additional limits on
671
identifying reliable within-participant change. 672
Age-related change in amygdala responses to fear faces over baseline seemed largely
673
the result of earlier trials in the task (see sFigures 32-34). While differences in task design and
674
contrast across studies have been highlighted as potential sources of discrepant findings on the
675
development of amygdala function (Killgore & Yurgelun-Todd, 2007; Lieberman et al., 2007;
676
Swartz et al., 2014), this result indicates that attention to trial structure and task duration may
677
also be necessary in comparing studies. Because the paradigm used in the current study
678
involved a task requiring participants to press for one face (‘neutral’) and not press for ‘fear’
679
faces, findings specific to fear faces over baseline under the current paradigm may also be
680
driven by behavioral task demands. Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Running head: AMYGDALA—MPFC AGE-RELATED CHANGE
Additionally, quadratic and inverse age models did not find consistent evidence for nonlinear
691
age-related change (see sFigures 45-48 & 58-61). 692
gPPI results were sensitive to whether a deconvolution step had been included in the
693
preprocessing pipeline, such that we mostly found age-related decreases in amygdala–mPFC
694
connectivity with a deconvolution step included, and age-related increases without it (although
695
most pipelines would not have been ‘statistically significant’ on their own, see Figure 4B). 696
While deconvolution has been argued to be a necessary step for event-related PPI analyses
697
(Gitelman et al., 2003), recent work has shifted guidelines on its use, and it may not be
698
recommended for block designs (Di et al., 2020; Di & Biswal, 2017). Because the true
699
‘neuronal’ signal underlying the BOLD timeseries within a given ROI cannot be directly
700
measured, deconvolution algorithms are difficult to validate. Further, deconvolution may cause
701
PPI results to be driven by baseline connectivity if task regressors are not centered (Di et al.,
702
2017), although such centering did not have a major influence on age-related change results
703
in the present analyses (see sFigure 44). Within the current study, small tweaks to AFNI’s
704
3dTfitter algorithm for deconvolution resulted in vastly different regressors (see sFigure 36),
705
suggesting the potential for high analytic variability even between gPPI analyses ostensibly
706
using deconvolution. While the present study does not provide evidence that can inform
707
whether or not deconvolution is recommended, further work is needed to optimize and
708
validate applications of gPPI methods and selection of appropriate task designs. gPPI may be
709
better equipped for block-designs and particularly ill-posed for rapid event-related tasks due to
710
both difficulties in resolving which times within the BOLD timeseries reflect functional
711
connectivity evoked by rapid (350ms) events and low statistical power in estimating such task-
712
evoked connectivity (see sFigures 35-37; O’Reilly et al., 2012). Concurrent with previous work,
713 Running head: AMYGDALA—MPFC AGE-RELATED CHANGE
Additionally, quadratic and inverse age models did not find consistent evidence for nonlinear
691
age-related change (see sFigures 45-48 & 58-61). Insert Figure 6 about here CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint Insert Figure 6 about here 681
682
Amygdala–mPFC Functional Connectivity
683
We did not find evidence for our second hypothesis, as neither gPPI nor BSC analyses
684
indicated consistent evidence of age-related change in amygdala–mPFC functional
685
connectivity (see Table 1 Aims 2a-2b, Figures 4-5). Thus, the age-related changes in task-
686
evoked amygdala–mPFC connectivity identified in prior work (Gee et al., 2013; Kujawa et al.,
687
2016; Wu et al., 2016) was not identified here, consistent with (Zhang et al., 2019). Crucially,
688
however, our specification curves did not find strong evidence against such age-related
689
change, as we did not observe precise and consistent ‘null’ estimates across specifications. 690 Age-related change in amygdala responses to fear faces over baseline seemed largely
673
the result of earlier trials in the task (see sFigures 32-34). While differences in task design and
674
contrast across studies have been highlighted as potential sources of discrepant findings on the
675
development of amygdala function (Killgore & Yurgelun-Todd, 2007; Lieberman et al., 2007;
676
Swartz et al., 2014), this result indicates that attention to trial structure and task duration may
677
also be necessary in comparing studies. Because the paradigm used in the current study
678
involved a task requiring participants to press for one face (‘neutral’) and not press for ‘fear’
679
faces, findings specific to fear faces over baseline under the current paradigm may also be
680
driven by behavioral task demands. 681 Amygdala–mPFC Functional Connectivity
683
We did not find evidence for our second hypothesis, as neither gPPI nor BSC analyses
684
indicated consistent evidence of age-related change in amygdala–mPFC functional
685
connectivity (see Table 1 Aims 2a-2b, Figures 4-5). Thus, the age-related changes in task-
686
evoked amygdala–mPFC connectivity identified in prior work (Gee et al., 2013; Kujawa et al.,
687
2016; Wu et al., 2016) was not identified here, consistent with (Zhang et al., 2019). Crucially,
688
however, our specification curves did not find strong evidence against such age-related
689
change, as we did not observe precise and consistent ‘null’ estimates across specifications. 690 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint . Running head: AMYGDALA—MPFC AGE-RELATED CHANGE 692
gPPI results were sensitive to whether a deconvolution step had been included in the
693
preprocessing pipeline, such that we mostly found age-related decreases in amygdala–mPFC
694
connectivity with a deconvolution step included, and age-related increases without it (although
695
most pipelines would not have been ‘statistically significant’ on their own, see Figure 4B). 696
While deconvolution has been argued to be a necessary step for event-related PPI analyses
697
(Gitelman et al., 2003), recent work has shifted guidelines on its use, and it may not be
698
recommended for block designs (Di et al., 2020; Di & Biswal, 2017). Because the true
699
‘neuronal’ signal underlying the BOLD timeseries within a given ROI cannot be directly
700
measured, deconvolution algorithms are difficult to validate. Further, deconvolution may cause
701
PPI results to be driven by baseline connectivity if task regressors are not centered (Di et al.,
702
2017), although such centering did not have a major influence on age-related change results
703
in the present analyses (see sFigure 44). Within the current study, small tweaks to AFNI’s
704
3dTfitter algorithm for deconvolution resulted in vastly different regressors (see sFigure 36),
705
suggesting the potential for high analytic variability even between gPPI analyses ostensibly
706
using deconvolution. While the present study does not provide evidence that can inform
707
whether or not deconvolution is recommended, further work is needed to optimize and
708
validate applications of gPPI methods and selection of appropriate task designs. gPPI may be
709
better equipped for block-designs and particularly ill-posed for rapid event-related tasks due to
710
both difficulties in resolving which times within the BOLD timeseries reflect functional
711
connectivity evoked by rapid (350ms) events and low statistical power in estimating such task-
712
evoked connectivity (see sFigures 35-37; O’Reilly et al., 2012). Concurrent with previous work,
713
beta series correlation analyses may have higher statistical power for identifying task-related
714 Additionally, quadratic and inverse age models did not find consistent evidence for nonlinear
691
age-related change (see sFigures 45-48 & 58-61). 692 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint . Running head: AMYGDALA—MPFC AGE-RELATED CHANGE CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Running head: AMYGDALA—MPFC AGE-RELATED CHANGE g
Age-related change estimates for amygdala—mPFC BSC showed somewhat higher
716
robustness to analytic decisions compared to gPPI. For BSC analyses, choice of mPFC ROI
717
contributed most to variability in age-related change estimates (see Figure 5B, sFigures 55-
718
57). While a global signal correction (post-hoc distribution centering) greatly decreased
719
average amygdala—mPFC BSC connectivity (see Figure 5D, sFigure 52) for both fear and
720
neutral faces, this analytical step did not impact age-related change estimates as heavily
721
(sFigures 55-57). The fact that global signal correction so dramatically decreased average
722
estimated amygdala—mPFC BSC may indicate that, like with resting-state fMRI analyses,
723
positive functional connectivity values are due in part to motion and physiology-related
724
confounds (Gratton et al., 2020; Power et al., 2019). Supporting this, BSC estimates were
725
correlated with mean framewise displacement across scans for the fear > baseline and neutral >
726
baseline contrasts only when a global signal correction was not applied (see sFigure 62). In
727
addition, while test-retest reliability for all BSC measures was poor, BSC estimates from
728
pipelines including a global signal correction step mostly demonstrated somewhat higher ICC
729
(Figure 6). While these results are consistent with prior work indicating that correcting for the
730
global signal can mitigate artifacts (Ciric et al., 2017; Satterthwaite et al., 2012), other work
731
indicates that such corrections also remove meaningful biological signals (Belloy et al., 2018;
732
Glasser et al., 2018; Yousefi et al., 2018). 733 Glasser et al., 2018; Yousefi et al., 2018). 733
734
Amygdala–mPFC circuitry and separation anxiety
735
We did not find associations between any task-related amygdala–mPFC measures
736
(reactivity or functional connectivity) and separation anxiety behaviors (see Table 1 Aim 3;
737
Figure 6). This finding stands in contrast to associations between amygdala–mPFC
738
connectivity and anxiety identified in previous developmental work (Gee et al., 2013;
739 . Running head: AMYGDALA—MPFC AGE-RELATED CHANGE CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE
Jalbrzikowski et al., 2017; Kujawa et al., 2016; Qin et al., 2014). However, given that analyses
740
of brain-behavior associations may require imaging cohorts much larger than the current
741
sample (especially considering the low reliability of the measures used; Grady et al., 2020;
742
Marek et al., 2020), the absence of relationships here may not be strong evidence against the
743
existence of potential associations between amygdala–mPFC circuitry and developing
744
anxiety-related behaviors. 745
746
Advantages and pitfalls of the multiverse approach
747
Our findings contribute to a body of work demonstrating that preprocessing and
748
modeling choices can meaningfully influence results (Botvinik-Nezer et al., 2020). Indeed,
749
most studies involving many analytical decision points could benefit from multiverse analyses. 750
Such specification curves can help to examine the stability of findings in both exploratory and
751
confirmatory research (Flournoy et al., 2020). Particularly when methodological ‘gold
752
standards’ have not been determined, specification curves may be informative for examining
753
the impacts of potential analysis decisions (Bridgeford et al., 2020; Dafflon et al., 2020). 754
Further, wider use of specification curves might help to resolve discrepancies between study
755
findings stemming from different analysis pipelines. 756
While specification curve analyses may benefit much future research we also note
757
. CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is mad
py g
p
p
p
y
p
g
p
p Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Our findings contribute to a body of work demonstrating that preprocessing and
748
modeling choices can meaningfully influence results (Botvinik-Nezer et al., 2020). Indeed,
749
most studies involving many analytical decision points could benefit from multiverse analyses. Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Running head: AMYGDALA—MPFC AGE-RELATED CHANGE
difficult. To this end, we created interactive visualizations for visual exploration of individual
765
analysis specifications. 766
Computational resources are a relevant concern when conducting multiverse analyses
767
as well. In the current study, preprocessing (registration in particular) was the most
768
computationally intensive step, taking an estimated 4 hours of compute time per scan per
769
pipeline using 4 cores on a Linux-based institutional research computing cluster. However,
770
specification curve analyses themselves were relatively less intensive, with all group-level
771
models of amygdala reactivity completing in a total of 48 hours using 4 cores on a laboratory
772
Linux-based server. Specification curves using maximum likelihood models (lme4 in R; Bates
773
& Bolker, 2011) were even more efficient, with thousands of models running within minutes
774
using a 2019 MacBook Pro (2.8 GHz Intel Core i7). 775 Running head: AMYGDALA—MPFC AGE-RELATED CHANGE 750
Such specification curves can help to examine the stability of findings in both exploratory and
751
confirmatory research (Flournoy et al., 2020). Particularly when methodological ‘gold
752
standards’ have not been determined, specification curves may be informative for examining
753
the impacts of potential analysis decisions (Bridgeford et al., 2020; Dafflon et al., 2020). 754
Further, wider use of specification curves might help to resolve discrepancies between study
755
findings stemming from different analysis pipelines. 756 Further, wider use of specification curves might help to resolve discrepancies between study
755
findings stemming from different analysis pipelines. 756 While specification curve analyses may benefit much future research, we also note
757
that multiverses are only as comprehensive as the included specifications (Steegen et al.,
758
2016), and such analyses alone do not solve problems related to unmodeled confounds,
759
design flaws, inadequate statistical power, circular analyses, or non-representative sampling. 760
Further, unless all specifications are decided a priori, analyses are vulnerable to problems of
761
analytic flexibility (Gelman & Loken, 2014), and inclusion of less justified specifications can
762
bias results (Del Giudice & Gangestad, 2021). Because specification curves can include
763
hundreds or thousands of individual analyses, rigorous evaluation of individual models can be
764 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE
difficult. To this end, we created interactive visualizations for visual exploration of individual
765
analysis specifications. 766
Computational resources are a relevant concern when conducting multiverse analyse
767
as well. In the current study, preprocessing (registration in particular) was the most
768
computationally intensive step, taking an estimated 4 hours of compute time per scan per
769
pipeline using 4 cores on a Linux-based institutional research computing cluster. Running head: AMYGDALA—MPFC AGE-RELATED CHANGE However,
770
specification curve analyses themselves were relatively less intensive, with all group-level
771
models of amygdala reactivity completing in a total of 48 hours using 4 cores on a laboratory
772
Linux-based server. Specification curves using maximum likelihood models (lme4 in R; Bates
773
& Bolker, 2011) were even more efficient, with thousands of models running within minutes
774
using a 2019 MacBook Pro (2.8 GHz Intel Core i7). 775
776
Limitations
777
The present study is subject to several limitations that may be addressed in future
778
investigations. Perhaps most crucially, our conclusions (along with those of many
779
developmental fMRI studies) are limited by the poor test-retest reliability of the fMRI data. 780
Because amygdala—mPFC measures showed low reliability across study visits, the statistica
781
power of our analyses of age-related changes is likely low (Elliott et al., 2020; Zuo et al., 2019
782
Low-powered studies can yield increased rates of both false positive and false negative
783
results (as well as errors of the sign and magnitude of estimates; Button et al., 2013; Gelman
784
& Carlin, 2014); therefore we caution against interpretation of our developmental findings (an
785
brain-behavior associations) beyond the cohort studied in the present investigation. In
786
particular, the low statistical power of our rapid event-related task design may be a major
787
contributor to the low test-retest reliability and variance in outcomes across analysis
788
specifications. That being said, achieving high-powered studies presents a challenge for
789
. CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is difficult. To this end, we created interactive visualizations for visual exploration of individual
765
analysis specifications.
766 ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Running head: AMYGDALA—MPFC AGE-RELATED CHANGE studying populations that cannot tolerate lengthy fMRI sessions. Both findings that were more
790
robust to analytical decisions (amygdala reactivity) and findings that were less so (amygdala—
791
mPFC connectivity, associations with separation anxiety) may be most valuable in meta-
792
analytic contexts where greater aggregate statistical power can be achieved. In particular,
793
future work on amygdala—mPFC development will benefit from optimization of measures both
794
on robustness to analytic variability (Li et al., 2021) and reliability (Kragel et al., 2021). 795
Present findings are also limited by the number of participants studied (Bossier et al.,
796
2020; Marek et al., 2020), the number of longitudinal study sessions per participant (King et al.,
797
2018), and the duration of the task (Nee, 2019). Work with larger sample sizes, more study
798
sessions per participant, and more task data collected per session will be necessary for
799
charting functional amygdala–mPFC development and examining heterogeneity across
800
individuals (although collecting task-based fMRI will continue to be challenging for studies
801
including younger children). The generalizability of the current findings may also be limited by
802
the fact that this cohort was skewed towards high incomes and not racially or ethnically
803
representative of the Los Angeles or United States population. 804
Findings are also somewhat limited by the fact that the present study is not wholly
805
confirmatory, despite preregistration. Because our multiverse analysis approaches expanded
806 studying populations that cannot tolerate lengthy fMRI sessions. Both findings that were more
790
robust to analytical decisions (amygdala reactivity) and findings that were less so (amygdala—
791
mPFC connectivity, associations with separation anxiety) may be most valuable in meta-
792
analytic contexts where greater aggregate statistical power can be achieved. In particular,
793
future work on amygdala—mPFC development will benefit from optimization of measures both
794
on robustness to analytic variability (Li et al., 2021) and reliability (Kragel et al., 2021). 795 Present findings are also limited by the number of participants studied (Bossier et al.,
796
2020; Marek et al., 2020), the number of longitudinal study sessions per participant (King et al.,
797
2018), and the duration of the task (Nee, 2019). difficult. To this end, we created interactive visualizations for visual exploration of individual
765
analysis specifications.
766 difficult. To this end, we created interactive visualizations for visual exploration of individual
765
analysis specifications. 766 Computational resources are a relevant concern when conducting multiverse analyses
767
as well. In the current study, preprocessing (registration in particular) was the most
768
computationally intensive step, taking an estimated 4 hours of compute time per scan per
769
pipeline using 4 cores on a Linux-based institutional research computing cluster. However,
770
specification curve analyses themselves were relatively less intensive, with all group-level
771
models of amygdala reactivity completing in a total of 48 hours using 4 cores on a laboratory
772
Linux-based server. Specification curves using maximum likelihood models (lme4 in R; Bates
773
& Bolker, 2011) were even more efficient, with thousands of models running within minutes
774
using a 2019 MacBook Pro (2.8 GHz Intel Core i7). 775 investigations. Perhaps most crucially, our conclusions (along with those of many
779
developmental fMRI studies) are limited by the poor test-retest reliability of the fMRI data. 780
Because amygdala—mPFC measures showed low reliability across study visits, the statistical
781
power of our analyses of age-related changes is likely low (Elliott et al., 2020; Zuo et al., 2019). 782
Low-powered studies can yield increased rates of both false positive and false negative
783
results (as well as errors of the sign and magnitude of estimates; Button et al., 2013; Gelman
784
& Carlin, 2014); therefore we caution against interpretation of our developmental findings (and
785
brain-behavior associations) beyond the cohort studied in the present investigation. In
786
particular, the low statistical power of our rapid event-related task design may be a major
787
contributor to the low test-retest reliability and variance in outcomes across analysis
788
specifications. That being said, achieving high-powered studies presents a challenge for
789 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. difficult. To this end, we created interactive visualizations for visual exploration of individual
765
analysis specifications.
766 Work with larger sample sizes, more study
798
sessions per participant, and more task data collected per session will be necessary for
799
charting functional amygdala–mPFC development and examining heterogeneity across
800
individuals (although collecting task-based fMRI will continue to be challenging for studies
801
including younger children). The generalizability of the current findings may also be limited by
802
the fact that this cohort was skewed towards high incomes and not racially or ethnically
803
representative of the Los Angeles or United States population. 804 Findings are also somewhat limited by the fact that the present study is not wholly
805
confirmatory, despite preregistration. Because our multiverse analysis approaches expanded
806
significantly beyond the methods we preregistered, most of the present analyses, while
807
hypothesis-driven, must be considered exploratory (Flournoy et al., 2020). The fact that some
808
specifications used data included in previous similar analyses of the same cohort(Gee et al.,
809
2013) also limits the confirmatory power of the present study (Kriegeskorte et al., 2009). This
810
may be especially true because longitudinal models could not identify within-person change
811
as distinct from between-participant differences (see Figure 2D), indicating that our age-
812
related change estimates may be influenced by cross-sectional information similar to that
813
investigated by Gee et al. (Gee et al., 2013). 814 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Running head: AMYGDALA—MPFC AGE-RELATED CHANGE
Though the current study aimed to estimate longitudinal age-related changes in
815
amygdala–mPFC functional circuitry evoked by fear and neutral faces, the current findings
816
may not be specific to these stimuli (Hariri et al., 2002). difficult. To this end, we created interactive visualizations for visual exploration of individual
765
analysis specifications.
766 Because our task did not include non-
817
face foils or probe specific emotion-related processes, results may be driven by attention,
818
learning, or visual processing, rather than affective or face processing. In particular, because
819
participants were instructed to press a button for neutral faces and withhold a button press for
820
fear faces, observed amygdala—mPFC responses may in part reflect response inhibition (for
821
fear faces; Menon et al., 2001) and target detection processes (for neutral faces; Jonkman et
822
al., 2003). Findings for the fear faces > baseline and neutral > baseline contrasts also may not
823
be valence-specific in the absence of a different emotional face as part of the contrast. 824
Further, because all faces were adult White women, the current results may not generalize to
825
faces more broadly (Richeson et al., 2008; Telzer et al., 2012). Additionally, because face
826
stimuli were the same across study visits, exposure effects across sessions may confound
827
longitudinal findings (although exposure effects may be possible any time a task is repeated,
828
even if stimuli are unique), particularly age-related decreases in amygdala responses (Telzer
829
et al., 2018). While within-session amygdala habituation effects have been shown across
830
several paradigms (Geissberger et al., 2020; Hare et al., 2008; Hein et al., 2018), between-
831
session habituation effects are unlikely beyond 2-3 weeks (Geissberger et al., 2020; Johnstone
832
et al., 2005; Plichta et al., 2014; Spohrs et al., 2018). 833 Though the current study aimed to estimate longitudinal age-related changes in
815
amygdala–mPFC functional circuitry evoked by fear and neutral faces, the current findings
816
may not be specific to these stimuli (Hariri et al., 2002). Because our task did not include non-
817
face foils or probe specific emotion-related processes, results may be driven by attention,
818
learning, or visual processing, rather than affective or face processing. In particular, because
819
participants were instructed to press a button for neutral faces and withhold a button press for
820
fear faces, observed amygdala—mPFC responses may in part reflect response inhibition (for
821
fear faces; Menon et al., 2001) and target detection processes (for neutral faces; Jonkman et
822
al., 2003). Findings for the fear faces > baseline and neutral > baseline contrasts also may not
823
be valence-specific in the absence of a different emotional face as part of the contrast. difficult. To this end, we created interactive visualizations for visual exploration of individual
765
analysis specifications.
766 824 Finally, our findings on age-related change in amygdala and mPFC function may be
834
biased or confounded by age-related differences in head motion (Ciric et al., 2017), anatomical
835
image quality and alignment (Gilmore et al., 2020; Rorden et al., 2012), signal dropout, and
836
physiological artifacts (Boubela et al., 2015; Fair et al., 2020; Gratton et al., 2020). While our
837
multiverse analyses included preprocessing and group-level modeling specifications designed
838 Finally, our findings on age-related change in amygdala and mPFC function may be
834
biased or confounded by age-related differences in head motion (Ciric et al., 2017), anatomical
835
image quality and alignment (Gilmore et al., 2020; Rorden et al., 2012), signal dropout, and
836
physiological artifacts (Boubela et al., 2015; Fair et al., 2020; Gratton et al., 2020). While our
837
multiverse analyses included preprocessing and group-level modeling specifications designed
838 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE
to minimize some of such potential issues, future work is still needed to optimize
839
discrimination of developmental changes of interest from such potential confounds. 840
Despite these limitations, the present study concords with prior investigations in
841
demonstrating the value of multiverse approaches to quantify sensitivity to researcher
842
decisions. The results highlight key analytic considerations for future studies of age-related
843
changes in amygdala—mPFC function, as well as for studies of human brain development
844
more broadly. 845
846
. CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Running head: AMYGDALA—MPFC AGE-RELATED CHANGE o minimize some of such potential issues, future work is still needed to optimize In addition, we provide publicly available simulated data structured similarly to the
869
study data on amygdala reactivity, such that interested readers can view multiverse analysis
870
walkthroughs (https://pab2163.github.io/amygdala_mpfc_multiverse) and experiment with
871
analysis code. Additional materials, including MNI space masks and preregistration
872
documentation, are available at https://osf.io/hvdmx/
873
874
875
876
877
878
879
880 o minimize some of such potential issues, future work is still needed to optimize Despite these limitations, the present study concords with prior investigations in
841
demonstrating the value of multiverse approaches to quantify sensitivity to researcher
842
decisions. The results highlight key analytic considerations for future studies of age-related
843
changes in amygdala—mPFC function, as well as for studies of human brain development
844
more broadly. 845 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Running head: AMYGDALA—MPFC AGE-RELATED CHANGE
CRediT Author Statement:
847
848
Paul Alexander Bloom: Conceptualization, Methodology, Formal Analysis, Writing - Original
849
Draft, Visualization Michelle VanTieghem: Methodology, Writing – Review & Editing Laurel
850
Gabard-Durnam: Investigation, Methodology, Writing – Review & Editing, Dylan Gee:
851
Investigation, Methodology, Writing – Review & Editing Jessica Flannery: Investigation, Writing
852
– Review & Editing Christina Caldera: Investigation, Writing – Review & Editing Bonnie Goff:
853
Investigation, Writing – Review & Editing Eva Telzer: Investigation, Writing – Review & Editing
854
Kathryn L. Humphreys: Investigation, Writing – Review & Editing Dominic Fareri:
855
Investigation, Writing – Review & Editing Mor Shapiro: Investigation, Writing – Review &
856
Editing Sameah Algharazi: Validation, Writing – Review & Editing Niall Bolger: Methodology,
857
Formal Analysis, Writing – Review & Editing Mariam Aly: Methodology, Formal Analysis,
858
Supervision, Writing – Review & Editing Nim Tottenham: Conceptualization, Methodology,
859
Investigation, Resources, Data Curation, Writing - Original Draft, Supervision, Funding
860
Acquisition
861
862
863
Code Availability
864
865
Code for preprocessing, analysis, and data visualizations for this manuscript is
866
available at https://github.com/pab2163/amygdala_mpfc_multiverse. While unfortunately this
867
code cannot be run as written without data, we have attempted to document analysis steps
868
clearly. Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Running head: AMYGDALA—MPFC AGE-RELATED CHANGE
References
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904 Code Availability Code for preprocessing, analysis, and data visualizations for this manuscript is
available at https://github.com/pab2163/amygdala_mpfc_multiverse. While unfortunately this
code cannot be run as written without data, we have attempted to document analysis steps
clearly. In addition, we provide publicly available simulated data structured similarly to the
study data on amygdala reactivity, such that interested readers can view multiverse analysis
walkthroughs (https://pab2163.github.io/amygdala_mpfc_multiverse) and experiment with
analysis code. Additional materials, including MNI space masks and preregistration
documentation, are available at https://osf.io/hvdmx/ . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
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this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is ma
py g
p
p
p
y
,
;
p
g
p
p Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Age-related
change in amygdala–
mPFC functional
connectivity (FC) to
fear faces, as
measured by
generalized
psychophysiological
interaction (gPPI)
Amygdala–mPFC
FC will decrease as
a function of age
such that as age
increases, the
valence of FC will
shift from positive to
negative. Multiverse gPPI analysis with anatom
defined bilateral amygdala seed and m
target ROIs using multilevel linear
regression at the group level. Multiverse decision points:
Deconvolution step, mPFC ROI defin
contrast estimate type (t-stat vs. beta
estimate), group-level model covariat
2b. Age-related
change in amygdala–
mPFC functional
connectivity to fear
faces, as measured by
beta-series correlation
(BSC)
None for BSC
specifically
Multiverse BSC analysis between
amygdala and mPFC using multilevel
regression at the group level. Multiverse decision points:
Global signal subtraction, amygdala R
definition, mPFC ROI definition, group
model covariates
3. Associations of
amygdala reactivity,
change in amygdala
reactivity across trials,
or amygdala–mPFC
FC with separation
anxiety
None
Multiverse multilevel linear regression
brain measures as predictors for sepa
anxiety behaviors, controlling for age
Multiverse decision points:
Separation anxiety measure, FC mea
mPFC ROI (FC only), amygdala ROI,
contrast, deconvolution step (gPPI on 1369
Table 1: Summary of main aims, hypotheses, methods, and findings
1370
1371
1372
Aim
Preregistered
Hypothesis
Analysis Methodology
Key Findings
1a. Age-related
change in amygdala
reactivity to fear faces
Amygdala reactivity
to fearful faces will
decrease with age,
such that younger
children will have
more positive
amygdala reactivity
(higher BOLD
response to fear
faces relative to
implicit baseline)
than older youth. Multiverse amygdala ROI (anatomically-
defined) analysis using multilevel linear
regression at the group level. Multiverse decision points:
Preprocessing software, GLM software,
GLM nuisance regressors, amygdala ROI
definition, contrast estimate type (t-stat vs. beta estimate), HRF shape, group-level
model covariates, exclusion of previously
analyzed scans
• Across decision points, weak but
consistent negative age-related
change in amygdala reactivity to
fear > baseline and neutral >
baseline contrasts
• No consistent evidence for age-
related change in fear > neutral
contrast
• Longitudinal models could identify
consistent between-participant
differences but not within-participant
age-related change
1b. Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Age-related
change in patterns of
amygdala responses
across task trials
None
Multiverse analysis of slopes of amygdala
reactivity across trials, and amygdala
reactivity in each half of trials using
multilevel linear regression at the group
level, single trial models
Multiverse decision points:
Global signal subtraction, amygdala ROI
definition, group-level model covariates
• On average, amygdala reactivity
decreased across trials (for both
fear and neutral faces)
• Amygdala reactivity for earlier trials
was higher at younger ages
• Age-related change in amygdala
reactivity to fear faces in the first
half of trials, but not the second half
• Similar, but somewhat weaker age-
related change for neutral faces
2a. Age-related
change in amygdala–
mPFC functional
connectivity (FC) to
fear faces, as
measured by
generalized
psychophysiological
interaction (gPPI)
Amygdala–mPFC
FC will decrease as
a function of age
such that as age
increases, the
valence of FC will
shift from positive to
negative. Multiverse gPPI analysis with anatomically
defined bilateral amygdala seed and mPFC
target ROIs using multilevel linear
regression at the group level. Multiverse decision points:
Deconvolution step, mPFC ROI definition,
contrast estimate type (t-stat vs. beta
estimate), group-level model covariates
• No consistent evidence for age-
related change in gPPI for any
contrast
• gPPI estimates extremely sensitive
to deconvolution step in creation of
regressors
2b. Age-related
change in amygdala–
mPFC functional
connectivity to fear
faces, as measured by
beta-series correlation
(BSC)
None for BSC
specifically
Multiverse BSC analysis between
amygdala and mPFC using multilevel linear
regression at the group level. Multiverse decision points:
Global signal subtraction, amygdala ROI
definition, mPFC ROI definition, group-level
model covariates
• No consistent evidence for age-
related change in BSC for any
condition
• Amygdala–mPFC BSC was most
sensitive to selection of mPFC ROI
• Global signal subtraction reduced
average amygdala–mPFC BSC, but
impacts on age-related changes
were small
• BSC estimates were not strongly
associated with gPPI estimates
3. Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Xu, J., Hao, L., Chen, M., He, Y., Jiang, M., Tian, T., Wang, H., Wang, Y., Wang, D., Han, Z. R.,
1340
Tan, S., Men, W., Gao, J., He, Y., Tao, S., Dong, Q., & Qin, S. (2021). Developmental
1341
Sex Differences in Negative Emotion Decision-Making Dynamics: Computational
1342
Evidence and Amygdala-Prefrontal Pathways. Cerebral Cortex, bhab359. 1343
https://doi.org/10.1093/cercor/bhab359
1344 Xu, J., Hao, L., Chen, M., He, Y., Jiang, M., Tian, T., Wang, H., Wang, Y., Wang, D., Han, Z. R.,
1340
Tan, S., Men, W., Gao, J., He, Y., Tao, S., Dong, Q., & Qin, S. (2021). Developmental
1341
Sex Differences in Negative Emotion Decision-Making Dynamics: Computational
1342
Evidence and Amygdala-Prefrontal Pathways. Cerebral Cortex, bhab359. 1343
https://doi.org/10.1093/cercor/bhab359
1344 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE 1369
Table 1: Summary of main aims, hypotheses, methods
1370
1371
1372
Aim
Preregistered
Hypothesis
Analysis Methodology
1a. Age-related
change in amygdala
reactivity to fear faces
Amygdala reactivity
to fearful faces will
decrease with age,
such that younger
children will have
more positive
amygdala reactivity
(higher BOLD
response to fear
faces relative to
implicit baseline)
than older youth. Multiverse amygdala ROI (anatomica
defined) analysis using multilevel line
regression at the group level. Multiverse decision points:
Preprocessing software, GLM softwar
GLM nuisance regressors, amygdala
definition, contrast estimate type (t-sta
beta estimate), HRF shape, group-lev
model covariates, exclusion of previo
analyzed scans
1b. Age-related
change in patterns of
amygdala responses
across task trials
None
Multiverse analysis of slopes of amyg
reactivity across trials, and amygdala
reactivity in each half of trials using
multilevel linear regression at the grou
level, single trial models
Multiverse decision points:
Global signal subtraction, amygdala R
definition, group-level model covariate
2a. Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Table 2: Summary of forking pipelines used in analyses for each aim1
1374
Aim/Analysis
Decision Point
Choices
1a. Age-related change in
amygdala reactivity to fear faces >
baseline
Preprocessing Software
FSL FEAT, C-PAC
GLM Software
FSL FEAT, AFNI 3dDeconvolve
Hemodynamic Response Function
Double Gamma, Single Gamma
Nuisance Regressors
24 motion regressors, 6 motion regressors, 18
motion regressors + WM + CSF
Low-frequency artifact removal
High-pass filter (.01Hz), Quadratic drift regressor
First-level GLM Estimates
Beta Estimates, T-statistics
Native vs. Standard MNI Space
Native Space (Freesurfer), Harvard-Oxford Atlas
in MNI
Amygdala ROI
Bilateral, Left, Right, High Signal, Low Signal
Inclusion of 45 previously analyzed scans
Include, Exclude
Outlier treatment
Exclude +/-3SD from mean, Exclude +/-3SD
from mean + robust regression
Group-level model covariates
Mean FD, Mean FD + run, Mean FD + scanner,
Mean FD + run + scanner
Group-level model quadratic term
Yes, No
Group-level model random slopes
Yes, No
1b. Age-related change in
patterns of amygdala responses
across task trials
FSL preproc & GLM, high-pass
filter, 24 motion regressors, 2G
HRF, beta estimates, included
previously analyzed scans, and
robust group-level regression
Method of quantifying within-scan change
Slopes across trials, trials split into halves, single-
trial models
Global Signal Subtraction
Yes, No
Amygdala ROI (all MNI space)
Bilateral, Left, Right
Group-level model covariates
Mean FD, Mean FD + run, Mean FD + scanner,
Mean FD + run + scanner
Group-level model quadratic term
Yes, No
Group-level model random slopes
Yes, No
2a Age-related change in
amygdala–mPFC functional
connectivity (FC) to fear faces >
baseline, as measured by (gPPI)
FSL preproc & GLM, high-pass
filter, 24 motion regressors, 2G
HRF, bilateral amygdala ROI in
MNI space
Deconvolution step
Yes, No
mPFC ROI (all MNI space)
3 different 5mm spheres, large vmPFC mask
Outlier treatment
Exclude +/-3SD from mean, Exclude +/-3SD
from mean + robust regression
Inclusion of 45 previously analyzed scans
Include, Exclude
Group-level model covariates
Mean FD, Mean FD + run, Mean FD + scanner,
Mean FD + run + scanner
Group-level model quadratic term
Yes, No
Group-level model random slopes
Yes, No
2b. Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Associations of
amygdala reactivity,
change in amygdala
reactivity across trials,
or amygdala–mPFC
FC with separation
anxiety
None
Multiverse multilevel linear regressions with
brain measures as predictors for separation
anxiety behaviors, controlling for age
Multiverse decision points:
Separation anxiety measure, FC measure,
mPFC ROI (FC only), amygdala ROI,
contrast, deconvolution step (gPPI only)
• No evidence that amygdala
reactivity, amygdala–mPFC
connectivity, or change in amygdala
reactivity across trials were
associated with separation anxiety
behaviors • On average, amygdala reactivity
decreased across trials (for both
fear and neutral faces) • Age-related change in amygdala
reactivity to fear faces in the first
half of trials, but not the second half • Similar, but somewhat weaker age-
related change for neutral faces • Amygdala–mPFC BSC was most
sensitive to selection of mPFC ROI • Global signal subtraction reduced
average amygdala–mPFC BSC, but
impacts on age-related changes
were small • No evidence that amygdala
reactivity, amygdala–mPFC
connectivity, or change in amygdala
reactivity across trials were
associated with separation anxiety
behaviors . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Age-related change in
amygdala–mPFC functional
connectivity to fear faces >
baseline, as measured by (BSC)
FSL preproc & GLM, high-pass
filter, 24 motion regressors, 2G
HRF, beta estimates, robust
group-level regression, included
previously analyzed scans
Amygdala ROI (all MNI space)
Bilateral, Left, Right
mPFC ROI (all MNI space)
3 different 5mm spheres, large vmPFC mask
Global Signal Subtraction
Yes, No
Group-level model covariates
Mean FD, Mean FD + run, Mean FD + scanner,
Mean FD + run + scanner
Group-level model quadratic term
Yes, No
Group-level model random slopes
Yes, No
3. Associations of amygdala
reactivity, change in amygdala
reactivity across trials, or
amygdala–mPFC FC with
separation anxiety
See supplemental methods p. 29
for details on included pipelines. Brain measure
Amygdala reactivity, amygdala reactivity slopes,
amygdala–mPFC gPPI, amygdala–mPFC BSC
Global Signal Subtraction (amygdala
reactivity slopes & BSC only)
Yes, No
Deconvolution step (gPPI only)
Yes, No
mPFC ROI (all MNI space, gPPI & BSC
only)
3 different 5mm spheres, large vmPFC mask
Separation anxiety outcome variable
RCADS, SCARED raw scores, SCARED t-scores
1Bolded choices indicate those most closely matching preregistered pipelines. 1375 1374 1375 1Bolded choices indicate those most closely matching preregistered pipelines. . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE
1376
1377
Figure 1. A. Schematic showing study inclusion criteria. B. Included scans at each study wave,
1378
with each dot representing one scan, and horizontal lines connecting participants across study
1379
waves. 1380
1381
1382
1383
1384
1385
1386
1387
1388
. CC-BY 4.0 International license
available under a Running head: AMYGDALA—MPFC AGE-RELATED CHANGE 1376 1376 1376
1377
Figure 1. A. Schematic showing study inclusion criteria. B. Included scans at each study wave,
1378
with each dot representing one scan, and horizontal lines connecting participants across study
1379
waves. 1380 Figure 1. A. Schematic showing study inclusion criteria. B. Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Included scans at each study wave
with each dot representing one scan, and horizontal lines connecting participants across study
waves. 387
1388
1389 8 1389 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Figure 2. Multiverse analyses of age-related change in amygdala reactivity. A. Specification
1390
curve of age-related change in fear > baseline amygdala reactivity. Points represent estimated
1391
linear age-related change and lines are corresponding 95% posterior intervals (PIs). Models are
1392
ordered by age-related change estimates, with the dotted line representing the median estimate
1393
across all specifications. Color indicates sign of beta estimates and whether respective posterior
1394
intervals include 0 (red = negative excluding 0; blue = negative including 0, green = positive
1395
including 0, black = median across all specifications). B. Model specification information
1396
corresponding to each model in A. Variables on the y-axis represent analysis choices,
1397
corresponding color-coded marks indicate that a choice was made, and blank space indicates
1398
that the choice was not made in a given analysis. Within each category panel (amygdala ROI,
1399
Group-Level Model, and Participant-Level Model), decision points are ordered from top to
1400
bottom by the median model rank when the corresponding choice is made (i.e. choices at the
1401
top of each panel tend to have more negative age-related change estimates). Black points with
1402
error bars represent the median and IQR ranks of specifications making the choice indicated on
1403
the corresponding line. C. Example participant-level data and model predictions for age-related
1404
related change in amygdala reactivity for both the fear > baseline (green) and neutral-baseline
1405
(orange) contrasts. Data are shown for a preregistered pipeline using a native space bilateral
1406
amygdala mask, 24 motion regressors, t-statistics, high-pass filtering, and participant-level
1407
GLMs in FSL. Points represent participant-level estimates, light lines connect estimates from
1408
participants with multiple study visits, and dark lines with shaded area represent model
1409
predictions and 95% posterior intervals. D. Specification curves for a subset of models
1410
separately parametrizing within-participant (right) vs. Running head: AMYGDALA—MPFC AGE-RELATED CHANGE between-participant (left) age-related
1411
change for both the fear > baseline (green) and neutral > baseline (orange) contrasts, as well as
1412
the median across specifications (black). See
1413
h
b
h
d
f
f
1414 ps://pbloom.shinyapps.io/amygdala_mpfc_multiverse/ for interactive visualizations. . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE
417
Figure 3. Age-related change in amygdala reactivity across trials. A. An example model of
418
estimated age-related change in slopes of beta estimates across both fear (green) and neutral
419
(orange) trials. Negative slopes represent higher amygdala activity in earlier trials relative to
420
later trials. B. Example models of estimated age-related change in amygdala reactivity for the
421
fear > baseline (left) and neutral > baseline (right) contrasts for both the first (red) and second
422
(blue) halves of trials. In both A and B, points represent participant-level estimates, light lines
423
connect estimates from participants with multiple study visits, and dark lines with shaded area
424
represent model predictions and 95% posterior intervals. C. Example single-trial model
425
predictions of estimated amygdala reactivity for fear (left) and neutral (right) faces as a function
426
of age and trial number. Age was modeled as a continuous variable, and average predictions for
427
participants of age 6 (red), 12 (green) and 18 (blue) years are shown for visualization purposes. 428
All estimates in A-C shown are from an example analysis pipeline using bilateral amygdala
429
estimates and without global signal correction. D. Specification curve for age-related change in
430
slopes across fear trials (i.e., many parallel analyses for the fear trials in subplot B). E. 431
Specification curve for age-related change in slopes across neutral trials (i.e., neutral trials in
432
plot B). GSS = global signal correction using post-hoc mean centering. For both D and E, color
433
indicates sign of beta estimates and whether respective posterior intervals include 0 (green =
434
positive including 0, purple = positive excluding 0, black = median across all specifications), and
435
horizontal dotted lines represent median estimates across all analysis decisions. Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Variables on
436
the y-axis represent analysis choices, corresponding color-coded marks indicate that a choice
437
was made, and blank space indicates that the choice was not made in a given analysis. 438 Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Running head: AMYGDALA—MPFC AGE-RELATED CHANGE 7
Figure 3. Age-related change in amygdala reactivity across trials. A. An example model of
8
estimated age-related change in slopes of beta estimates across both fear (green) and neutral
9
(orange) trials. Negative slopes represent higher amygdala activity in earlier trials relative to
0
later trials. B. Example models of estimated age-related change in amygdala reactivity for the
1
fear > baseline (left) and neutral > baseline (right) contrasts for both the first (red) and second
2
(blue) halves of trials. In both A and B, points represent participant-level estimates, light lines
3
connect estimates from participants with multiple study visits, and dark lines with shaded area
4
represent model predictions and 95% posterior intervals. C. Example single-trial model
5
predictions of estimated amygdala reactivity for fear (left) and neutral (right) faces as a function
6
of age and trial number Age was modeled as a continuous variable and average predictions for
7 1417
Figure 3. Age-related change in amygdala reactivity across trials. A. An example model of
1418
estimated age-related change in slopes of beta estimates across both fear (green) and neutral
1419
(orange) trials. Negative slopes represent higher amygdala activity in earlier trials relative to
1420
later trials. B. Example models of estimated age-related change in amygdala reactivity for the
1421
fear > baseline (left) and neutral > baseline (right) contrasts for both the first (red) and second
1422
(blue) halves of trials. In both A and B, points represent participant-level estimates, light lines
1423
connect estimates from participants with multiple study visits, and dark lines with shaded area
1424
represent model predictions and 95% posterior intervals. C. Example single-trial model
1425
predictions of estimated amygdala reactivity for fear (left) and neutral (right) faces as a function
1426
of age and trial number. Age was modeled as a continuous variable, and average predictions for
1427
participants of age 6 (red), 12 (green) and 18 (blue) years are shown for visualization purposes. 1428
All estimates in A-C shown are from an example analysis pipeline using bilateral amygdala
1429
estimates and without global signal correction. D. Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Specification curve for age-related change in
1430
slopes across fear trials (i.e., many parallel analyses for the fear trials in subplot B). E. 1431
Specification curve for age-related change in slopes across neutral trials (i.e., neutral trials in
1432
plot B). GSS = global signal correction using post-hoc mean centering. For both D and E, color
1433
indicates sign of beta estimates and whether respective posterior intervals include 0 (green =
1434
positive including 0, purple = positive excluding 0, black = median across all specifications), and
1435
horizontal dotted lines represent median estimates across all analysis decisions. Variables on
1436
the y-axis represent analysis choices, corresponding color-coded marks indicate that a choice
1437
was made, and blank space indicates that the choice was not made in a given analysis. 1438 . Age-related change in amygdala reactivity across trials. A. An example model of 1417
Figure 3. Age-related change in amygdala reactivity across trials. A. An example model of
1418
estimated age-related change in slopes of beta estimates across both fear (green) and neutral
1419
(orange) trials. Negative slopes represent higher amygdala activity in earlier trials relative to
1420
later trials. B. Example models of estimated age-related change in amygdala reactivity for the
1421
fear > baseline (left) and neutral > baseline (right) contrasts for both the first (red) and second
1422
(blue) halves of trials. In both A and B, points represent participant-level estimates, light lines
1423
connect estimates from participants with multiple study visits, and dark lines with shaded area
1424
represent model predictions and 95% posterior intervals. C. Example single-trial model
1425
predictions of estimated amygdala reactivity for fear (left) and neutral (right) faces as a function
1426
of age and trial number. Age was modeled as a continuous variable, and average predictions for
1427
participants of age 6 (red), 12 (green) and 18 (blue) years are shown for visualization purposes. 1428
All estimates in A-C shown are from an example analysis pipeline using bilateral amygdala
1429
estimates and without global signal correction. D. Specification curve for age-related change in
1430
slopes across fear trials (i.e., many parallel analyses for the fear trials in subplot B). E. 1431
Specification curve for age-related change in slopes across neutral trials (i.e., neutral trials in
1432 Figure 3. Age-related change in amygdala reactivity across trials. A. Running head: AMYGDALA—MPFC AGE-RELATED CHANGE It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Running head: AMYGDALA—MPFC AGE-RELATED CHANGE 1440
1441
1442
1443
Figure 4. Multiverse analyses of age-related change in amygdala–mPFC connectivity using
1444
gPPI methods. A. MNI space mPFC ROIs used in connectivity analyses. B. Example
1445
participant-level data and model predictions for age-related related change in amygdala–mPFC
1446
gPPI for analysis pipelines with a deconvolution step (red), or without (blue) for each of the four
1447
regions shown in A. Although deconvolution changed the sign of age-related change estimates,
1448
the estimates are not 'statistically significant' for each pipeline alone, except for mPFC ROIs 1 &
1449
2 without deconvolution. C. Specification curve of age-related change in fear > baseline
1450
amygdala–mPFC gPPI. Points represent estimated linear age-related change and lines are
1451
corresponding 95% posterior intervals. Models are ordered by age-related change estimates,
1452
and the dotted line represents the median estimate across all specifications. Color indicates
1453
sign of beta estimates and whether respective posterior intervals include 0 (blue = negative
1454
including 0, green = positive including 0, purple = positive excluding 0, black = median across all
1455
specifications). Black points with error bars represent the median and IQR ranks of
1456
specifications making the choice indicated on the corresponding line. D. Model specification
1457
information corresponding to each model in C. Variables on the y-axis represent analysis
1458
choices, corresponding color-coded marks indicate that a choice was made, and blank space
1459
indicates that the choice was not made in a given analysis. Within each category (Group-Level
1460
Model, mPFC ROI, and Participant-Level Model) respectively, decision points are ordered from
1461
top to bottom by the median model rank when the corresponding choice is made (i.e., choices at
1462
the top of each panel tend to have more negative age-related change estimates). See
1463
https://pbloom.shinyapps.io/amygdala_mpfc_multiverse/ for interactive visualizations. 1464
1465 1443
Figure 4. Multiverse analyses of age-related change in amygdala–mPFC connectivity using
1444
gPPI methods. A. Running head: AMYGDALA—MPFC AGE-RELATED CHANGE An example model of
418
estimated age-related change in slopes of beta estimates across both fear (green) and neutral
419
(orange) trials. Negative slopes represent higher amygdala activity in earlier trials relative to
420
later trials. B. Example models of estimated age-related change in amygdala reactivity for the
421
fear > baseline (left) and neutral > baseline (right) contrasts for both the first (red) and second
422
(blue) halves of trials. In both A and B, points represent participant-level estimates, light lines
423
connect estimates from participants with multiple study visits, and dark lines with shaded area
424
represent model predictions and 95% posterior intervals. C. Example single-trial model
425
predictions of estimated amygdala reactivity for fear (left) and neutral (right) faces as a function
426
of age and trial number. Age was modeled as a continuous variable, and average predictions for
427
participants of age 6 (red), 12 (green) and 18 (blue) years are shown for visualization purposes. 428
All estimates in A-C shown are from an example analysis pipeline using bilateral amygdala
429
estimates and without global signal correction. D. Specification curve for age-related change in
430
slopes across fear trials (i.e., many parallel analyses for the fear trials in subplot B). E. 431
Specification curve for age-related change in slopes across neutral trials (i.e., neutral trials in
432
plot B). GSS = global signal correction using post-hoc mean centering. For both D and E, color
433 p
(
y p
y
p
)
Specification curve for age-related change in slopes across neutral trials (i.e., neutral trials in
432
plot B). GSS = global signal correction using post-hoc mean centering. For both D and E, color
433
indicates sign of beta estimates and whether respective posterior intervals include 0 (green =
434
positive including 0, purple = positive excluding 0, black = median across all specifications), and
435
horizontal dotted lines represent median estimates across all analysis decisions. Variables on
436
the y-axis represent analysis choices, corresponding color-coded marks indicate that a choice
437
was made, and blank space indicates that the choice was not made in a given analysis. 438 Running head: AMYGDALA—MPFC AGE-RELATED CHANGE
. CC-BY 4.0 International license
available under a
which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Running head: AMYGDALA—MPFC AGE-RELATED CHANGE MNI space mPFC ROIs used in connectivity analyses. B. Example
1445
participant-level data and model predictions for age-related related change in amygdala–mPFC
1446
gPPI for analysis pipelines with a deconvolution step (red), or without (blue) for each of the four
1447
regions shown in A. Although deconvolution changed the sign of age-related change estimates,
1448
the estimates are not 'statistically significant' for each pipeline alone, except for mPFC ROIs 1 &
1449
2 without deconvolution. C. Specification curve of age-related change in fear > baseline
1450
amygdala–mPFC gPPI. Points represent estimated linear age-related change and lines are
1451
corresponding 95% posterior intervals. Models are ordered by age-related change estimates,
1452
and the dotted line represents the median estimate across all specifications. Color indicates
1453
sign of beta estimates and whether respective posterior intervals include 0 (blue = negative
1454
including 0, green = positive including 0, purple = positive excluding 0, black = median across all
1455
specifications). Black points with error bars represent the median and IQR ranks of
1456
specifications making the choice indicated on the corresponding line. D. Model specification
1457
information corresponding to each model in C. Variables on the y-axis represent analysis
1458
choices, corresponding color-coded marks indicate that a choice was made, and blank space
1459
indicates that the choice was not made in a given analysis. Within each category (Group-Level
1460
Model, mPFC ROI, and Participant-Level Model) respectively, decision points are ordered from
1461
top to bottom by the median model rank when the corresponding choice is made (i.e., choices at
1462
the top of each panel tend to have more negative age-related change estimates). See
1463
https://pbloom.shinyapps.io/amygdala_mpfc_multiverse/ for interactive visualizations. 1464 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Running head: AMYGDALA—MPFC AGE-RELATED CHANGE 1466
Figure 5. Multiverse analyses of age-related change in amygdala–mPFC connectivity using
1467
beta-series correlation (BSC) methods. A. Specification curve of age-related change in
1468
amygdala–mPFC BSC for fear trials. Points represent estimated linear age-related change and
1469
lines are corresponding 95% posterior intervals. Running head: AMYGDALA—MPFC AGE-RELATED CHANGE 1474
Variables on the y-axis represent analysis choices, corresponding color-coded marks indicate
1475
that a choice was made, and blank space indicates that the choice was not made in a given
1476
analysis. Within each category (amygdala ROI, group-level model, global signal subtraction, and
1477
mPFC ROI) respectively, decision points are ordered from top to bottom by the median model
1478
rank when the corresponding choice is made (i.e., choices at the top of each panel tend to have
1479
more negative age-related change estimates). Black points with error bars represent the median
1480
and IQR ranks of specifications making the choice indicated on the corresponding line. GSS =
1481
global signal correction using post-hoc mean centering. C. Example model predictions for age-
1482
related change in amygdala–mPFC BSC for fear trials for analysis pipelines with a global signal
1483
subtraction (GSS, post-hoc mean centering) step (red), or without (blue) for each of the four
1484
mPFC regions (see Figure 4A) with the left and right amygdala. Pipelines shown have random
1485
slopes, no covariates for task block or scanner, and no quadratic age term. D. Between-scan
1486
rank-order correlations between amygdala–mPFC connectivity measures. All gPPI measures
1487
are for the fear > baseline contrast, and BSC measures are for fear trials. See
1488
https://pbloom.shinyapps.io/amygdala_mpfc_multiverse/ for interactive visualizations. 1489
1490 1466
Figure 5. Multiverse analyses of age-related change in amygdala–mPFC connectivity using
1467
beta-series correlation (BSC) methods. A. Specification curve of age-related change in
1468
amygdala–mPFC BSC for fear trials. Points represent estimated linear age-related change and
1469
lines are corresponding 95% posterior intervals. Models are ordered by age-related change
1470
estimates, and the dotted line represents the median estimate across all specifications. Color
1471
indicates sign of beta estimates and whether respective posterior intervals include 0 (blue =
1472
negative including 0, green = positive including 0, purple = positive excluding 0, black = median
1473
across all specifications). B. Model specification information corresponding to each model in A. 1474
Variables on the y-axis represent analysis choices, corresponding color-coded marks indicate
1475
that a choice was made, and blank space indicates that the choice was not made in a given
1476
analysis. Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Models are ordered by age-related change
1470
estimates, and the dotted line represents the median estimate across all specifications. Color
1471
indicates sign of beta estimates and whether respective posterior intervals include 0 (blue =
1472
negative including 0, green = positive including 0, purple = positive excluding 0, black = median
1473
across all specifications). B. Model specification information corresponding to each model in A. 1474
Variables on the y-axis represent analysis choices, corresponding color-coded marks indicate
1475
that a choice was made, and blank space indicates that the choice was not made in a given
1476
analysis. Within each category (amygdala ROI, group-level model, global signal subtraction, and
1477
mPFC ROI) respectively, decision points are ordered from top to bottom by the median model
1478
rank when the corresponding choice is made (i.e., choices at the top of each panel tend to have
1479
more negative age-related change estimates). Black points with error bars represent the median
1480
and IQR ranks of specifications making the choice indicated on the corresponding line. GSS =
1481
global signal correction using post-hoc mean centering. C. Example model predictions for age-
1482
related change in amygdala–mPFC BSC for fear trials for analysis pipelines with a global signal
1483
subtraction (GSS, post-hoc mean centering) step (red), or without (blue) for each of the four
1484
mPFC regions (see Figure 4A) with the left and right amygdala. Pipelines shown have random
1485
slopes, no covariates for task block or scanner, and no quadratic age term. D. Between-scan
1486
rank-order correlations between amygdala–mPFC connectivity measures. All gPPI measures
1487
are for the fear > baseline contrast, and BSC measures are for fear trials. See
1488
https://pbloom.shinyapps.io/amygdala_mpfc_multiverse/ for interactive visualizations. 1489
1490 1466
Figure 5. Multiverse analyses of age-related change in amygdala–mPFC connectivity using
1467
beta-series correlation (BSC) methods. A. Specification curve of age-related change in
1468
amygdala–mPFC BSC for fear trials. Points represent estimated linear age-related change and
1469
lines are corresponding 95% posterior intervals. Models are ordered by age-related change
1470
estimates, and the dotted line represents the median estimate across all specifications. Color
1471
indicates sign of beta estimates and whether respective posterior intervals include 0 (blue =
1472
negative including 0, green = positive including 0, purple = positive excluding 0, black = median
1473
across all specifications). B. Model specification information corresponding to each model in A. Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Separate
497
specification curves for associations of amygdala reactivity (left), amygdala–mPFC connectivity
498
(both gPPI and BSC; center two panels), and amygdala reactivity slopes across trials (right) with
499
the three separation anxiety outcomes shown in A. Points represent estimated associations
500
between brain measures and separation anxiety (controlling for mean framewise displacement
501
and age) and lines are corresponding 95% posterior intervals. Models are ordered by beta
502
estimates, and the dotted line represents the median estimate across all specifications. Color
503
indicates sign of beta estimates and whether respective posterior intervals include 0 (red =
504
negative excluding 0, blue = negative including 0, green = positive including 0). Scores on each
505
separation anxiety outcome were z-scored for comparison. C. Example model predictions for
506
associations between fear > baseline amygdala–mPFC gPPI and each separation anxiety
507
measure. Predictions and 95% posterior intervals are plotted for each separation anxiety
508
measure separately for each mPFC region, and for gPPI pipelines with and without a
509
deconvolution step. Pipelines shown use robust regression, have random slopes, no covariates
510
for task block or scanner, and no quadratic age term. 511
512 1492
Figure 6. Multiverse analyses of associations between amygdala–mPFC circuitry and
1493
separation anxiety. A. Age-related change in SCARED and RCADS raw and t-scores for parent-
1494
reported separation anxiety subscales. The red dotted line in the middle panel represents the
1495
clinical threshold for the standardized RCADS measure (because this T-score measure is
1496
standardized based on age and gender, no age-related change is expected). B. Separate
1497
specification curves for associations of amygdala reactivity (left), amygdala–mPFC connectivity
1498
(both gPPI and BSC; center two panels), and amygdala reactivity slopes across trials (right) with
1499
the three separation anxiety outcomes shown in A. Points represent estimated associations
1500
between brain measures and separation anxiety (controlling for mean framewise displacement
1501
and age) and lines are corresponding 95% posterior intervals. Models are ordered by beta
1502
estimates, and the dotted line represents the median estimate across all specifications. Color
1503
indicates sign of beta estimates and whether respective posterior intervals include 0 (red =
1504
negative excluding 0, blue = negative including 0, green = positive including 0). Scores on each
1505
separation anxiety outcome were z-scored for comparison. C. Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Within each category (amygdala ROI, group-level model, global signal subtraction, and
1477
mPFC ROI) respectively, decision points are ordered from top to bottom by the median model
1478
rank when the corresponding choice is made (i.e., choices at the top of each panel tend to have
1479
more negative age-related change estimates). Black points with error bars represent the median
1480
and IQR ranks of specifications making the choice indicated on the corresponding line. GSS =
1481
global signal correction using post-hoc mean centering. C. Example model predictions for age-
1482
related change in amygdala–mPFC BSC for fear trials for analysis pipelines with a global signal
1483
subtraction (GSS, post-hoc mean centering) step (red), or without (blue) for each of the four
1484
mPFC regions (see Figure 4A) with the left and right amygdala. Pipelines shown have random
1485
slopes, no covariates for task block or scanner, and no quadratic age term. D. Between-scan
1486
rank-order correlations between amygdala–mPFC connectivity measures. All gPPI measures
1487
are for the fear > baseline contrast, and BSC measures are for fear trials. See
1488
https://pbloom.shinyapps.io/amygdala_mpfc_multiverse/ for interactive visualizations. 1489
1490 Multiverse analyses of age-related change in amygdala–mPFC connectivity using . CC-BY 4.0 International license
available under a
which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Running head: AMYGDALA—MPFC AGE-RELATED CHANGE 492
Figure 6. Multiverse analyses of associations between amygdala–mPFC circuitry and
493
separation anxiety. A. Age-related change in SCARED and RCADS raw and t-scores for parent-
494
reported separation anxiety subscales. The red dotted line in the middle panel represents the
495
clinical threshold for the standardized RCADS measure (because this T-score measure is
496
standardized based on age and gender, no age-related change is expected). B. Running head: AMYGDALA—MPFC AGE-RELATED CHANGE Example model predictions for
1506
associations between fear > baseline amygdala–mPFC gPPI and each separation anxiety
1507
measure. Predictions and 95% posterior intervals are plotted for each separation anxiety
1508
measure separately for each mPFC region, and for gPPI pipelines with and without a
1509
deconvolution step. Pipelines shown use robust regression, have random slopes, no covariates
1510
for task block or scanner, and no quadratic age term. 1511 92
Figure 6. Multiverse analyses of associations between amygdala–mPFC circuitry and
93
separation anxiety. A. Age-related change in SCARED and RCADS raw and t-scores for parent-
94
reported separation anxiety subscales. The red dotted line in the middle panel represents the
95
clinical threshold for the standardized RCADS measure (because this T-score measure is
96
standardized based on age and gender, no age-related change is expected). B. Separate
97
specification curves for associations of amygdala reactivity (left), amygdala–mPFC connectivity
98
(both gPPI and BSC; center two panels), and amygdala reactivity slopes across trials (right) with
99
the three separation anxiety outcomes shown in A. Points represent estimated associations
00
between brain measures and separation anxiety (controlling for mean framewise displacement
01
and age) and lines are corresponding 95% posterior intervals. Models are ordered by beta
02
estimates, and the dotted line represents the median estimate across all specifications. Color
03
indicates sign of beta estimates and whether respective posterior intervals include 0 (red =
04
negative excluding 0, blue = negative including 0, green = positive including 0). Scores on each
05
separation anxiety outcome were z-scored for comparison. C. Example model predictions for
06
associations between fear > baseline amygdala–mPFC gPPI and each separation anxiety
07
measure. Predictions and 95% posterior intervals are plotted for each separation anxiety
08
measure separately for each mPFC region, and for gPPI pipelines with and without a
09
deconvolution step. Pipelines shown use robust regression, have random slopes, no covariates
10
for task block or scanner, and no quadratic age term. 11 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 22, 2022. ;
https://doi.org/10.1101/2021.10.08.463601
doi:
bioRxiv preprint Running head: AMYGDALA—MPFC AGE-RELATED CHANGE 1514
Figure 7. Longitudinal test-retest Bayesian ICC estimates. ICC values are shown for amygdala
1515
reactivity (A), slopes of amygdala reactivity betas across trials (B), amygdala—mPFC functional
1516
connectivity using both gPPI and BSC methods (C), and separation anxiety and in-scanner
1517
head motion measurements (D). Shaded background colors depict whether ICC estimates are
1518
categorized as poor (< 0.4), fair (0.4 - 0.6), or good (0.6 – 0.75) reliability. No ICC estimates met
1519
the threshold for excellent reliability ( >0.75). Bayesian ICC estimates were calculated through a
1520
variance decomposition based on posterior predictive distributions. Negative values indicate
1521
higher posterior predictive variances not conditioned on random effect terms than conditioned
1522
on random effects terms. 1523
1524 1514
Figure 7. Longitudinal test-retest Bayesian ICC estimates. ICC values are shown for amygdala
1515
reactivity (A), slopes of amygdala reactivity betas across trials (B), amygdala—mPFC functional
1516
connectivity using both gPPI and BSC methods (C), and separation anxiety and in-scanner
1517
head motion measurements (D). Shaded background colors depict whether ICC estimates are
1518
categorized as poor (< 0.4), fair (0.4 - 0.6), or good (0.6 – 0.75) reliability. No ICC estimates met
1519
the threshold for excellent reliability ( >0.75). Bayesian ICC estimates were calculated through a
1520
variance decomposition based on posterior predictive distributions. Negative values indicate
1521
higher posterior predictive variances not conditioned on random effect terms than conditioned
1522
on random effects terms. 1523
1524
1525
1526 1514
Figure 7. Longitudinal test-retest Bayesian ICC estimates. ICC values are shown for amygdala
1515
reactivity (A), slopes of amygdala reactivity betas across trials (B), amygdala—mPFC functional
1516
connectivity using both gPPI and BSC methods (C), and separation anxiety and in-scanner
1517
head motion measurements (D). Shaded background colors depict whether ICC estimates are
1518
categorized as poor (< 0.4), fair (0.4 - 0.6), or good (0.6 – 0.75) reliability. No ICC estimates met
1519
the threshold for excellent reliability ( >0.75). Bayesian ICC estimates were calculated through a
1520
variance decomposition based on posterior predictive distributions. Negative values indicate
1521
higher posterior predictive variances not conditioned on random effect terms than conditioned
1522
on random effects terms. 1523
1524
1525
1526 1514
Figure 7. Longitudinal test-retest Bayesian ICC estimates. ICC values are shown for amygdala
1515
reactivity (A), slopes of amygdala reactivity betas across trials (B), amygdala—mPFC functional
1516
connectivity using both gPPI and BSC methods (C), and separation anxiety and in-scanner
1517
head motion measurements (D). Shaded background colors depict whether ICC estimates are
1518
categorized as poor (< 0.4), fair (0.4 - 0.6), or good (0.6 – 0.75) reliability. No ICC estimates met
1519
the threshold for excellent reliability ( >0.75). Bayesian ICC estimates were calculated through a
1520
variance decomposition based on posterior predictive distributions. Negative values indicate
1521
higher posterior predictive variances not conditioned on random effect terms than conditioned
1522
on random effects terms. 1523
1 24
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https://openalex.org/W3113181251
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http://remcb-puce.edu.ec/remcb/article/download/369/308
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es
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La agremiación médica
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Revista Ecuatoriana de Medicina y Ciencias Biológicas (REMCB)/Revista Ecuatoriana de Medicina y Ciencias Biológicas (REMCB) (En línea)
| 2,017
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cc-by-sa
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REVISTA ECUATORIANA DE
A:i<O II
Abril·· Junio
EDITORIAL
LA AGREMIACION MEDICA
Problema viejo y largamente discutido -ha sido éste, el de la agremi&ción obLigatoria de los médicos ecuatorianos. Desde !a época de Espejo, el
protomédico ·quiteño,· hasta nuest,ros días, han sido numerosos los intentos
de asociar a todos !os médicos en una sola entidad clasista. La· realización
más efectiva lo constituye la Federación Médica dcl Ec1wdor, la misma que
en l.os últi-mos años, gracias al prestigio y actividad· de sus dirigentes se ha
robustecido grandemente y ha logrado e! que l.as instituciones públicas la
reconozcan -de hecho- como la verdadera 1·epresentante de la clase mé'
dica del país.
Con motivo de un proyecto de ley, según el cual todos los médicos del
Ecuador se convertirían en miernbros de la Federación Médica, la discusión
se ha actualizado, pero sobre todo en e! sentido de 1'econocer las mwltiploes
ventajas de q1w la totalidad de pmfesiona!es constituyan· una sola asociación
clasista. La Federación Médica, con ser !a entidad que· ha logrado reunir,
en su seno, el 'mayor número de médicos no h-a logrado, especialmente, en
!as dos ciudades más populosas del país, afilia?' sino a un 50 o 70o/0 de profesionales.
Este hecho lim.ita su fuerza, su capacidad pam impedir que el mérlico
sea atropellado -por razones políticas} por ejemplo- en sus derechos, en su
estabilidad en el trabajo, en su dignidad profesional.
El Código Sanitario establece muchos de los de>·echos del médico, pero
en ausencia de un organismo sólido y poderoso que 1·espalde al pmfesional,
tales derechos no pasan de MT sino declmnaciones en el vacío.
Biblioteca Nacional del Ecuador "Eugenio Espejo"
70
REVISTA ECUATORIANA DE MEDICINA. VOL. II, N9 2, 1964
Por otra parte, como ha sucedido ya en muchos paíse~, es necesario que
alguna. entidad 1>e!e permanentemente po'l' !a idoneidad científica y moral del
médico. E! título profesional concedido por las uni1>ersidades de! país, cierte<mente,
el primero e indispensable paso. Pero las ciencias médicas 1W
son -ni !ejanamente- e! limitado acer1>o de conocimientos que el estudiante
adquiere en las aulas universitarias. Tampoco la medicina e.• estática. Por e!
contrario, evoluciona y progresa con un<L m.pidez asombmsa. Alguna entidad
médic<L debe responsabilizarse porque sus asociados sigan el ¡·itmo de prog>·eso de los conocimientos y del desarrollo de las ciencias; porque el médico,
lejos de convertirse en un peligro social po1· mal ejercicio profesional, siga
siendo el sctlvaguardia de la salud y de! bienestar de los ciudadanos.
es
Es necesario, asimismo, que una entidad médica regute las relaciones
entre profesionales, entre éstos y los pacientes, entre el médico y !as clínicas
u hospitales, entre los pacientes y estas instituciones. Hay sutiles relaciones
que escapan al ámbito de una ley general, como el Código Sanitario.
Todo esto debería esta,. en manos de una Federación Médica que agrupe a todos los profesionales y la cual podría contar con secciones especiales,
con funciones específicas, como el Colegio Médico, para efectos de la Licencia
de eje1·cicio de la medicina, el tribunal de honor, para el fallo sobre ciertos
conflictos profesionales, etc.
Por circunstancias de carácter histórico, la Federación Médica ha abarcado no sólo a médicos sino a los profesionales de ciencias afines como:
Odontología, Farmacia, Enfermería, etc. Esta época puede y debe ser superada. La Federación Médica debe asociar sólo a médicos, pues sus problemas
e intereses profesionales son específicos, en cambio puede formarse uná Confederación que asocie en un grande y amplio organismo a las varias Fede1'aciones Nacionale8 de médicos, odontólogos, bioquímicos, farmacéuticos, etc.
Biblioteca Nacional del Ecuador "Eugenio Espejo"
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https://openalex.org/W4320064016
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https://brill.com/downloadpdf/journals/jocp/aop/article-10.1163-26660393-bja10064/article-10.1163-26660393-bja10064.pdf
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English
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Corpus Linguistics for Pragmatics: A guide for research, written by Rühlemann, Christoph
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Contrastive pragmatics
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cc-by
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Book Review ∵ Rühlemann, Christoph, Corpus Linguistics for Pragmatics: A guide for research. London and New York: Routledge, 2019. 206 pages, ISBN: 978-0-429-45107-2 (ebk) This book is the first comprehensive research guide on corpus pragmatics. It
utilizes the corpus linguistic approaches to address core issues in pragmatics. Focus studies from current corpus linguistic research and practical applica
tions of using corpora are included. A companion website with the original
data for the practical assignments makes this book a user-friendly manual for
both seasoned researchers in pragmatics and undergraduate and graduate stu
dents interested in corpus pragmatics research. The book has eight chapters. The first and last chapters introduce and summarize the volume. The other
chapters (Chapters 2–7) discuss critical areas of corpus pragmatics research;
each of these chapters starts with an introduction of the critical area, followed
by one or two focus studies in corpus linguistics, and ends with a guided exer
cise and additional application activities. A review of each chapter in this book
is included below. This book is the first comprehensive research guide on corpus pragmatics. It
utilizes the corpus linguistic approaches to address core issues in pragmatics. Focus studies from current corpus linguistic research and practical applica
tions of using corpora are included. A companion website with the original
data for the practical assignments makes this book a user-friendly manual for
both seasoned researchers in pragmatics and undergraduate and graduate stu
dents interested in corpus pragmatics research. The book has eight chapters. The first and last chapters introduce and summarize the volume. The other
chapters (Chapters 2–7) discuss critical areas of corpus pragmatics research;
each of these chapters starts with an introduction of the critical area, followed
by one or two focus studies in corpus linguistics, and ends with a guided exer
cise and additional application activities. A review of each chapter in this book
is included below. Chapter 1 introduces the two parent fields of corpus pragmatics, corpus
linguistics and pragmatics, and the new interdisciplinary field, corpus prag
matics. One parent field, corpus linguistics, involves quantitative research with
frequencies and statistics. Although annotations are often time-consuming to
complete, annotated corpora are more convenient than raw text corpora for
researchers to search for more abstract linguistic patterns. The other parent
field, pragmatics, concerns the use of language in communicative contexts. The key to pragmatic analysis is the interpretation of different contextual vari
ables. © Dongmei Cheng, 2022 | doi:10.1163/26660393-bja10064
This is an open access article distributed under the terms of the CC BY 4.0 license.
Downloaded from Brill.com 10/24/2024 04:41:00AM
via Open Access. This is an open access article distributed under the terms
of the CC BY 4.0 license.
https://creativecommons.org/licenses/by/4.0/ Contrastive PragmaticS (2022) 1–5 Contrastive PragmaticS (2022) 1–5 icense.
Downloaded from Brill.com 10/24/2024 04:41:00AM
This is an open access article distributed under the terms
of the CC BY 4.0 license.
https://creativecommons.org/licenses/by/4.0/ Book Review Both verbal and nonverbal clues are used in pragmatic research, which
is traditionally qualitative and is done through careful reading of a small sam
ple of texts in contexts. Corpus pragmatics consists of both the quantitative
nature of corpus linguistics and the qualitative nature of pragmatic analysis. In this introductory chapter, the author includes a comprehensive overview of
the three fields; however, when introducing annotated corpora, it would have Book Review 2 been better if the author had noted the challenges associated with pragmatic
annotations, such as the challenges of segmenting the text and ensuring the
accuracy of the labels used in annotations (Archer, Aijmer, & Wichmann, 2012). y
(
j
)
Chapter 2 examines the study of speech acts using corpus approaches. Indirect speech acts are ones that speakers use to communicate more than
what they say verbally. Accurately interpreting indirect speech acts depends
on the mutually shared background between the speakers and the hearers. Brown and Levinson’s (1987) politeness theory explains the speakers’ motiva
tion to use indirect speech acts. The chapter’s focused study demonstrates how
to conduct corpus research on the speech act expression “why don’t you” in
the Cambridge and Nottingham Corpus of Discourse in English (CANCODE). Two types of speech acts are identified through analyzing the concordance
examples: a request for information (e.g., why don’t you address the envelope?)
and a suggestion for action (e.g., why don’t you come with us?). A study on the
collocates reveals the differences between these two speech acts. For example,
when “why don’t you” functions as a suggestion, a reporting verb (e.g., said) often
introduces it as part of direct speech not to threaten the addressee’s negative
face. The author’s thorough discussion of the classic works on speech acts is
helpful for those who need a refreshing review; his detailed interpretation of
the speaker’s preference of using indirect requests based on the politeness
theory provides a model for readers who seek the same type of theory-based
interpretations for their own speech act corpus data. Readers also had an
opportunity to apply the knowledge they learned in this chapter to a guided
task that compares the use of the “why not + V ” pattern in a corpus with the
“why don’t you” pattern analyzed in the focus study. Chapter 3 discusses the concept of deixis and the corresponding corpus
pragmatic studies. 10.1163/26660393-bja10064 | Contrastive PragmaticS (2022) 1–5
Downloaded from Brill.com 10/24/2024 04:41:00AM
via Open Access. This is an open access article distributed under the terms
of the CC BY 4.0 license.
https://creativecommons.org/licenses/by/4.0/ Book Review Deixis is situational coordinates that speakers use, verbally
and nonverbally, to communicate information regarding the context. Social
deictics can change depending on the formality of the situation, e.g., “eat
with someone at McDonald’s” vs. “dine with the monarch” (Rühlemann, 2019,
p. 66). The focused study included in this chapter presents the case of the dia
chronic change in the usage of the modal verb “must.” Results from this study
showed that across 80 years (1923–2006) of the data from the TIME magazine,
the deontic usage of “must” to express obligations decreased while the epis
temic use of “must” to convey limited knowledge increased (Rühlemann, 2019,
p. 68). As “deontic ‘must’ is a social deictic [which] encodes a social power
relationship,” Rühlemann (2019) attributes the decline of its frequency to
people’s changing perceptions of acceptable social power relations (p. 68). The authentic dialogue examples presented in this chapter and the author’s
detailed interpretations greatly facilitate readers’ understanding of deixis as Book Review 3 a context-dependent pragmatic concept. It would have been better, however,
if the author had included a clear list and definitions of the different catego
ries of deixis in this chapter, e.g., person deixis, social deixis, place (spatial)
deixis, emphatic deixis, time (temporal) deixis, and discourse deixis (Archer
et al., 2012). Chapter 4 discusses pragmatic markers and the associated corpus research
in this area. Rühlemann (2019) explains that pragmatic markers are short
navigation words that help people with effective communication. Corpus
research on pragmatic markers has been extensive because pragmatic mark
ers are “both high-frequent in and characteristic of spontaneous conversation”
(Rühlemann, 2019, p. 83). Research shows that the most frequent pragmatic
markers include single words such as “okay,” “well,” “right,” “actually,” “like,” “so,”
and “cos” and multi-word expressions such as “I (would) like,” “you see,” “I don’t
know,” “and stuff, ” “or something,” and “and things like that” (Rühlemann, 2019,
p. 83). The focused study of this chapter is on the acoustic properties of “well,”
which are affected by the different functions of the word. When “well” serves
as a pragmatic marker to indicate dispreference, restarting and quoting, it is
“toneless, reduced, and short,” different from the acoustic properties of this
word when it fulfills a non-pragmatic syntactic function (e.g., as an adverb
or an adjective) (Rühlemann, 2019, p. 98). Contrastive PragmaticS (2022) 1–5 | 10.1163/26660393-bja10064
Downloaded from Brill.com 10/24/2024 04:41:00AM
via Open Access. This is an open access article distributed under the terms
of the CC BY 4.0 license.
https://creativecommons.org/licenses/by/4.0/ Book Review The author’s presentation of this
chapter’s content reflects the nature of corpus pragmatics research by incor
porating both the corpus frequency data and qualitative functional analyses of
featured pragmatic markers. Readers should also appreciate the author’s inclu
sion of multifunctional pragmatic markers, “well” and “BE like,” in the focus
study and application tasks, as they are ubiquitous in both corpus research and
daily conversation encounters. Chapter 5 presents the concept of evaluation of corpus linguistics. Evaluation
is “the expression, overt or covert, implicit or explicit, verbal or nonverbal, of
the speaker’s stance” (Rühlemann, 2019, p. 110). Thus, evaluation expressions
are also referred to as stance markers (Bibler et al., 1999). Stance markers are
frequently used in storytelling as a way of persuasion. The author includes
a focused study on evaluative prosody (also called semantic prosody or dis
course prosody) in this chapter. Evaluative prosody in collocation means that
the keyword and its collocate share the same positive or negative evaluative
polarity (Partington, 2015). For example, the concordance examples from the
British National Corpus (BNC) showed that the phrasal verb “set in” is followed
by unpleasant things or events, such as “war years,” “skiddy weather,” “panic,”
“shock” and “depression” (Rühlemann, 2019, p. 121). Researching evaluative
prosody is difficult as it is “often hidden to the naked eye” (Rühlemann, 2019,
p. 124). The author sets another good example when presenting the content Contrastive PragmaticS (2022) 1–5 | 10.1163/26660393-bja10064
via Open Access. This is an open acces
https://cr Contrastive PragmaticS (2022) 1–5 | 10.1163/26660393-bja10064
via Open Access. This is an open access article distributed under the terms
of the CC BY 4.0 license
https://creativecommons org/licenses/by/4 0/ Book Review 4 of this chapter following the corpus pragmatics research approach. The fre
quency data on collocates of the targeted phrasal verbs, combined with the
qualitative analyses of the meanings of these collocates, aid a comprehen
sive understanding of the concept of evaluative prosody and how it works in
authentic language contexts. g
g
Chapter 6 analyzes turn-taking features in social interaction via corpus
and conversational analysis. Turning-taking is a universal behavior in con
versation. Rühlemann (2019) estimates that a typical speaker of the English
language takes about 1,200 turns and speaks about 13,000 words per day. The focused study included in this chapter is on the adjacency relationship
in the corpus. 10.1163/26660393-bja10064 | Contrastive PragmaticS (2022) 1–5
Downloaded from Brill.com 10/24/2024 04:41:00AM
via Open Access. This is an open access article distributed under the terms
of the CC BY 4.0 license.
https://creativecommons.org/licenses/by/4.0/ Book Review The term “adjacency pair” is used by conversational analysts
to “refer to coupled turns where the first part of the pair predicts what will
[happen] in the next part.” Typical adjacency pairs are “greeting-greeting,
offers/invitation-acceptance/rejection, and question-answer” (Archer et al.,
2012, p. 67). In conversational structure, speakers have preferences regard
ing the type of response included in the second pair of an adjacency pair. For example, the preferred reaction to request is granting, and dispreferred
response is denying (Archer et al., 2012). Rühlemann (2019) studied the con
versational structures in storytelling and discovered that storytellings “are
preference-organized” with “the preferred response [as the] one that mirrors
the teller’s stance towards the events” and such “stance convergence” is real
ized via “backchannels” (p. 153). Again, the detailed discussions and corpus
examples in this chapter facilitate readers’ understanding of conversational
structure in the English language. Chapter 7 explores the area of multimodality in corpus research. Rühlemann
(2019) states that a multimodal corpus is a “system in which the verbal, the
vocal, and the kinetic modalities are integrated into a unified whole” (p. 176). Although multimodal corpus linguistics is a relatively new area of study,
it is an important area in linguistics as “human (linguistic) communication
is basically multimodal” (Allwood, 2008, p. 223). The focused study of this
chapter investigates multimodality in storytelling. Gaze is a “key resource for
next-speaker selection” in storytelling (Rühlemann, 2019, p. 180). For example,
a speaker turned away his gaze from the interlocutor to keep the floor and fixed
his gaze on the interlocutor to elicit help when searching for a word (Allwood,
2008). “Alternating gaze” and “a heightened use of vocal and bodily resources”
are shown to be critical resources that storytellers use to guide the audience to
the climax of the stories (Rühlemann, 2019, p. 188). This chapter addresses an
important yet less researched area in corpus pragmatics by bringing the analy
ses of different language modalities together on specific pragmatic features. Book Review 5 Chapter 8 concludes the whole book. It reiterates the book’s primary goals
of introducing readers to the key areas and sample works of corpus prag
matics research and offering application tasks for readers to conduct their
corpus-pragmatic research projects. The author also emphasized that the five
core areas introduced in this book (i.e., speech acts, deixis, pragmatic markers,
evaluation, conversational structure, and multimodality) are deeply intercon
nected. Book Review Future research directions are also provided in this concluding chapter. For example, “the annotation of speech acts in a multimodal corpus” is a prom
ising direction because many speech functions can be performed nonverbally
and verbally (Rühlemann, 2019, p. 199). Despite the challenges of doing corpus
pragmatic research in multimodality, the author still views this area as a sig
nificant gap to fill in future research. This research helps us understand the
multimodal nature of conversational interactions. This volume is a good resource and practice book for researchers and stu
dents interested in conducting corpus research on pragmatics. Those looking
to explore a new interdisciplinary research area should benefit from learning
the author’s introductions to the corpus approaches on the core issues in prag
matics, examining the focus tasks, and practicing the application tasks. Dongmei Cheng | ORCID: 0000-0001-5207-6654
Associate Professor of Applied Linguistics, Department of Literature
and Languages, Texas A&M University Commerce, Commerce, TX,
United States
dongmei.cheng@tamuc.edu Contrastive PragmaticS (2022) 1–5 | 10.1163/26660393-bja10064
Downloaded from Brill.com 10/24/2024 04:41:00AM
via Open Access. This is an open access article distributed under the terms
of the CC BY 4.0 license.
https://creativecommons.org/licenses/by/4.0/ References Allwood, Jens. 2008. Multimodal corpora. In: Anke Lüdeling and Merja Kytŏ (eds.),
Corpus Linguistics: An International Handbook. Berlin: Mouton de Gruyter. 207–225. Archer, Dawn, Karin Aijmer, and Anne Wichmann. 2012. Pragmatics: An Advanced
Resource Book for Students. New York: Routledge. Allwood, Jens. 2008. Multimodal corpora. In: Anke Lüdeling and Merja Kytŏ (eds.),
Corpus Linguistics: An International Handbook. Berlin: Mouton de Gruyter. 207–225. Archer, Dawn, Karin Aijmer, and Anne Wichmann. 2012. Pragmatics: An Advanced
Resource Book for Students. New York: Routledge. Biber, Douglas, Stig Johansson, Geoffrey Leech, Susan Conrad, and Edward Finegan. 1999. Longman Grammar of Spoken and Written English. Harlow: Pearson. Brown, Penelope, and Stephen C. Levinson. 1987. Politeness: Some Universals in Language
Use. Cambridge: Cambridge University Press. Partington, Alan. 2015. Evaluative prosody. In: Karin Aijmer and Christoph Rühlemann
(eds.), Corpus Pragmatics: A Handbook. Cambridge: Cambridge University Press. 279–303. f
g
Biber, Douglas, Stig Johansson, Geoffrey Leech, Susan Conrad, and Edward Finegan. 1999. Biber, Douglas, Stig Johansson, Geoffrey Leech, Susan Conrad, and Edward Finegan. 1999. Longman Grammar of Spoken and Written English. Harlow: Pearson. Biber, Douglas, Stig Johansson, Geoffrey Leech, Susan Conrad, and Edward Finegan. 1999. Longman Grammar of Spoken and Written English. Harlow: Pearson. Longman Grammar of Spoken and Written English. Harlow: Pearson. Brown Penelope andStephenC Levinson 1987 Politeness:SomeUniversalsinLanguage Longman Grammar of Spoken and Written English. Harlow: Pearson. Brown, Penelope, and Stephen C. Levinson. 1987. Politeness: Some Universals in Language
Use Cambridge: Cambridge University Press Brown, Penelope, and Stephen C. Levinson. 1987. Politeness: Some Universals in Language
Use. Cambridge: Cambridge University Press. Brown, Penelope, and Stephen C. Levinson. 1987. Politeness: Some Universals in Language
Use. Cambridge: Cambridge University Press. Brown, Penelope, and Stephen C. Levinson. 1987. Politeness: Some Universals in Language
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(eds.), Corpus Pragmatics: A Handbook. Cambridge: Cambridge University Press. 279–303. Partington, Alan. 2015. Evaluative prosody. In: Karin Aijmer and Christoph Rühlemann
(eds.), Corpus Pragmatics: A Handbook. Cambridge: Cambridge University Press. 279–303. Contrastive PragmaticS (2022) 1–5 | 10.1163/26660393-bja10064
via Open Access. This is an open acces
htt
// Contrastive PragmaticS (2022) 1–5 | 10.1163/26660393-bja10064
via Open Access. This is an open access article distributed under the terms
of the CC BY 4.0 license. https://creativecommons org/licenses/by/4 0/ Contrastive PragmaticS (2022) 1–5 | 10.1163/26660393-bja10064
via Open Access. This is an open access
https://crea
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https://openalex.org/W3020356371
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https://europepmc.org/articles/pmc7189750?pdf=render
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English
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Development of an Improved Rotational Orthosis for Walking With Arm Swing and Active Ankle Control
|
Frontiers in neurorobotics
| 2,020
|
cc-by
| 8,155
|
Development of an Improved
Rotational Orthosis for Walking With
Arm Swing and Active Ankle Control
Zaile Mu 1, Qiuju Zhang 1, Guo-Yuan Yang 2, Le Xie 3 and Juan Fang 1,2*†
1 School of Mechanical Engineering, Jiangnan University, Wuxi, China, 2 Med-X Research Institute and School of Biomedical
Engineering, Shanghai Jiao Tong University, Shanghai, China, 3 Institute of Medical Robotics, Shanghai Jiao Tong University,
Shanghai, China Based on interlimb neural coupling, gait robotic systems should produce walking-like
movement in both upper and lower limbs for effective walking restoration. Two orthoses
were previously designed in our lab to provide passive walking with arm swing. However,
an active system for walking with arm swing is desirable to serve as a testbed for
investigation of interlimb neural coupling in response to voluntary input. Given the
important function of the ankle joint during normal walking, this work aimed to develop
an improved rotational orthosis for walking with arm swing, which is called ROWAS
II, and especially to develop and evaluate the algorithms for active ankle control. After
description of the mechanical structure and control schemes of the overall ROWAS II
system, the closed-loop position control and adjustable admittance control algorithms
were firstly deduced, then simulated in Matlab/Simulink and finally implemented in the
ROWAS II system. Six able-bodied participants were recruited to use the ROWAS II
system in passive mode, and then to estimate the active ankle mechanism. It was
showed that the closed-loop position control algorithms enabled the ROWAS II system
to track the target arm-leg walking movement patterns well in passive mode, with the
tracking error of each joint <0.7◦. The adjustable admittance control algorithms enabled
the participants to voluntarily adjust the ankle movement by exerting various active force. Higher admittance gains enabled the participants to more easily adjust the movement
trajectory of the ankle mechanism. The ROWAS II system is technically feasible to
produce walking-like movement in the bilateral upper and lower limbs in passive mode,
and the ankle mechanism has technical potential to provide various active ankle training
during gait rehabilitation. This novel ROWAS II system can serve as a testbed for further
investigation of interlimb neural coupling in response to voluntary ankle movement and
is technically feasible to provide a new training paradigm of walking with arm swing and
active ankle control. Keywords: interlimb neural coupling, adjustable admittance control, pole-placement design, active ankle control,
gait rehabilitation ORIGINAL RESEARCH
published: 22 April 2020
doi: 10.3389/fnbot.2020.00017 †Present address:
Juan Fang,
Institute for Rehabilitation and
Performance Technology (IRPT), Bern
University of Applied Sciences,
Burgdorf, Switzerland Edited by:
Alois C. Knoll,
Technical University of
Munich, Germany Reviewed by:
Ning Sun,
Nankai University, China
Jose De Jesus Rubio,
National Polytechnic Institute of
Mexico, Mexico Reviewed by:
Ning Sun,
Nankai University, China
Jose De Jesus Rubio,
National Polytechnic Institute of
Mexico, Mexico *Correspondence:
Juan Fang
juan.fang@bfh.ch †Present address:
Juan Fang,
Institute for Rehabilitation and
Performance Technology (IRPT), Bern
University of Applied Sciences,
Burgdorf, Switzerland Received: 03 December 2019
Accepted: 10 March 2020
Published: 22 April 2020 INTRODUCTION 2003). Saglia et al. developed admittance control algorithms for
active ankle rehabilitation (Saglia et al., 2010). Taherifar et al. designed a smart assist-as-needed control system with a variable
admittance control strategy which reduced the interaction energy
between the user and the exoskeleton device (Taherifar et al.,
2018). Zhang et al. developed an admittance controller with
EMG-based torque prediction and achieved multiple assistive
gait patterns (Gui et al., 2017). Admittance control algorithms
promote active participation of the users during training in the
rehabilitation robotic systems. In parallel with the traditional therapeutic techniques for walking
rehabilitation, robot-assisted gait training reduces the working
load of therapists and shortens the rehabilitation cycle for
patients (Lum et al., 2002). Rehabilitation robotic systems can
provide repetitive and intensive training in various modalities
(Orrell et al., 2006; Muramatsu and Takano, 2007; Chang and
Kim, 2013). Accordingly, they are widely used clinically to assist
rehabilitation of walking. Human walking is a synchronous movement of upper and
lower limbs. Arm swing helps to stabilize gait and to reduce
energy expenditure (Bruijn et al., 2010). The coordinated
movement between upper and lower limbs during human
walking is considered to be neutrally coordinated (Dietz
et al., 2010). Based on the interlimb neural coupling, gait
robotic systems should produce synchronous movement of
upper and lower limbs for effective walking restoration (Ferris
et al., 2006). Two rotational orthoses for walking with arm
swing were designed in our lab, called ROWAS (Fang et al.,
2017a) and aROWAS (Fang et al., 2017b). The drives were
installed bilaterally on the shoulder, hip, knee, and ankle joints. Both ROWAS and aROWAS achieved walking-like coordinated
movement in the bilateral upper and lower limbs in passive mode. However, an active system for arm-leg walking is desirable to
serve as a testbed for investigation of interlimb neural coupling
in response to voluntary input. Our previous ROWAS and aROWAS systems only provided
passive training (Fang et al., 2017a,b). Given the important
function of the ankle joint during normal walking, it is desirable
to implement active ankle movement training in the arm-leg
synchronized walking. Furthermore, such a robotic system can
serve as an effective tool for investigation of interlimb neural
coupling in response to voluntary input. This work aimed to
develop an improved rotational orthosis for walking with arm
swing, which is called ROWAS II, and especially to develop and
evaluate the algorithms for active ankle control. Citation: Mu Z, Zhang Q, Yang G-Y, Xie L and
Fang J (2020) Development of an
Improved Rotational Orthosis for
Walking With Arm Swing and Active
Ankle Control. Front. Neurorobot. 14:17. doi: 10.3389/fnbot.2020.00017 April 2020 | Volume 14 | Article 17 Frontiers in Neurorobotics | www.frontiersin.org Development of ROWAS II and Active Ankle Control Mu et al. INTRODUCTION Our previous
work briefly described the admittance control algorithms which
produced active ankle movement with a fixed admittance gain
(Mu et al., 2019). This work explained this investigation in detail
by firstly assessing the functionality of the overall ROWAS II
system, and then evaluating the adjustable admittance control
algorithms for the ankle mechanism in the ROWAS II system. Rehabilitation robotic systems often have passive and active
movement modes to provide various training modalities for
patients with different motor functions. Passive training, which
allows patients to follow a fixed reference trajectory, is suitable
for those in the early post-injury stage. Cai et al. observed that
the spinal-cord-transected mice that performed assist-as-needed
training regained better movement abilities than the mice that
performed training with fixed trajectories (Cai et al., 2006). Active training is often used by the patients who can perform
certain voluntary movement (Tian et al., 2007). Furthermore,
the ankle joint plays a key role in human walking (Kepple
et al., 1997; Mcnealy and Gard, 2008), such as swing initiation,
weight support and forward progression. So active training of
the ankle joint is essential for gait restoration for the patients
with impaired walking ability. However, there are few gait
rehabilitation robotics in the market that integrate active ankle
training (Pratt, 2000; Susanna et al., 2008; Esquenazi and Packel,
2012). Frontiers in Neurorobotics | www.frontiersin.org METHODS The mechanical development of the ROWAS II system was
performed using SolidWorks (Version 2016, Dassault Systèmes
SolidWorks Corporation, Massachusetts, USA). After the overall
control scheme of the ROWAS II system was described, the
control algorithms for passive and active training were firstly
deduced, then simulated in Matlab/Simulink (MathWorks Inc.,
2015a, USA) and finally implemented in the ROWAS II system. Six able-bodied participants were recruited to evaluate the
functionality of the ROWAS II system and especially the active
ankle mechanism. Mechanical Development p
The ROWAS II system is mainly composed of a bed frame, a
body weight support (BWS) system, mechanisms for the upper
and lower limbs and a ground-simulation plate (Figure 1). The
bed frame is composed of square steels, while the mechanisms for
the upper and lower limbs are made of aluminum alloy. Using
two linear actuators (1013CPC, Moteck Co., Ltd., China), the
bed frame can be tilted from a horizontal position to a vertical
position to provide training in different positions. Actuated by a
stepper motor (86HS45, Leadshine Technology Co., Ltd., China)
with a gearbox (ratio of 10:1), the BWS system can support a
user of 100 kg. In addition, the system can adjust the lengths
of the upper and lower limbs so as to fit users with different
heights between 1.50 and 1.80 m. The mechanical stops constrain
the mechanical range of motion (ROM) for each joint. The There are several control approaches, such as the state-
space method in modern control (Rubio et al., 2019a), and its
successful application in electric vehicle control (Gao et al., 2020),
perturbations attenuation (Rubio et al., 2019b) and manipulator
control (Rubio, 2018). Admittance algorithms in classical control
are often used and successfully applied in many rehabilitation
systems for active training. Compared with impedance control
(Hogan, 1985), admittance control does not require an accurate
plant model, and can be implemented by adding a force sensor
on the link position (Richardson et al., 2003). Richardson et al. applied an admittance scheme to achieve a flexible control of
a pneumatic physiotherapy robotic system (Richardson et al., April 2020 | Volume 14 | Article 17 2 Development of ROWAS II and Active Ankle Control Mu et al. FIGURE 1 | The ROWAS II system: (A) the CAD model and (B) the prototype with a test participant. (1) Bed frame, (2) BWS system, (3) mechanism for the upper
limb, (4) mechanisms for the lower limbs, (5) linear actuator, and (6) ground-simulation plate. Adapted from our previous paper (Mu et al., 2019) with permission
(license:4797140179422) from IEEE. FIGURE 1 | The ROWAS II system: (A) the CAD model and (B) the prototype with a test participant. (1) Bed frame, (2) BWS system, (3) mechanism for the upper
limb, (4) mechanisms for the lower limbs, (5) linear actuator, and (6) ground-simulation plate. Adapted from our previous paper (Mu et al., 2019) with permission
(license:4797140179422) from IEEE. Control Development
Overall Control Scheme hip, knee, and ankle joints to enable passive walking-like training
in the ROWAS II system. The nominal plant Po(s) linking the input voltage V to the
hardware controller and the actual angle, was expressed as: To control the eight motors for synchronized movement in
the bilateral upper and lower limbs, eight hardware controllers
(maxon motor, Switzerland) were used and set in speed mode
(Figure 3). As one NI data acquisition card (PCI 6221, National
Instruments, USA) has only two analog-output channels, one PCI
card can transfer data for two hardware controllers. Therefore,
four PCI cards were used for the eight hardware controllers
and were inserted into three computers. One computer [Lenovo
(Beijing) Co., Ltd., China] has two PCI slots while the other
two computers [Dell (China) Co., Ltd., China] have one PCI
slot. These three computers established synchronization via a
digital trigger sent by these PCI cards. The angle sensors (Xinle
Co., Ltd., Shanghai, China) measured the movement of the eight
joints, which was used to develop the closed-loop position control
algorithms. The force sensors provided the force information
from the participants, which was used to develop the adjustable
admittance control algorithms. Part of the following control
algorithms was adapted from our previous paper (Mu et al., 2019)
with permission (license:4797130595869) from IEEE. V →θ : Po(s) = Bo(s)
Ao(s) = k
s , na = 1,
(1) (1) where na is the degree of the polynomial Ao(s), and the steady-
state gain k was obtained empirically using system identification. The feedback controller Cfb(s) was obtained using the pole-
placement approach (Aström and Murray, 2010), which was a
linear, time invariant and strictly-proper transfer function, θ*-θ →V : Cfb(s) = G(s)
H(s),
(2) (2) where the polynomials G(s) and H(s) can be identified as G(s) = gngsng + gng−1sng−1 + · · · + g0,
(3)
H(s) = snh + hnh−1snh−1 + · · · + h0. (4) (3)
(4) (4) Mechanical Development FIGURE 2 | The models of the mechanisms for the right upper and lower limbs. (A) Upper limb model: (1) shoulder motor (EC 90), (2) angle sensor, (3) gearbox (ratio
of 100:1), and (4) shoulder adjusting bars. (B) Lower limb model: (1) hip assisting bar, (2), (5) and (10) angle sensor, (3) hip motor (RE 50), (4) and (11) force sensor, (6)
knee motor (RE 40) with a gearbox (ratio of 3.5:1), (7) knee assisting bar, (8) ankle assisting bar, (9) ankle motor (RE 40), and (12) shoe platform. FIGURE 2 | The models of the mechanisms for the right upper and lower limbs. (A) Upper limb model: (1) shoulder motor (EC 90), (2) angle sensor, (3) gearbox (ratio
of 100:1), and (4) shoulder adjusting bars. (B) Lower limb model: (1) hip assisting bar, (2), (5) and (10) angle sensor, (3) hip motor (RE 50), (4) and (11) force sensor, (6)
knee motor (RE 40) with a gearbox (ratio of 3.5:1), (7) knee assisting bar, (8) ankle assisting bar, (9) ankle motor (RE 40), and (12) shoe platform. emergency stop button for the control system can ensure users’
safety in the ROWAS II system. which were installed behind the hip and knee mechanisms and
in front of the ankle mechanism. The series linear actuator
composed of a motor and a ball screw assembly (TBI Motion
Technology Co., Ltd., China). It retracted or extended to actuate
the hip, knee, and ankle joints. The force sensor (Jnsensor Co.,
Ltd., China) at the end of the actuator can measure the force to
pull and push the leg segment. The mechanisms of the upper and lower limbs (Figure 2) were
actuated by servo drives (maxon motor, Switzerland). Different
from the previous ROWAS systems which had the motors
mounted on bilateral sides of the hip, knee, and ankle joints (Fang
et al., 2017a,b), the ROWAS II system used series linear actuators, April 2020 | Volume 14 | Article 17 Frontiers in Neurorobotics | www.frontiersin.org 3 Development of ROWAS II and Active Ankle Control Mu et al. FIGURE 3 | Control scheme. FIGURE 3 | Control scheme. Closed-Loop Position Control for the ROWAS II
System The transfer function of the overall system To(s) is The transfer function of the overall system To(s) is Frontiers in Neurorobotics | www.frontiersin.org April 2020 | Volume 14 | Article 17 System The closed-loop position control algorithms were developed to
make the actual angle θ track the target angle θ∗(Figure 4A), and
were implemented in the bilateral mechanisms of the shoulder, θ∗→θ : To(s) =
CfbPo
1 + CfbPo
=
BoG
AoH + BoG. (5) (5) April 2020 | Volume 14 | Article 17 Frontiers in Neurorobotics | www.frontiersin.org 4 Development of ROWAS II and Active Ankle Control Mu et al. FIGURE 4 | Control algorithms. Adapted from our previous paper (Mu et al., 2019) with permission (license:4797130595869) from IEEE. (A) Closed-loop position
control and (B) adjustable admittance control. FIGURE 4 | Control algorithms. Adapted from our previous paper (Mu et al., 2019) with permission (license:4797130595869) from IEEE. (A) Closed-loop position
control and (B) adjustable admittance control. So, the order of To(s) n1 and the number of the unknown
parameters n2, are expressed as
Therefore, the feedback controller Cfb(s) is
2 (6)
(7)
Cfb(s) =
g0
s + h0
=
ω2
n
k
s + 2ξωn
. (14) p
2,
p
n1 = na + nh,
(6)
n2 = ng + 1 + nh,
(7)
Cfb(s) =
g0
s + h0
=
ω2
n
k
s + 2ξωn
. (14) n1 = na + nh,
(6)
n2 = ng + 1 + nh,
(7)
Cfb(s) =
g0
s + h0
=
ω2
n
k
s + 2ξωn
. n1 = na + nh,
(6)
n2 = ng + 1 + nh,
(7) (14) (7) In the closed-loop position control algorithms, the critical
damping ratio ζ = 1 was employed when the system has no
overshoot in response to a step input. In this case, the parameter
ωn is approximately ωn = 3.35/tr (Nise, 2000), where tr, which
is the rise time of the closed-loop system, was defined here as
tr = 0.8 s. To obtain a unique solution to the controller Cfb(s), n1 should be
equal to n2, thus, na = ng + 1. (8) (8) Equations (1) and (8) yield ng = 0. Therefore, the transfer
functions of Cfb(s) and To(s) are: Frontiers in Neurorobotics | www.frontiersin.org Adjustable Admittance Control for the Ankle Joint Cfb(s) = G(s)
H(s)=
g0
s + h0
,
(9)
To(s) =
CfbPo
1 + CfbPo
=
kg0
s2 + h0s + kg0
. (10) (9) The adjustable admittance control algorithms provided various
degrees of active movement control, and were implemented in
the right ankle mechanism. The adjustable admittance control
(Figure 4B) differed from the closed-loop position control
(Figure 4A) in that the target position θ∗was modified by θamt,
which depended on the active force F (Figure 4B) exerted by
the participant and the adjustable admittance gain Ga, which are
described now. In the closed-loop position control algorithms,
the target position θ∗was the reference ankle trajectory, while
in the adjustable admittance control algorithms, the target
position θ∗was the reference ankle trajectory plus the admittance
angle θamt. (10) The relationship between the target angle θ∗to the actual angle θ
of the overall system in the time domain is: The relationship between the target angle θ∗to the actual angle θ
of the overall system in the time domain is: θ∗=
¨θ + h0 ˙θ + kg0 θ
kg0
. (11) (11) Setting To(s) to a standard 2nd order transfer function To(s) =
ω2
n
s2 + 2ξωns + ω2n
,
(12) The admittance control algorithms used a stiffness-damping-
inertial system to represent the relationship between the adjusted
angle θamt and active force F, giving (12) where ξ and ωn are the damping ratio and natural frequency,
respectively. Comparison between Equations (10) and (12)
results in F = M ¨θamt + P ˙θamt + Qθamt,
(15) (15) where M, P, and Q are, respectively, the inertia, damping
and stiffness of the admittance control strategy. By Laplace g0 = ω2
n
k , h0 = 2ξωn. (13) (13) April 2020 | Volume 14 | Article 17 Frontiers in Neurorobotics | www.frontiersin.org Mu et al. Development of ROWAS II and Active Ankle Control FIGURE 5 | Simulation model. FIGURE 5 | Simulation model. transformation, the admittance controller Camt was yielded as the current study. A saturation block from Matlab/Simulink was
included to restrict the motor speed within the limit. Camt(s) = θamt(s)
F(s)
=
1
Ms2 + Ps + Q . (16)
Camt(s) = 1
Q ×
Q
M
s2 + P
M s + Q
M
. (17) Camt(s) = θamt(s)
F(s)
=
1
Ms2 + Ps + Q . Simulation of the Control Algorithms g
A
simulation
model
was
developed
in
Matlab/Simulink
to evaluate the control algorithms (Figure 5). Po was the
experimentally obtained transfer function, while Cfb and C′amt
were, respectively, the control algorithms deduced previously. The model simulated the closed-loop position control algorithms
in passive mode when the switch was connected to a. Updating
Po to the transfer functions for the mechanisms of bilateral
shoulder, hip, knee, and ankle joints yielded the simulation
results of synchronous movement in the overall ROWAS
II system running in passive mode. When the switch was
connected to b, the model simulated the adjustable admittance
control algorithms in active mode, where Po was the transfer
function of the right ankle mechanism. The active force was
obtained based on the experimental force measurement. The
model simulated at a sampling frequency of 200 Hz. The
simulated angle was used to calculate the root-mean-square
tracking error (RMSE) in section Experimental Evaluation of the
ROWAS II System. Camt(s) = GaC′amt,
(18)
C′amt(s) =
Q
M
s2 + P
M s + Q
M
. (19) (18) (19) Setting C′amt(s) to a standard second-order transfer function as C′amt(s) =
ω2
n
s2 + 2ξωns + ω2n
,
(20) (20) where ξ and ωn are the damping ratio and natural frequency
respectively. Comparison between Equations (19) and (20)
results in ωn =
r
Q
M , ξ =
P
2√MQ. (21) (21) Therefore Therefore Q = Mωn2, P = 2ξ
p
MQ. (22) (22) Adjustable Admittance Control for the Ankle Joint (16) Although the adjustable admittance controller in Equation
(20) and the transfer function of the overall position control
system To(s) in Equation (12) coincidently used the same
response criteria such as the damping ratio and rise time,
which largely depended upon the mechanical setup of the
ROWAS II system, the adjustable admittance controller and the
position controller were totally different in their functions and
physical meanings. (16) Thus Camt(s) = 1
Q ×
Q
M
s2 + P
M s + Q
M
. (17) (17) To develop the adjustable admittance control algorithms, an
admittance gain Ga was introduced. So To develop the adjustable admittance control algorithms, an
admittance gain Ga was introduced. So Frontiers in Neurorobotics | www.frontiersin.org Experimental Evaluation of the ROWAS II
System The mechanical structure of the ankle mechanism yielded the
inertial component M = 2 Ns2/◦. Using the same parameters ξ
= 1, tr = 0.8 s as adopted in the position control, Equation (22)
yielded the admittance parameters P = 16.65 Ns/◦, Q = 34.92 N/◦. Six able-bodied participants were recruited to evaluate the
system functionality (Table 1). In this experiment, the bed
frame was tilted up to 75◦relative to the ground, and
the walking cycle of the ROWAS II system was 7 s. The
sampling
frequency
of
the
control
system
was
200 Hz. Besides, the joints angle profiles for the bilateral shoulder,
hip, knee, and ankle joints, which were recorded in a
standard normal gait analysis experiment (Fang et al., 2015), In the admittance control algorithms the admittance gain Ga
was tuned. Actuator saturation is often observed in movement
control (Sun et al., 2020). Inclusion of saturation functions in the
control system can effectively reduce the tracking error brought
by input saturation (Sun et al., 2019), and can be also applied in April 2020 | Volume 14 | Article 17 Frontiers in Neurorobotics | www.frontiersin.org 6 Development of ROWAS II and Active Ankle Control Mu et al. were used as the reference trajectories θr for the ROWAS
II system. the bilateral shoulder, hip, and knee joints and the left ankle
mechanism were fixed so that the participant was in a standing
position. The right ankle mechanism ran firstly in passive
mode, where the closed-loop position control algorithms were
implemented. The participant supported their weight on their
left foot, and followed the movement produced by the right ankle
mechanism. Then the right ankle mechanism ran in active mode,
where low admittance (Ga = 0.6) and high admittance (Ga = 1.6)
gains were implemented. Four different movement cases were
tested, which were: The overall ROWAS II system was evaluated in passive
mode, where the eight joints ran the closed-loop position
control algorithms (section Closed-Loop Position Control for
the ROWAS II System). Supported by the BWS system, the
participant put the feet on the shoe platform (Figure 1B). The mechanisms of the bilateral shoulder, hip, knee, and
ankle joints in the ROWAS II system were alighted to
the corresponding joints of the participant. The participants
passively followed the movement produced by the ROWAS II
system and repeated each walking subsession three times. Experimental Evaluation of the ROWAS II
System Please
see the Supplementary Video “Test of the ROWAS II System”
to see the performance of a representative participant during
the experiment. (1) The participant always lifted up the right foot voluntarily
during the whole gait cycle; (2) The participant lifted up and pushed down the right
foot voluntarily within 30–60% and 60–80% of the gait
cycle, respectively; In the subtests of the ankle mechanism, the participant
still used the overall ROWAS II system. The mechanisms of (3) The participant pushed down and lifted up the right
foot voluntarily within 30–60% and 60–80% of the gait
cycle, respectively; TABLE 1 | Participant information. Participant
P1
P2
P3
P4
P5
P6
Age range (yrs)
20–25
20–25
20–25
25–30
30–35
20–25
Body mass (kg)
55
48
59
62
55
74
Height (m)
1.65
1.60
1.72
1.66
1.60
1.76 y
p
y
(4) The participant always pushed down the right foot
voluntarily during the whole gait cycle. The four cases tested the response of the ankle mechanism to
active ankle dorsiflexion and plantarflexion, which are often
used ankle functions in normal walking (Winter, 2009). The
participants repeated each ankle movement case three times in
the experiment. Please see the Supplementary Video “Test of the FIGURE 6 | The joint performance of the representative participant P1 using the ROWAS II in passive mode. (A) Shoulder joint. (B) Hip joint. (C) Knee joint. (D)
Ankle joint. FIGURE 6 | The joint performance of the representative participant P1 using the ROWAS II in passive mode. (A) Shoulder joint. (B) Hip joint. (C) Knee joint. (D)
Ankle joint. FIGURE 6 | The joint performance of the representative participant P1 using the ROWAS II in passive mode. (A) Shoulder joint. (B
Ankle joint. April 2020 | Volume 14 | Article 17 Frontiers in Neurorobotics | www.frontiersin.org 7 Development of ROWAS II and Active Ankle Control Mu et al. TABLE 2 | RMSE values of all participants using the ROWAS II system running in
passive mode. TABLE 2 | RMSE values of all participants using the ROWAS II system running in
passive mode. ROWAS II System” to see the performance of a representative
participant during the experiment. RMSE is the normal value to represent the track error (Rubio,
2018; Rubio et al., 2019a,b; Gao et al., 2020). Therefore, the
tracking error of the passive and active control algorithms was
based on the RMSE for each joint movement: θpRMSE =
v
u
u
t 1
N
N
X
i=1
(θps(i) −θ(i))2,
(23)
θaRMSE =
v
u
u
t 1
N
N
X
i=1
(θas(i) −θ(i))2,
(24) θpRMSE =
v
u
u
t 1
N
N
X
i=1
(θps(i) −θ(i))2,
(23)
θaRMSE =
v
u
u
t 1
N
N
X
i=1
(θas(i) −θ(i))2,
(24) (23) FIGURE 7 | The performance of P1 using the ankle mechanism in passive
mode. (A) Ankle joint trajectories. (B) Input voltage. (C) Passive force. In (C),
negative values mean that the force sensor was pulled. (24) Evaluation of Active Ankle Control in the
ROWAS II System where N is the number of data points during one gait cycle. θps
and θas were the simulated angle in passive and active modes,
which were obtained using the simulated model (Figure 5). In the passive subtest of the ankle mechanism, the force
sensor measured the force required to achieve the target ankle
movement (Figure 7C), which was important to calculate the
voluntary input from the participant in the following active
subtest. Figure 7B shows the voltage to the motor, which is
proportional to the motor speed. In passive mode, the closed-
loop position control algorithms enabled the ankle mechanism
to track the target trajectories well (Figure 7A), with θpRMSE for
all participants <0.5◦(the sixth row in Table 2). Experimental Evaluation of the ROWAS II
System Test
θpRMSE
P1
P2
P3
P4
P5
P6
Overall ROWAS II
Shoulder (◦)
0.10
0.10
0.10
0.10
0.10
0.10
Hip (◦)
0.49
0.33
0.28
0.40
0.35
0.34
Knee (◦)
0.58
0.48
0.50
0.59
0.52
0.54
Ankle (◦)
0.41
0.63
0.47
0.51
0.50
0.67
Ankle mechanism
Ankle (◦)
0.45
0.42
0.40
0.46
0.45
0.46 FIGURE 7 | The performance of P1 using the ankle mechanism in passive
mode. (A) Ankle joint trajectories. (B) Input voltage. (C) Passive force. In (C),
negative values mean that the force sensor was pulled. ROWAS II System” to see the performance of a representative
participant during the experiment. ROWAS II System” to see the performance of a representative
participant during the experiment. Evaluation of the ROWAS II System in
Passive Mode The closed-loop position control algorithms were implemented
in the bilateral joint mechanisms of the ROWAS II system, but
this paper only presented the results from the right side for sake
of simplicity. After three tests were performed to the eight joints
running in open loop, the steady-state gain for the shoulder (ks),
hip (kh), knee (kk), and ankle (ka) mechanisms of the ROWAS II
system were evaluated as: p
p
(
)
In active mode, the participants finished four different
experiment cases with the admittance gain Ga = 0.6 and
Ga = 1.6, respectively. The results of P1 were presented in
Figures 8–11, where the simulation result of passive movement
was plotted to provide comparison. It was shown that P1
voluntarily adjusted the ankle movement. The active force
F (Figures 8–11E,F) was obtained by subtracting the force
measurement in passive mode (e.g., Figure 7C) from that
recorded in active mode. The negative values of the active
force mean that the participant pushed the foot downward. The
more force P1 actively exerted to lift up the foot, the more
upward movement the ankle mechanism generated (compared
Figures 8A,E with Figures 9A,E, and compared Figures 8B,F
with Figures 9B,F). This also applied when the participant
pushed the foot downward. The more force P1 pushed down
the foot, the more downward movement the ankle mechanism
produced (compared Figures 10A,E with Figures 11A,E, and
compared Figures 10B,F with Figures 11B,F). Besides, different
admittance gains allowed the ankle mechanism to have different
degrees of adjustment in response to active force. Compared (25) (25) ks = −0.18; kh = 1.05; kk = −0.27; ka = 0.60. (25) Using the closed-loop position control algorithms, the ROWAS
II system produced synchronized walking with arm swing. A
representative participant P1 (Figure 6) showed that ROMs of
the shoulder, hip, knee, and ankle joints were in the normal
range during human walking (Winter, 2009). The experimental
values almost coincided with the simulated values with a
RMSE of the shoulder, hip, knee, and ankle as 0.10◦, 0.49◦,
0.58◦, and 0.41◦, respectively. The ROWAS II system tracked
the target trajectory well in passive mode, with θpRMSE of all
joints for all participants <0.7◦(from the second to the fifth
rows in Table 2). April 2020 | Volume 14 | Article 17 Frontiers in Neurorobotics | www.frontiersin.org 8 Development of ROWAS II and Active Ankle Control Mu et al. FIGURE 8 | The performance of P1 when he lifted up his foot voluntarily during the whole gait cycle (Case 1). (A,B) Ankle joint trajectories. (C,D) Input voltage. (E,F)
Active force. FIGURE 9 | The performance of P1 when he lifted up and pushed down his foot voluntarily within 30–60% and 60–80% of the gait cycle, respectively (Case 2). (A,B)
Ankle joint trajectories. (C,D) Input voltage. (E,F) Active force. Frontiers in Neurorobotics | www frontiersin org
9
April 2020 | Volume 14 | Article 17 FIGURE 8 | The performance of P1 when he lifted up his foot voluntarily during the whole gait cycle (Case 1). (A,B) Ankle joint trajectories. (C,D) Input voltage. (E,F)
Active force. FIGURE 8 | The performance of P1 when he lifted up his foot voluntarily during the whole gait cycle (Case 1). (A,B) Ankle joint trajectories. (C,D) Input voltage. (E,F)
Active force. FIGURE 9 | The performance of P1 when he lifted up and pushed down his foot voluntarily within 30–60% and 60–80% of the gait cycle, respectively (Case 2). (A,B)
Ankle joint trajectories. (C,D) Input voltage. (E,F) Active force. FIGURE 9 | The performance of P1 when he lifted up and pushed down his foot voluntarily within 30–60% and 60–80% of the gait cycle, respectively (Case 2). (A,B)
Ankle joint trajectories. (C,D) Input voltage. (E,F) Active force. April 2020 | Volume 14 | Article 17 9 Development of ROWAS II and Active Ankle Control Mu et al. (25) FIGURE 10 | The performance of P1 when he pushed down and lifted up his foot voluntarily within 30–60% and 60–80% of the gait cycle, respectively (Case 3). (A,B)
Ankle joint trajectories. (C,D) Input voltage. (E,F) Active force. FIGURE 10 | The performance of P1 when he pushed down and lifted up his foot voluntarily within 30–60% and 60–80% of the gait cycle, respectively (Case 3). (A,B)
Ankle joint trajectories. (C,D) Input voltage. (E,F) Active force. the algorithms for active ankle control. The closed-loop position
control algorithms were implemented in the ROWAS II system in
passive mode, while the adjustable admittance control algorithms
were implemented in the ankle mechanism in active mode. Evaluated by six able-bodied participants, the ROWAS II system
was technically feasible to produce walking-like movement in the
bilateral upper and lower limbs in passive mode, and the ankle
mechanism had technical potential to provide active training,
where the ROM of the ankle joint was variously adjusted by the
voluntary input from the users. The novel ROWAS II system
can serve as a testbed for further investigation of interlimb
neural coupling in response to voluntary ankle movement and is
technically feasible to provide a new training paradigm of walking
with arm swing and active ankle control. with the experiments of the low admittance gain, P1 produced a
significantly adjusted ROM of the ankle joint in the experiments
of the high admittance gain [compared (A) with (B) in Figures 8–
11, respectively]. Similar results were observed in all participants. The small
errors between the experimental angle and the simulated active
angle θaRMSE during the four active movement cases proved
that the control algorithms yielded good tracking performance
(θaRMSE < 1.0◦, shown with dashed lines in Figure 12). The
relatively large differences between the experimental angle and
the simulated passive angle θpRMSE (solid lines in Figure 12)
of all participants during the four active movement cases
demonstrated that the participants voluntarily adjusted their
ankle movement trajectories. No matter whether the participant
lifted up or pushed down the foot, the ROM was more adjusted
when the higher admittance gain was implemented (Figures 12,
13). Using the adjustable admittance control algorithms, all
participants adjusted the ROM of the ankle mechanism by their
voluntary input. The ROWAS II system differed from the previous systems
(Fang et al., 2017a,b) in both mechanical structure and control
system. (25) The ROWAS and aROWAS systems had the rotary
motors for the lower limbs mounted on bilateral sides. However,
the ROWAS II system used series linear actuators which were
installed behind the hip and knee mechanisms and in front
of the ankle mechanism. This mechanical arrangement left
the space around lateral hip joints free, therefore allowing
natural arm swing. Furthermore, the force sensor at the
end of the actuator provided the active force information,
which enabled implementation of the adjustable admittance Frontiers in Neurorobotics | www.frontiersin.org Frontiers in Neurorobotics | www.frontiersin.org CONCLUSIONS The novel ROWAS II system was mechanically designed,
manufactured
and
implemented
with
passive
and
active
movement control algorithms. After evaluated by six able-bodied
participants, the ROWAS II system produced synchronized
walking movement with arm swing in passive mode. The
adjustable admittance control algorithms enabled the ankle
mechanism to be adjusted by the voluntary input from the user,
with a higher admittance gain producing a larger degree of
adjustment. The experimental evaluation demonstrated that the
ROWAS II system and the control algorithms were technically
feasible. The ROWAS II system has potential to serve as a
testbed for further investigation of interlimb neural coupling in
response to voluntary ankle movement and is technically feasible
to provide a new training paradigm of walking with arm swing
and active ankle control. After development of an overall active
ROWAS II system, future work will focus on using the ROWAS
II system to investigate the interlimb neural coupling in patients
with neurological impairments. The limitation of this study was that only six participants
were recruited. More participants are desirable to obtain a more
detailed evaluation of the ROWAS II prototype. Nevertheless,
six participants provided enough results for the technical
evaluation of the system functionality. Furthermore, to allow
an inter-individual comparison, a big screen should have
been used so that the participant would have known how
much active force was provided. Since the knee actuators
also have force sensors, the adjustable admittance control
algorithms will be implemented in both ankle and knee
joints of the ROWAS II system. This study will be followed
by the development of an overall active ROWAS II system DISCUSSION The steady gains of the April 2020 | Volume 14 | Article 17 Frontiers in Neurorobotics | www.frontiersin.org 11 Development of ROWAS II and Active Ankle Control Mu et al. FIGURE 13 | The difference of the peak ankle angle between the experimental and passive simulated results produced by different active force. The left subplot
shows the results when the participants voluntarily pushed down the foot within 60–80% of the gait cycle. The right subplot shows the results when the participants
voluntarily lifted up the ankle joint within 30–60% of the gait cycle. FIGURE 13 | The difference of the peak ankle angle between the experimental and passive simulated results produced by different active force. The left subplot
shows the results when the participants voluntarily pushed down the foot within 60–80% of the gait cycle. The right subplot shows the results when the participants
voluntarily lifted up the ankle joint within 30–60% of the gait cycle. ROWAS II system produced walking-like movement with
arm
swing,
and
has
technical
potential
to
be
applied
in
rehabilitation
of
walking
for
patients
in
the
early
post-injury stage. and an investigation of the interlimb neural coupling in
response to voluntary movement. Future work will apply the
active ROWAS II system in clinical tests on patients with
neurological impairments. Apart from passive training, the ROWAS II system achieved
active training in the ankle mechanism after implementation
of the adjustable admittance control algorithms. In active
mode, the participants voluntarily performed four different
ankle movements, which corresponded to the often used ankle
function in normal walking (Winter, 2009). The relatively large
difference between θpRMSE and θaRMSE (Figure 12) demonstrated
that the participants voluntarily adjusted their ankle movement. The higher admittance gain enabled the participants to more
easily change the movement trajectory of the ankle mechanism
(Figures 12, 13). This novel ROWAS II system can serve as a
testbed for further investigation of interlimb neural coupling in
response to voluntary ankle movement and is technically feasible
to provide a new training paradigm of walking with arm swing
and active ankle control. DISCUSSION This work aimed to develop an improved rotational orthosis for
walking with arm swing, and especially to develop and evaluate April 2020 | Volume 14 | Article 17 Frontiers in Neurorobotics | www.frontiersin.org 10 Development of ROWAS II and Active Ankle Control Mu et al. FIGURE 11 | The performance of P1 when he pushed down his foot voluntarily during the whole gait cycle (Case 4). (A,B) Ankle joint trajectories. (C,D) Input voltage. (E,F) Active force. FIGURE 11 | The performance of P1 when he pushed down his foot voluntarily during the whole gait cycle (Case 4). (A,B) Ankle joint trajectories. (C,D) Input voltage. (E F) Active force FIGURE 11 | The performance of P1 when he pushed down his foot voluntarily during the whole gait cycle (Case 4). (A,B) Ankle joint trajectories. (C,D) Input voltage. (E,F) Active force. FIGURE 12 | The average RMSE of all participants in four active ankle movement cases. control algorithms, as demonstrated in the active control
of the ankle mechanism in the ROWAS II system. The
improved system ROWAS II provided more functions, which are
discussed below. The ROWAS II system produced synchronized arm-leg
walking movement in passive mode. The steady gains of the
shoulder, hip, knee, and ankle mechanisms were estimated
using
system
identification. Based
on
the
approximate
gains,
the
closed-loop
position
control
algorithms
were
developed
using
pole-placement
approach. The
control
algorithms allowed the ROWAS II system to track the
target trajectories very well. This demonstrated that the FIGURE 12 | The average RMSE of all participants in four active ankle movement cases. FIGURE 12 | The average RMSE of all participants in four active ankle movement cases. FIGURE 12 | The average RMSE of all participants in four active ankle movement cases. shoulder, hip, knee, and ankle mechanisms were estimated
using
system
identification. Based
on
the
approximate
gains,
the
closed-loop
position
control
algorithms
were
developed
using
pole-placement
approach. The
control
algorithms allowed the ROWAS II system to track the
target trajectories very well. This demonstrated that the control algorithms, as demonstrated in the active control
of the ankle mechanism in the ROWAS II system. The
improved system ROWAS II provided more functions, which are
discussed below. The ROWAS II system produced synchronized arm-leg
walking movement in passive mode. Frontiers in Neurorobotics | www.frontiersin.org REFERENCES Hogan, N. (1985). Impedance control - an approach to manipulation. I - Theory. II
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1481–1489. FUNDING qualified researcher at https://drive.google.com/drive/folders/1_
uUu4-yCYDK7bp5fQDxzHVjJtTFm9qdV?usp=sharing. This work was supported in part by the National Natural
Science Foundation of China (JF: Grant No. 81401856),
a
research
funding
from
Jiangsu
Province
(JF:
Grant
No. 1076010241170110), and a Postgraduate Research &
Practice Innovation Program of Jiangsu Province (ZM: Grant
No. KYCX19_1880). This work was supported in part by the National Natural
Science Foundation of China (JF: Grant No. 81401856),
a
research
funding
from
Jiangsu
Province
(JF:
Grant
No. 1076010241170110), and a Postgraduate Research &
Practice Innovation Program of Jiangsu Province (ZM: Grant
No. KYCX19_1880). ACKNOWLEDGMENTS The authors acknowledge Mr. Zhonghua Xu and Mr. Longfei
Chen from Jiangnan University for their assistance in the
mechanical development of the ROWAS II system. ETHICS STATEMENT The studies involving human participants were reviewed and
approved by Med-X Research Institute, Shanghai Jiao Tong
University, Shanghai, China (Ethical approval No. 2019051). The participants provided their written informed consent to
participate in this study. SUPPLEMENTARY MATERIAL JF and ZM designed and manufactured the system, conducted
the experiments, and analyzed the data. ZM drafted the
manuscript. JF modified it critically. QZ supervised the
experiments, analyzed the data, and checked this paper. G-YY
and LX helped to provide the design concepts and checked
this manuscript. The Supplementary Material for this article can be found
online
at:
https://www.frontiersin.org/articles/10.3389/fnbot. 2020.00017/full#supplementary-material Supplementary Video | Test of the ROWAS II System. DATA AVAILABILITY STATEMENT The raw data supporting the conclusions of this article are
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distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the
original author(s) and the copyright owner(s) are credited and that the original
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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000480 April 2020 | Volume 14 | Article 17 Frontiers in Neurorobotics | www.frontiersin.org 14
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Magnetic resonance elastography in nonlinear viscoelastic materials under load
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Biomechanics and modeling in mechanobiology
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Abstract Characterisation of soft tissue mechanical properties is a topic of increasing interest in translational and clinical research. Magnetic resonance elastography (MRE) has been used in this context to assess the mechanical properties of tissues in vivo
noninvasively. Typically, these analyses rely on linear viscoelastic wave equations to assess material properties from measured
wave dynamics. However, deformations that occur in some tissues (e.g. liver during respiration, heart during the cardiac cycle,
or external compression during a breast exam) can yield loading bias, complicating the interpretation of tissue stiffness from
MRE measurements. In this paper, it is shown how combined knowledge of a material’s rheology and loading state can be used
to eliminate loading bias and enable interpretation of intrinsic (unloaded) stiffness properties. Equations are derived utilising
perturbation theory and Cauchy’s equations of motion to demonstrate the impact of loading state on periodic steady-state
wave behaviour in nonlinear viscoelastic materials. These equations demonstrate how loading bias yields apparent material
stiffening, softening and anisotropy. MRE sensitivity to deformation is demonstrated in an experimental phantom, showing a
loading bias of up to twofold. From an unbiased stiffness of 4910.4±635.8 Pa in unloaded state, the biased stiffness increases
to 9767.5± 1949.9Pa under a load of ≈34% uniaxial compression. Integrating knowledge of phantom loading and rheology
into a novel MRE reconstruction, it is shown that it is possible to characterise intrinsic material characteristics, eliminating
the loading bias from MRE data. The framework introduced and demonstrated in phantoms illustrates a pathway that can
be translated and applied to MRE in complex deforming tissues. This would contribute to a better assessment of material
properties in soft tissues employing elastography. Keywords Elastic waves · Tissue mechanics · Nonlinear mechanics · Magnetic resonance elastography · Biorheology Keywords Elastic waves · Tissue mechanics · Nonlinear mechanics · Magnetic resonance elastograp Magnetic resonance elastography in nonlinear viscoelastic materials
under load Adela Capilnasiu1
· Myrianthi Hadjicharalambous1,2 · Daniel Fovargue1 · Dharmesh Patel4 · Ondrej Holub1 ·
Lynne Bilston5,6 · Hazel Screen4 · Ralph Sinkus1,7 · David Nordsletten1,3 Adela Capilnasiu1
· Myrianthi Hadjicharalambous1,2 · Daniel Fovargue1 · Dharmesh Patel4 · Ondrej Holub1 ·
Lynne Bilston5,6 · Hazel Screen4 · Ralph Sinkus1,7 · David Nordsletten1,3 Received: 21 May 2018 / Accepted: 10 August 2018
© The Author(s) 2018 ORIGINAL PAPER
Magnetic resonance elastography in nonlinear viscoelastic materials
under load ORIGINAL PAPER Biomechanics and Modeling in Mechanobiology
https://doi.org/10.1007/s10237-018-1072-1 Biomechanics and Modeling in Mechanobiology
https://doi.org/10.1007/s10237-018-1072-1 ORIGINAL PAPER 1 Introduction Harmonic wave motion is then related to local
tissue properties, in most cases, through linear viscoelastic
wave equations (Glaser et al. 2012). MRE has been suc-
cessfully employed for a range of tissues—including brain
(Klatt et al. 2007; Green et al. 2008; Schregel et al. 2012),
liver (Huwart et al. 2006, 2007, 2008, Klatt et al. 2007) and
breast (Sinkus et al. 2000, 2005a, 2007, Houten et al. 2003)—
showing contrast between diseased and healthy tissue as well
as providing resolution of apparent tissue anisotropy (Qin
et al. 2013). More recently, it has also been applied to heart
(Elgeti et al. 2008, Kolipaka et al. 2009, Robert et al. 2009,
Kolipaka et al. 2010, Couade et al. 2011, Kolipaka et al. 2012, Elgeti and Sack 2014), skeletal muscles (Green et al. 2012, 2013), and other soft tissues, reviewed in Manduca
et al. (2001), Mariappan et al. (2010) and Fovargue et al. (2018b). ate measure of the intrinsic material properties and current
kinematic state. For instance, the nonlinearity arising from
compressivestrainwasquantifiedinexvivobovineliversam-
ples using MRE measurements (Clarke et al. 2011). Hence,
mixtures of patient-specific kinematics and intrinsic tissue
properties are non-trivial and presumably result in a reduc-
tion in clinical specificity. In this paper, the harmonic perturbation of Cauchy’s
equations of motion for a general incompressible nonlin-
ear viscoelastic material is examined, in order to understand
the links between intrinsic properties and kinematics in
MRE. From this analysis, a set of governing equations are
derived, illustrating the general impact of large deformation
on the motion of harmonic waves. Importantly, these equa-
tions enable the elimination of loading bias and therefore
retrieval of intrinsic properties when large-scale deforma-
tion and constitutive
behaviour
are known. Theoretical
test cases are presented, showing the ability of deforma-
tion to yield apparent increase, decrease and anisotropy
in materials. In order to demonstrate the applicability of
these perturbed equations, experiments were performed in
polyvinyl alcohol (PVA) phantoms. Rheological tests were
performed to characterise material behaviour, and loaded
phantoms were imaged using MRE. Results show the capac-
ity of these equations to correct for the complex dynamics
of wave motion in deformed materials. To the authors’
knowledge, this is the first cross-validation study linking
mechanical characterisation of a material in rheology with
wave motion through the deformed material in MRE. 1 Introduction Diagnosis and therapy planning often depend on developing
an accurate understanding of the state of a patient’s dis-
ease.Complexalterationsoccurindiseasedtissue,depending
on a range of factors such as changes to protein isoforms,
alterations in protein density, extracellular matrix reorganisa-
tion, inflammation, etc. These modifications fundamentally
impact tissue mechanical properties, triggering changes in
stiffness, elasticity and viscosity (Yeh et al. 2002; Yin et al. 2009; Baiocchini et al. 2016). The ability to detect changes in
tissue mechanical properties in vivo has the potential to sig-
nificantlybenefitclinicalmedicine,enablingamorethorough
assessment of pathology as well as monitoring of treatment
progression. This potential has lead to the development of
a number of approaches for the noninvasive assessment of
tissue mechanics. D.N. acknowledges funding from the Engineering and Physical
Sciences (EP/N011554/1 and EP/R003866/1). L.B. acknowledges
funding from the Australian Research Council Discovery Grant
(DP160100061) and an NHMRC senior research fellowship
(APP1077934). This work is funded by the King’s College London
and Imperial College London EPSRC Centre for Doctoral Training in
Medical Imaging (EP/L015226/1), and CDT industrial sponsor
Siemens Healthcare. This work was supported by the Wellcome
EPSRC Centre for Medical Engineering at Kings College London
(WT 203148/Z/16/Z) and by the National Institute for Health
Research (NIHR) Biomedical Research Centre award to Guy and St
Thomas’ NHS Foundation Trust in partnership with King’s College
London. The views expressed are those of the authors and not
necessarily those of the NHS, the NIHR or the Department of Health. B Adela Capilnasiu
adela.capilnasiu@kcl.ac.uk Extended author information available on the last page of the article 12 3 3 A. Capilnasiu et al. Manual palpation is a classical approach of inspecting
tissue for changes in elasticity in accessible organs (Weiss
2003, Nguyen et al. 2014). Advancements in medical imag-
ing have led to the development of elastography—a modern
quantitative technique for assessing the mechanical prop-
erties of internal organs. Elastography imaging is usually
achieved using Ultrasound or Magnetic Resonance Imaging
(MRI) (see Carey and Carey 2010 for a review), enabling
the determination of tissue properties in vivo. Elastography
imaging focuses on observing waves as they traverse through
tissues, thus allowing quantification of strain response due
to an excitation. In the case of magnetic resonance elas-
tography (MRE), low-amplitude harmonic waves (10–1000,
30–80Hz in vivo) are produced on the body and subsequently
imaged in three-dimensions (Manduca et al. 2001, Fovargue
et al. 2018b). 2 Materials and methods Here, ∇0 refers to the gradient operator taken with respect
to coordinates of Ω0, so that (∇0)k = ∂/∂Xk. For later use,
J = J(F) is implied. 2.1 Harmonic wave motion in deformed nonlinear
viscoelastic materials When relating kinematics and kinetics, the right or left
Cauchy Green strains MRE relies on the analysis of small amplitude harmonic
waves traversing through materials. As materials undergo
deformation, wave behaviour is impacted (Thurston and
Brugger 1964). One option to understand the relation-
ship between large-scale deformation and small-scale wave
behaviour is perturbation analysis. After covering nota-
tion 2.1.1, this section reviews perturbation results for
Cauchy’s equations (Sect. 2.1.2) considering the particular
case of a nonlinear viscoelastic material. Example cases are
then highlighted (Sect. 2.1.3), illustrating the impact of load
on the apparent stiffness characteristics stemming from MRE
waves. C = FT F,
B = FFT are often considered. These may also be defined in terms of
their isochoric variants (Ogden 1997; Bonet and Wood 2008)
as are often considered. These may also be defined in terms of
their isochoric variants (Ogden 1997; Bonet and Wood 2008)
as as ˆF = J −1/d F,
ˆC = ˆF
T ˆF,
ˆB = ˆF ˆF
T , which are invariant to changes in volumetric properties of
the material, i.e. ˆC(aF) = ˆC(F) for a real positive scalar
a ∈R+ (Holzapfel 2000). A set of invariants are associated
with these quantities. Here, the first and second invariants of
any second-order tensor A ∈Rm×m are defined as in Bonet
and Wood (2008) 2 Only dimensionality m was used as this case is relevant to this work,
particularly m = 3. 1 Introduction The
developments presented provide a key step towards improv-
ing the estimation of material properties in soft tissues, using
elastography. While interpreting stiffness measures in organs, such as
the liver, has shown diagnostic value, a challenge yet to
be addressed is the impact of macroscale deformations on
wave propagation behaviour. Broadly, by looking at the 1D
wave equation ∂2u/∂t2 = c2∂2u/∂x2, term c = √T /ρ
depends on tension T and linear density ρ and is scaling the
spatial second derivative of displacement u. The resultant
force term on the right hand side balances the acceleration
term on the left hand side, leading to the phase velocity
increasing with higher tension. Elastic, hyperelastic and vis-
coelastic nonlinear materials would exhibit this effect. This
is relevant in the context of tissues that undergo natural
deformations (e.g: the heart muscle during the cardiac cycle
(Asner et al. 2017), the liver during respiration (Kang et al. 2012), etc.) and tissues that undergo imposed deformations
(e.g. breast during MRE scans Sinkus et al. 2005a, 2007). As many tissues are known to exhibit significant nonlinear-
ity (Nash and Hunter 2000; Taber 2004; Liu et al. 2006;
Holzapfel and Ogden 2009; Gao et al. 2010; Nordsletten
et al. 2011b; Goenezen et al. 2012) under physiological con-
ditions, both natural and imposed deformation can result
in loading bias—an apparent stiffness that is a conglomer- As a starting point, Sect. 2.1 presents perturbation the-
ory employed within Cauchy’s equations of motion. The
perturbed equations are analysed in the context of a gen-
eral viscoelastic material under load, further leading to the
derivation of apparent stiffness moduli influenced by large
deformations and material constitutive law. For exemplifica-
tion purposes, an idealised case of harmonic wave motion
traversing a uniformly compressed Neo-Hookean material
is presented. Experiments are employed to test the theoreti-
cal ground developed. In Sect. 2.2, PVA phantoms are tested
in a rheological setup, to determine the material governing
law. In Sect. 2.3, MRE data are acquired in PVA phantoms at
different uniaxial compression levels. The knowledge on rhe-
ological behaviour and deformation is integrated into MRE
data analysis, in order to undo the loading bias. The exper-
imental results are presented in Sect. 3. Particularly, the
estimation of the intrinsic stiffness of PVA is presented in
Sect. 3.3. The theoretical framework presented in this study
could be extended to in vivo MRE data analysis. 1 Einstein summation over repetitive indices is assumed. 2.1.1 Kinematics, harmonic wave motion and notation Here, we briefly review the basic kinematics in large defor-
mation mechanics in order to later describe the macroscale
deformation of phantom and tissue material subjected to
MRE. For more complete dispositions, see for example
(Malvern 1969; Wang and Truesdell 1973; Graff 1991;
Holzapfel 2000; Bonet and Wood 2008). The motion of a
solid body, denoted by the region Ω0 ⊂Rd, is characterised
by the displacement field U : Ω0 × [0, T ] →Rd. In this
case, any reference point X ∈Ω0 may be deformed to its
physical position by the diffeomorphic Lagrangian mapping
L : Ω0×[0, T ] →Rd so that its position at a time t ∈[0, T ]
is given by IA = A : I,
I IA = A : A. For later clarity, the double contraction “:” is used as defined
in Bonet and Wood (2008), e.g. the contraction of second-
order square tensors A, B ∈Rm×m becomes a scalar1 A : B = Ai j Bi j, and the contraction of a fourth-order tensor A ∈Rm×m×m×m
and a second-order tensor B yields a second-order tensor in
Rm×m2 (A : B)i j = Ai jkl Bkl. x = L(X, t) = U(X, t) + X,
∀X ∈Ω0. For further use throughout the paper, the multiplication of
second- and fourth-order tensors AA or AB is defined to
take place over the last index of the first quantity and first
index of the second quantity, i.e. Due to the Lagrangian mapping,
note that any m-
dimensionalfunctionv : Ω0×[0, T ] →Rm canbewrittenas
a function of the deformed domain Ω(t), e.g. v : Ω →Rm,
with the understanding that v(x, t) = v(L−1(x, t), t). For
ease of notation, the · is dropped where there is clear dis-
tinction. (AA)i jkl = AisAsjkl,
(AB)i jkl = Ai jks Bsl. The stresses resulting from material deformation can be
separated into deviatoric and hydrostatic components. For
the latter, the effect is quantified using the hydrostatic pres-
sure P, which ensures some constraint on the volume or
pressure-volume relation. The deviatoric components are, in
turn, related to shape changes (or strains and strain history). Finally, the derivative of a second-order tensor with respect
to another is defined as (∇B A)i jkl = ∂Ai j
∂Bkl
. (∇B A)i jkl = ∂Ai j
∂Bkl
. 1 Introduction 123 Magnetic resonance elastography in nonlinear viscoelastic materials under load 2.1.1 Kinematics, harmonic wave motion and notation Important quantities for mechanics are the deformation
gradient tensor F and its determinant J: 1 Einstein summation over repetitive indices is assumed. 2 Only dimensionality m was used as this case is relevant to this work,
particularly m = 3. F = ∇0U + I,
J(F) = det F. F = ∇0U + I,
J(F) = det F. 12 3 A. Capilnasiu et al. (Wang et al. 2009, Nordsletten et al. 2011a, McCormick
et al. 2013, 2014, Hadjicharalambous et al. 2014), breast
(Rajagopal et al. 2010, Reynolds et al. 2011, Gamage et al. 2011), arterial wall (Holzapfel 2000, Gasser et al. 2006,
Hariton et al. 2007), etc.-where specific material response
is usually defined through biorheological experiments. The
simplest material models are purely elastic, based on spring
rheological elements. Complementary, pure viscosity is
based solely on dashpot elements. Biological tissues usu-
ally exhibit viscoelasticity; hence, a suitable tissue model
should consider a combination of these springs and dashpots. For instance, the Maxwell model is formed by considering a
spring and dashpot connected in series, whereas the Kelvin–
Voigt model is formed by considering a spring and dashpot
connected in parallel. These types of models have been anal-
ysed in literature (Liu and Bilston 2000, Bilston et al. 2001,
Banks et al. 2011), but have shortcomings in predicting creep
(Maxwell model) or stress relaxation (Kelvin–Voigt model)
(Liu et al. 2006, Banks et al. 2011). While the Maxwell
constitutive equation was found to be better suited for mod-
elling fluids (Houten et al. 2000; Sinkus et al. 2005a), the
Kelvin–Voigt constitutive equation was employed for pur-
poses similar to ours, modelling soft tissues subjected to
elastography testing (Sinkus et al. 2005a, Huwart et al. 2006,
Sinkus et al. 2007, Huwart et al. 2008) (albeit not consider-
ing the effects of large deformations on the wave behaviour). However, the Kelvin–Voigt model is not able to accurately
capture the power-law dependence on frequency that is usu-
ally observed in tissues (Chui et al. 2004, Sinkus et al. 2007,
Nicolle et al. 2010, Nicolle 2015). Thus, an adaptation of this
model is commonly used, which replaces the dashpot with a
springpot. This form was observed to describe tissue better
(Kiss et al. 2004). MRE relies on the propagation of shear waves through
the body, assumed to reach a harmonic steady state. Being
wave motion, these deformations are assumed to be linear
and small. 2.1.1 Kinematics, harmonic wave motion and notation The large deformation U is perturbed by these
small wave deformations uε : Ω0 × [0, T ] →Rd,
(1) (1) where the scale of the perturbations is much smaller than the
dominant length scale of the body or the large displacement it
undergoes. When the perturbations are periodic-in-time and
have reached a steady-state, the deformations and pressures
can be written as uε = Re{uceiωt},
pε = Re{pceiωt},
(2) (2) where uc = ur +iui (ur, ui : Ω0 →Rd) and pc = pr +ipi
(pr, pi : Ω0 →R) give the complex periodic steady-state
behaviour with real and imaginary parts given by subscripts
r and i, respectively. 2.1.2 Perturbation of Cauchy’s equations of motion For an incompressible body, at any time t ∈[0, T ], motion of
Ω0 satisfies Cauchy’s first law (conservation of momentum)
and conservation of mass shown in Eqs. 3a and 3b, respec-
tively (see Malvern 1969; Ogden 1997; Holzapfel 2000;
Bonet and Wood 2008): ρ J∂ttU −∇0 · (FS) = 0 on Ω0,
(3a)
J −1 = 0 on Ω0. (3b) (3a)
(3b) (3b) J −1 = 0 on Ω0. Here, ρ is the material density, ∂tt is the second time deriva-
tive, and Se = S(P, C) is the second Piola Kirchhoff (PK2)
stress tensor depending on the hydrostatic pressure, P, and
the right Cauchy Green tensor, C. To capture the viscoelastic behaviour exhibited generally
by tissues and polymers, we proceed by assuming that the
PK2 stress can be decomposed into an additive sum of elastic,
viscoelastic and hydrostatic components, i.e. The PK2 stress can be characterised as elastic, hyperelas-
tic, viscoelastic, etc., depending on the constitutive relation
that describes the material. A hyperelastic material, for exam-
ple, can be written as a function of C (or other strain
metrics) and space (X). In contrast, a viscoelastic material
additionally depends on time (t). We recall briefly that, for
hyperelastic materials—such as those commonly applied in
biomechanics—Se is given as the derivative of a governing
hyperelastic strain energy function, W = We(C) (Bonet and
Wood 2008), with respect to the right Cauchy Green tensor
C, i.e. S = Se + Sp + Dα
t Sv. (5) (5) The elastic part Se is defined through a hyperelastic strain
energy function We(C) as in Eq. 4, the hydrostatic part as
Sp = J PC−1, and the viscoelastic part is defined using the
Caputo formulation (Caputo 1967) of the fractional-order
derivative: Dα
t Sv =
1
Γ (1 −α)
t
0
1
(t −z)α ∂t Sv(z) dz. Se = 2∇CWe,
Se,i j = 2 ∂We
∂Ci j
. (4) (4) Here, Sv depends on a viscoelastic strain energy function
such as Sv = 2∇CWv. Considering the coefficient of deriva-
tion α to be 0, the viscoelastic stress Sv acts as a hyperelastic
term, while α = 1 indicates the first-order derivative ∂t Sv. 2.1.2 Perturbation of Cauchy’s equations of motion Fur-
thermore, the deformation can be undone, that is, the moduli
that reflect the undeformed state can be recovered. (6b) In order to investigate the effect of deformation on wave
behaviour, we consider the case where high-frequency/low-
amplitude harmonic waves (see Eq. 1) are imposed onto our
body that satisfies the set of Eq. 3. For our purposes, we
assume that the introduction of these micro-deformations uε
does not disrupt the natural state of macro-deformation U,
such that the total observed state (Uε, Pε) can be charac-
terised by Uε(X, t) = U(X, t) + uε(X, t),
Pε(X, t) = P(X, t) + pε(X, t),
(7) (7) 2.1.2 Perturbation of Cauchy’s equations of motion This
model
is
extensively
applied
in
nonlinear
biomechanics—for example in simulations of the heart 123 Magnetic resonance elastography in nonlinear viscoelastic materials under load G′
i jml = 1
J
F∇F(Se + ωα cos
πα
2
Sv
+ Sp) + IS
ismn Fln Fjs,
(10a)
G′′
i jml = ωα
J sin
πα
2
F∇F Sv
ismn Fln Fjs,
(10b) Intermediate values of α would lead to non-trivial transitional
results between Sv and ∂t Sv. Therefore, this formulation of
the viscoelastic term leads to a fractional nonlinear Kelvin–
Voigt type of model and the PK2 tensor for our viscoelastic
material becomes (10a) (10b) S = 2∇CWe + 2Dα
t (∇CWv) + J PC−1,
(6a)
Si j = 2 ∂We
∂Ci j
+ 2Dα
t
∂Wv
∂Ci j
+ J PC−1
i j . (6b) (6a) where F, Se and Sv depend on the unperturbed macro-
deformation U andhydrostaticpressureand P.Intheabsence
of deformation, under the assumption of an isotropic mate-
rial defined by a standard law (e.g. a Neo-Hookean model
for Se and Sv), these stiffness moduli are simply given by
G′ + iG′′ = G∗I, where G∗is the true complex shear mod-
ulus characterising the material and Ii jkl = δikδ jl. The wave
dynamics become symmetric, since I : ∇uc = ∇uc +∇uT
c . However, under deformation, the components of ∇uc are
scaled in a non-trivial way. By understanding the form of the
scaling influencing the G′ and G′′ moduli, the bias introduced
by deformation on the wave behaviour can be predicted. Fur-
thermore, the deformation can be undone, that is, the moduli
that reflect the undeformed state can be recovered. where F, Se and Sv depend on the unperturbed macro-
deformation U andhydrostaticpressureand P.Intheabsence
of deformation, under the assumption of an isotropic mate-
rial defined by a standard law (e.g. a Neo-Hookean model
for Se and Sv), these stiffness moduli are simply given by
G′ + iG′′ = G∗I, where G∗is the true complex shear mod-
ulus characterising the material and Ii jkl = δikδ jl. The wave
dynamics become symmetric, since I : ∇uc = ∇uc +∇uT
c . However, under deformation, the components of ∇uc are
scaled in a non-trivial way. By understanding the form of the
scaling influencing the G′ and G′′ moduli, the bias introduced
by deformation on the wave behaviour can be predicted. 2.1.3 Loading bias of planar shear waves in pure
compression 1 Planar waves through a loaded phantom; (Top left) Phantom
in undeformed state. (Top right) Phantom in compressed state, com-
pared against the undeformed state. (Bottom left) Planar wave created
by moving the front face of the phantom (in blue) along the e1 direc-
tion. (Bottom right) Planar wave created by moving the top face of the
phantom (in blue) along the e2 direction by moving the front face of the phantom (in blue) along the e1 direc-
tion. (Bottom right) Planar wave created by moving the top face of the
phantom (in blue) along the e2 direction Fig. 1 Planar waves through a loaded phantom; (Top left) Phantom
in undeformed state. (Top right) Phantom in compressed state, com-
pared against the undeformed state. (Bottom left) Planar wave created by moving the front face of the phantom (in blue) along the e1 direc-
tion. (Bottom right) Planar wave created by moving the top face of the
phantom (in blue) along the e2 direction height (e3 direction), imposing incompressibility, the defor-
mation U is described by with the stiffness modulus by removing the terms containing
δml and ˆBml from Eq. 11: U =
(1/
√
λ −1)X1, (1/
√
λ −1)X2, (λ −1)X3
T , G′ : ∇uc = 1
J
μe∇uc B + ∇uT
c
μeI ˆB
3
−P J
. where λ is the ratio of the final height over the initial height
(i.e. λ < 1). 2.1.3 Loading bias of planar shear waves in pure
compression with (U, P) satisfying the original Eq. 3 (without pertur-
bation conditions) and with (Uε, Pε) satisfying a perturbed
version of Eq. 3, i.e. For an intuitive illustration, let us consider the case of a sim-
ple isotropic Neo-Hookean material described by the strain
energy function ρ J ε∂ttUε −∇0 · (FεSε) = 0
on
Ω0,
(8a)
J ε −1 = 0
on
Ω0,
(8b) (8a)
(8b) We(P, F) = μe
2 (I ˆC −3), (
)
(8b) where μe is the elastic material parameter. The elastic stress
is derived to be where superscript ε indicates quantities dependent on the
perturbed state variables. A simplification of the set of Eq. 8 can be achieved by
expanding about the state variable U and then linearising
with respect to the small perturbations uε, pε (Thurston and
Brugger 1964). For completeness, details about the expan-
sion and linearisation processes can be found in Appendix 1. Considering the case of harmonic waves, a set of periodic
nonlinear viscoelastic wave equations can be derived in the
reference domain Ω0. By recasting the reference frame gra-
dient and divergence operators into their physical domain
counterparts, the set of Eq. 8 can be transformed into the
physical domain as: Se = μe
J 2/3
I −IC
3 C−1
. The viscous term is assumed to be zero, i.e. Sv = 0. Under
these assumptions, the real modulus G′ becomes G′
i jml = 1
J
μe ˆBljδim −2μe
3 ( ˆBi jδml + ˆBmlδi j)
+ δmlδi j
2μeI ˆB
9
+ P J
+ δmjδil
μeI ˆB
3
−P J
. (11) −ρω2uc −∇· [(G′ + iG′′) : ∇uc + pc I] = 0,
(9a)
∇· uc = 0,
(9b) (11) (9a)
(9b) (9b) To better understand the changes introduced by large
deformations U on the wave dynamics, we analyse the case
of plane waves through a material under pure compression. Thus, considering a cuboid that is compressed along the where ∇= ∂/∂x on Ω. Here, G′ and G′′ are the real and
imaginary stiffness moduli influencing the apparent wave
dynamics of uc and taking the form of fourth-order tensors: 12 3 A. Capilnasiu et al. Fig. 1 Planar waves through a loaded phantom; (Top left) Phantom
in undeformed state. (Top right) Phantom in compressed state, com-
pared against the undeformed state. (Bottom left) Planar wave created Fig. 2.1.3 Loading bias of planar shear waves in pure
compression In this case, the hydrostatic pressure is a constant
given by P = μe(1/λ −λ2)/3 and the kinematic tensors
describing this deformation are Additionally, under the divergence operator, ∇uT
c vanishes
(together with I ˆB, P and J, which are constant in space),
and thus, the elastic contribution can be further simplified to F =
⎛
⎜⎝
1
√
λ
0 0
0
1
√
λ 0
0
0 λ
⎞
⎟⎠, F =
⎛
⎜⎝
1
√
λ
0 0
0
1
√
λ 0
0
0 λ
⎞
⎟⎠, F =
⎛
⎜⎝
1
√
λ
0 0
0
1
√
λ 0
0
0 λ
⎞
⎟⎠,
B = ˆB = C = ˆC =
⎛
⎝
1
λ 0 0
0 1
λ 0
0 0 λ2
⎞
⎠,
(12) ∇· (G′ : ∇uc) = ∇· (μe∇uc B)
(14) (14) Let us now analyse particular examples of shear waves—
namely planar waves—travelling through the compressed
cuboid. In the first case, consider that the front face of the
cuboid is vibrated along the e1 direction (see Fig. 1), an
idealised wave of the form B = ˆB = C = ˆC =
⎛
⎝
1
λ 0 0
0 1
λ 0
0 0 λ2
⎞
⎠,
(12) (12) with J = 1. 2.2.1 PVA phantom preparation A material suitable for our purposes should be able to with-
stand large deformations without rupturing and to have low
wave attenuation. For this, polyvinyl alcohol cryogel (PVA-
C) was selected, which is suitable for mimicking soft tissue
properties and has a high MR signal (Surry et al. 2004,
Sinkus et al. 2005a). Following a protocol similar to the
one described in Xia et al. (2011), phantoms were created
by mixing polyvinyl alcohol powder (P1763 Sigma-Aldrich
Company Ltd., UK) with deionised water in a concentra-
tion of 7%. A magnetic stirrer was used for 2h, in order
to fully dissolve the powder into the water, while heating
the concoction to 90◦C. The mixture was covered through-
out the process, to avoid evaporation, left to cool down at
room temperature and poured into cuboid moulds of dimen-
sions 64 × 48 × 42 mm. The moulds were then placed in
a freezer at −20◦C and underwent three cycles of freez-
ing (14h) and thawing (10h) (F-T). These specifications
ensured that the material was suitable for testing under large
deformations. Laboratory experimentation with higher PVA
concentrations (e.g. 10%) led to phantoms that were not
easily deformable, whereas reducing the number of F-T
cycles led to unstable phantoms that would leak water under
loading. uc =
⎛
⎝
0
u2(x3)
0
⎞
⎠
(16) (16) are probing the cuboid in the direction in which it was orig-
inally compressed, since the only nonzero component of the
wave gradient is (∇uc)23. This results in a stiffness scaling
by λ2, i.e. μe∇uc B = λ2μe∇uc. With λ < 1, the biased stiffness λ2μe appears to be softer
than the material’s true stiffness μe. From analysing these two simple instances of planar
waves (Eqs. 15 and 16), we gain an understanding of how
the waves probing a material under compression experience
different loading bias—stiffer in the expanded direction e1
and softer in the compressed direction e3. Had the waves been
more complex, like in Eq. 13, then the effect of the loading
would yield During the freezing process, volumetric expansion
occurred. Therefore, the phantoms’ top was cut, in order to
achieve cuboid shapes. This led to differences in the phan-
toms’ height, which ranged between 32 and 42 mm. For a
meaningful variance testing, 14 homogeneous phantoms
were created, out of which 7 were used for rheological exper-
iments and 7 for MRE experiments. with J = 1. uc =
⎛
⎝
u1(x2)
0
0
⎞
⎠
(15) A pure shear wave which will perturb the macro-
deformation U can be written as (15) uc =
⎛
⎝
u1(x2, x3)
u2(x1, x3)
u3(x1, x2)
⎞
⎠. (13) (13) is created, with the only nonzero entry in the wave displace-
ment gradient being (∇uc)12. Therefore, the deformation
contribution in Eq. 14 simply becomes a scaling by 1/λ: This form ensures that ∇uc has only off-diagonal compo-
nents, i.e. ∇uc : I = 0, thus satisfying the incompressibility
assumption of the material. Since the deformation considered
employs no shearing (i.e. no off-diagonal components), then
also B : ∇uc = 0 and therefore this wave form allows for
simplifications when being contracted, as needed in Eq. 9a, This form ensures that ∇uc has only off-diagonal compo-
nents, i.e. ∇uc : I = 0, thus satisfying the incompressibility
assumption of the material. Since the deformation considered
employs no shearing (i.e. no off-diagonal components), then
also B : ∇uc = 0 and therefore this wave form allows for
simplifications when being contracted, as needed in Eq. 9a, μe∇uc B = μe
λ ∇uc. μe∇uc B = μe
λ ∇uc. As such, the loading bias is influencing the apparent stiffness
of the cuboid, which appears to be μe/λ, whereas the intrinsic 12 123 123 Magnetic resonance elastography in nonlinear viscoelastic materials under load then be later (Sect. 2.3.2) integrated with deformation and
MRE data. stiffnessisμe.Thus,underthissetupofthewave,thematerial
stiffness appears to be higher, since λ < 1. In the second case, consider a planar wave created by
vibrating the top face of the cuboid, this time along the e2
direction (see Fig. 1). Then, the idealised wave displace-
ments 2.2.2 Rheological testing of PVA phantoms The phantoms allow for suitable examination methods that
can test the theory developed in Sect. 2.1. It was seen that, in
order to account for the nonlinear complex stiffness moduli
G′ and G′′ (Eq. 10), understanding of the nonlinear behaviour
and frequency dependence of the material must be inferred. This can be done through rheological testing, which is used to
characterise material behaviour by quantifying its response
to different types of applied stress. As such, each of the six
phantoms created for rheology experiments was tested in a
Bose Electroforce 5500 test instrument. The setup, seen in
Fig.2,consistedofasupportingfixedplatenandanadjustable 2.2.1 PVA phantom preparation However, one phantom
was excluded in the rheological tests due to rupture. The time
between phantom fabrication and testing in either rheology
or MRE was within twoweeks, to avoid potential material
degradation. μe∇uc B = μe
⎛
⎜⎜⎜⎜⎜⎝
0
1
λ
∂u1
∂x2
λ2 ∂u1
∂x3
1
λ
∂u2
∂x1
0
λ2 ∂u2
∂x3
1
λ
∂u3
∂x1
1
λ
∂u3
∂x2
0
⎞
⎟⎟⎟⎟⎟⎠
. μe∇uc B = μe
⎛
⎜⎜⎜⎜⎜⎝
0
1
λ
∂u1
∂x2
λ2 ∂u1
∂x3
1
λ
∂u2
∂x1
0
λ2 ∂u2
∂x3
1
λ
∂u3
∂x1
1
λ
∂u3
∂x2
0
⎞
⎟⎟⎟⎟⎟⎠
. Thus, even under simplified conditions like an idealised shear
wave travelling through a Neo-Hookean material, we get a
feeling for how biased the wave propagation becomes under
pure compression. It can be presumed that, under less than
ideal conditions (more complex materials and deformations),
the harmonic wave motion becomes increasingly intricate. 2.2 Nonlinear viscoelastic characterisation of PVA Experiments done in controllable media, where shapes and
deformations are simple, constitute the first step in evalu-
ating the theory explained in the previous section. A first
key component is using a material with a known rheologi-
cal behaviour, as this governs the effective loading bias (e.g. Eq. 6). The following sections describe the fabrication pro-
cess of PVA material and its testing in a rheological setup. A viscoelastic material law is also formulated. This law will 12 3 A. Capilnasiu et al. Fig. 2 Illustration of experimental setup, protocol and data in rheolog-
ical tests. (Left) Phantom in the rheological instrument. The moving
platen is compressing the phantom and oscillates vertically, while the
loading cell records the force. (Right, top) The traction measurements
in one phantom for the six tests: four micro-oscillations and one macro-
oscillation (sweeping over frequencies) and a relaxation test. (Right,
bottom) Platen displacements, corresponding to phantom compression
levels. Zoomed panel: exemplification of a micro-oscillatory test dis-
placements, showing the frequency sweep; illustration of a cycle in the
lowest frequency regime—one period, starting from the lowest point in
the compression Fig. 2 Illustration of experimental setup, protocol and data in rheolog-
ical tests. (Left) Phantom in the rheological instrument. The moving
platen is compressing the phantom and oscillates vertically, while the
loading cell records the force. (Right, top) The traction measurements
in one phantom for the six tests: four micro-oscillations and one macro-
oscillation (sweeping over frequencies) and a relaxation test. (Right, bottom) Platen displacements, corresponding to phantom compression
levels. Zoomed panel: exemplification of a micro-oscillatory test dis-
placements, showing the frequency sweep; illustration of a cycle in the
lowest frequency regime—one period, starting from the lowest point in
the compression the frequency response of PVA, with the aim of extrapolat-
ing it to the higher frequencies used in MRE. In all tests, but
clearly noticeable in the macro-oscillatory one, the displace-
ments’ amplitude is decreasing with increasing frequency. This is probably due to the instrument’s limitation which,
in the faster frequency regime, cannot reach the instructed
displacements. upper platen, which was movable in the vertical direction. A
load cell of capacity 225N measured the force required to
ensure a user defined deformation. For our aims, three types of tests were employed, with
the purpose of assessing PVA’s nonlinear behaviour. 2.2 Nonlinear viscoelastic characterisation of PVA The
first test was designed to investigate the material’s response
to dynamic micro-oscillations around a state of large static
load. This design replicates closely the in vivo MRE sce-
nario, where low-amplitude harmonic waves are propagating
in an organ which may be subjected to large deformation. For reproducing the nonlinear behaviour, four different static
loading states were investigated. Thus, the moving platen was
programmed to compress the phantom by 2, 4, 6 and 8 mm,
respectively (∼5–20% uniaxial compression), and then to
oscillate with an amplitude of 0.15 mm (∼0.4%). This test
will be referred to as the micro-oscillatory test. At the higher frequencies (≥5 Hz), it was observed that
the moving platen gains acceleration and introduces a bias on
the data. As such, calibration data were acquired, where the
same protocol was followed, but with no sample in between
theplatens.Subtractingthecalibrationdatafromthephantom
data eliminated the platen’s momentum bias. A final stress relaxation test was conducted by subject-
ing the material to a constant large deformation, in order to
capture the specific viscoelastic effect. Hence, the phantoms
wereheldunderacompressionof11mm(∼28.5%)for5min. The second test was designed to investigate the nonlinear
large deformation response in time. As such, large oscil-
lations were imposed around a state of large deformation:
the platen compressed the phantom by 5.8 mm (∼15% uni-
axial compression) and then oscillations of 4 mm (∼10%)
amplitude were imposed. This test will be referred to as the
macro-oscillatory test. Overall, each phantom underwent six tests: four micro-
oscillations at different loads (with seven different oscillatory
states acquired), one macro-oscillation (with seven different
oscillatory states acquired) and one relaxation. In between
tests, the phantoms were left to rest in water for 15–20min,
since PVA is best stored in water (Surry et al. 2004), for
hydration reasons. This resting time allowed for an efficient
testing protocol of the phantoms, and its duration was suffi-
cient to observe reproducibility. In both tests, oscillatory tests were carried under a fre-
quency sweep, from 0.1 Hz to 10 Hz (the instrument’s limit):
0.1, 0.5, 1, 2, 5, 7.5 and 10 Hz. A clear depiction of the testing
protocol can be seen in Fig. 2. This was done to investigate 123 123 Magnetic resonance elastography in nonlinear viscoelastic materials under load 2.2.3 Constitutive modelling of PVA In broad terms, since α is the only
nonlinear parameter of the model, the parameterisation pro-
cess is done by iterating over fixed values of α and then
solving a minimisation problem which yields a best fit of
the remaining linear parameters. It is desired that all tests
(micro-, macro-oscillations and relaxation) carry the same
importance in the fitting process, and that the error is not
dominatedbycertaintests (e.g. thosedoneat higher compres-
sion levels, which employ larger tractions). As such, for each
of the six tests (four micro-oscillation, one macro-oscillation
and one relaxation), the traction data and model were stan-
dardised according to the maximum traction value in the test, As previously mentioned in Sect. 2.1.2, we consider the
viscous part of PK2 to depend on a fractional-order derivative
of a viscoelastic stress Sv, which is defined here as the sum
δ1Se1 + δ2Se2. Hence, the PK2 tensor becomes (19) S = C1Se1 + C2Se2 + Dα
t (δ1Se1 + δ2Se2) + Sp. (19) Here, α is the fractional derivative coefficient, with α = 0
preserving the viscoelastic stress (Dα
t Se = Se), which turns
into a purely elastic contribution, and α = 1 indicating the
first-order derivative, which becomes a purely viscous con-
tribution. The hydrostatic stress Sp = J PC−1 completes
the definition of our PK2 tensor. 2.2.3 Constitutive modelling of PVA As previously mentioned, the acquired rheological data
consistofforcemeasurementsrequiredtoensureapredefined
displacement, each corresponding to a point in time. This was
transformed into traction data in the e3 direction by dividing
the force readings by the cuboid’s top area adjusted to the
deformation, i.e. PVA and other hydrogels have been modelled, previously,
as hyperelastic materials, employing laws that are generally
used to describe rubber-like materials. Often, a Mooney–
Rivlin type of model, employing two parameters, was found
to be sufficient to capture the elastic behaviour of PVA
(Anseth et al. 1996, Pazos et al. 2009). Extension to vis-
coelasticity was sometimes accomplished by considering
Maxwellian elements (Anseth et al. 1996, King et al. 2011). As such, here the aim is to model the PVA material as vis-
coelastic, yet using springpot elements. Following previous
investigations, we start the modelling process by consider-
ing the elastic part to be defined by a Mooney–Rivlin type of
material law: trd(t) = F(t)
A(t),
A(t) = A0h0
h(t) . Here, trd(t) is the traction obtained from the data, F(t) is the
force reading, and A(t) is the area at time t, which depends
on the initial top area A0 and the ratio of initial cuboid height
h0 to the compressed height h(t) (with h(t) = h0 −d(t)). Likewise, the traction in the e3 direction arising from the
model (trm) was computed using We(F) = C1(I ˆC −3) + C2(I I ˆC −3)2,
(17) (17) trm(t) = (σ(t) · n)3,
σ = FSFT , where C1 and C2 (Pa) are material parameters. Considering
the definition of the elastic part of the PK2 stress given, gen-
erally, as Se = 2∇CWe, it follows that Se has two elastic
parts scaled by the material parameters C1 and C2: where σ is the Cauchy stress and n is the normal to the surface
(in our case, we are interested in the normal to the top surface,
i.e. n = [0, 0, 1]T ). Since the deformation at any point in
time is given by Se(C) = C1Se1 + C2Se2. U(t) =
⎛
⎜⎜⎜⎝
h(t)
h0 −1
X1
h(t)
h0 −1
X2
h0
h(t) −1
X3
⎞
⎟⎟⎟⎠, The elastic terms are derived to be Se1 =
2
J 2/3
I −IC
3 C−1
,
Se2 =
8
J 4/3
I I ˆC −3
C −I IC
3 C−1
. 2.2.3 Constitutive modelling of PVA the elastic and viscoelastic tensors Se1,2 and Dα
t Se1,2 can be
determined, together with their counterparts in the Cauchy
stress. They take the form of diagonal second-order ten-
sors. The hydrostatic pressure P can also be determined
at each time point by noticing that the traction in the e1,2
directions is 0. Hence, P counteracts the elastic and vis-
coelastic contributions in both the e1,2 directions and can be
used as such in computing the traction in the e3 direction. Thus, the only unknowns in the traction model are parame-
ters C1, C2, δ1, δ2, α. In broad terms, since α is the only
nonlinear parameter of the model, the parameterisation pro-
cess is done by iterating over fixed values of α and then
solving a minimisation problem which yields a best fit of
the remaining linear parameters. It is desired that all tests
(micro-, macro-oscillations and relaxation) carry the same
importance in the fitting process, and that the error is not
dominatedbycertaintests (e.g. thosedoneat higher compres-
sion levels, which employ larger tractions). As such, for each
of the six tests (four micro-oscillation, one macro-oscillation
and one relaxation), the traction data and model were stan-
dardised according to the maximum traction value in the test,
i the elastic and viscoelastic tensors Se1,2 and Dα
t Se1,2 can be
determined, together with their counterparts in the Cauchy
stress. They take the form of diagonal second-order ten-
sors. The hydrostatic pressure P can also be determined
at each time point by noticing that the traction in the e1,2
directions is 0. Hence, P counteracts the elastic and vis-
coelastic contributions in both the e1,2 directions and can be
used as such in computing the traction in the e3 direction. the elastic and viscoelastic tensors Se1,2 and Dα
t Se1,2 can be
determined, together with their counterparts in the Cauchy
stress. They take the form of diagonal second-order ten-
sors. The hydrostatic pressure P can also be determined
at each time point by noticing that the traction in the e1,2
directions is 0. Hence, P counteracts the elastic and vis-
coelastic contributions in both the e1,2 directions and can be
used as such in computing the traction in the e3 direction. Thus, the only unknowns in the traction model are parame-
ters C1, C2, δ1, δ2, α. 2.2.4 Rheology data analysis and model fitting The viscoelastic model derived in the previous section
(Eq. 19) needs to be tailored to the PVA material used in
the rheological experiments by finding appropriate parame-
ters C1, C2, δ1, δ2 (Pa) and α. i.e. i.e. 12 3 3 123 A. Capilnasiu et al. trd(t) =
trd(t)
max
t |trd(t)|,
trm(t) =
trm(t)
max
t |trd(t)| was done over the tests altogether, to facilitate the interpre-
tation of the error, which is finally outlined as err =
err1 + err2
2
. (20) where trd(t) and trm(t) are kept for simplicity, to avoid addi-
tion of further notation. (20) Three characteristics were considered to be important
when fitting the model to the data: the compression, relax-
ation and oscillatory response. Thus, the error to be min-
imised was designed to comprise two parts, the first one
dealing with the compression and relaxation behaviour, and
the second one with the oscillatory behaviour. For the first
part, in order to ensure that the error is not dominated by
compression only, but also by relaxation, it is important to
consider the data broken down into cycles, i.e. one full oscil-
lation starting from the lowest load state, equivalent to one
period (see Fig. 2). Each of these cycles is confined within a
generic time interval [t1, t2]. A mean traction value ( ¯trd, ¯trm
for data and model, respectively) is defined as the integral of
the traction over the cycle, e.g. This form yields 0% for a perfect fit and 100% when
C1, C2, δ1, δ2 = 0 Pa, irrespective of α. Having defined the error to be minimised, a more detailed
parameter fitting process can be described. The nonlinear
parameter α is iterated between 0.05 and 1 (close to the
elastic and viscous limits, respectively), with an incremental
step of 0.05. Since the PVA material used has a low vis-
coelastic response (and α = 0.05 was observed to be the
most suitable), the incremental step was refined to be 0.01
between 0.01 and 0.1. For each fixed α, the linear parameters
C1, C2, δ1, δ2 were searched by solving the least squares
problem corresponding to error Eq. 20, using the inbuilt func-
tion lsqnonlin of MATLAB and Statistics Toolbox Release
2015a, The MathWorks, Inc., Natick, Massachusetts, USA. The nonlinear solver was used in order to allow for a posi-
tivity constraint on the parameters. 2.2.4 Rheology data analysis and model fitting For each phantom, a full
set of parameters was determined. ¯trd
k =
t2
t1
trd(t) dt,
¯trm
k =
t2
t1
trm(t) dt, To ensure parameter uniqueness, once the best α value was
determined, a new parameter search was performed. Thus,
each individual linear parameter (C1, C2, δ1 and δ2, respec-
tively) was fit using the process described above, but while
imposing the remaining three linear parameters to be 0 Pa. This was done in order to gauge the value of each model
component and to identify potential parameter coupling. over generic cycle k. This mean value captures the compres-
sion level and, by considering locally every cycle in a test,
captures the relaxation behaviour. Hence, the first part of the
error deals with the sum, over all cycles, of the mean traction
value difference between the data and the model, normalised
by the data: err1 =
k( ¯trd
k −¯trm
k )2
k( ¯trd
k)2
. 2.3 Harmonic wave motion in deformed PVA After characterising the PVA material through a viscoelastic
model, the next essential element in estimating the intrinsic
material parameters and bypassing the loading bias is merg-
ing the information on deformation and rheology with MRE
data. Thus, in what follows, the MRE experimental features
used to obtain simple deformations are described. The core
component of integrating material behaviour and deforma-
tion knowledge into the data analysis process is outlined. The second part of the error is designed to measure the dif-
ference in the oscillatory behaviour between data and model. Thus, the traction amplitude corresponding to a generic time
point t j in a cycle k is found as the difference between the
traction value and the traction mean over the cycle, e.g. trd(tk
j ) −¯trd
k,
trm(tk
j ) −¯trm
k . 2.3.1 MRE experimental setup The difference in amplitudes between the model and the data
is investigated across all time points in a cycle, over all cycles,
and is subsequently normalised by the data, as When designing the MRE experiment, it was important to
obtain shear waves that would propagate through the whole
body of the phantom, while maintaining a fixed position
of the phantom. The setup used is illustrated in Fig. 3. A
transducer indenting the phantom transmits small amplitude
compressional waves. Since the PVA material is aqueous, it
could easily slide on a flat surface. For this reason, the phan-
tom’s back side was in contact with the support, ensuring no
penetration, hence converting the compressional waves into err2 =
k
j
(trd(tk
j ) −¯trd
k) −(trm(tk
j ) −¯trm
k )
2
k
j
trd(tk
j ) −¯trd
k
2
. We note that the standardisation of the data was done over
each of the six tests separately, to ensure that they carry
equal importance within the error, while the normalisation 123 Magnetic resonance elastography in nonlinear viscoelastic materials under load Fig. 3 Illustration of the MRE test setup. (Top) 3D view of the setup. A coil vibrating to the frequency established by the MRE sequence is
connected to a flexible lamella, which causes an attached rod to vibrate
longitudinally. A piston fit at the end of the rod is indenting the phan-
tom, thus generating compressional waves. The phantom is resting on
a smooth support and is in contact with the back support plate, which
prevents the phantom from slipping and thus helps converting the com-
pressional waves into shear waves. An upper plate compressing the
phantom is kept in place by bolts fixed to the side plates. Reception
coils are placed around the phantom, for signal enhancement. (Bottom)
2D top view of the setup prevents the phantom from slipping and thus helps converting the com-
pressional waves into shear waves. An upper plate compressing the
phantom is kept in place by bolts fixed to the side plates. Reception
coils are placed around the phantom, for signal enhancement. (Bottom)
2D top view of the setup Fig. 3 Illustration of the MRE test setup. (Top) 3D view of the setup. A coil vibrating to the frequency established by the MRE sequence is
connected to a flexible lamella, which causes an attached rod to vibrate
longitudinally. 2.3.1 MRE experimental setup A piston fit at the end of the rod is indenting the phan-
tom, thus generating compressional waves. The phantom is resting on
a smooth support and is in contact with the back support plate, which and a reference non-motion encoded image removing back-
ground phase shifts due to the MRI gradients. Examples of
the geometric and wave images can be seen in Fig. 4, for the
undeformed and deformed configurations. shear waves. A large uniaxial deformation could be obtained
by simply compressing the phantom with a smooth top plate,
as seen in Fig. 3. Due to the sliding feature of the PVA
material, the phantoms did not bulge under the uniaxial com-
pression just described. Using this setup inside the MR scanner, coronal wave data
were recorded using MRE in undeformed and deformed con-
figurations. Each phantom was scanned in the reference state
(uncompressed), first deformation state consisting of uniax-
ial compression of ∼5mm (∼14%) and second deformation
state consisting of uniaxial compression of ∼12mm (∼35%). MR acquisition consisted of two primary scans. First, a
geometric scan was acquired providing 55 cross-sectional
slices with a field of view (FOV) of 96×96 mm (resolution of
96×96 pixels and slice thickness of 1 mm). Scan parameters
wereadjustedtoenhancesignalofthePVAmaterial(TE/TR=
120.00 /5000.00 ms). Using this setup inside the MR scanner, coronal wave data
were recorded using MRE in undeformed and deformed con-
figurations. Each phantom was scanned in the reference state
(uncompressed), first deformation state consisting of uniax-
ial compression of ∼5mm (∼14%) and second deformation
state consisting of uniaxial compression of ∼12mm (∼35%). 2.3.2 MRE data analysis It can be observed in the wave displacements, b
the three rows, that the wavelength increases under compressi
wave images in the third, fourth and fifth columns have been c
around the phantom area to exclude the surrounding noise Fig. 4 Images corresponding to the uncompressed (top row) and
compressed (middle and bottom rows) phantoms (here illustrated in
phantom 12). (First column) Phantom depicted at the different defor-
mation states. The uncompressed state is kept for reference. The piston
(grey bar) is indenting the phantom perpendicularly during the MRE
scan. Slices are acquired in the coronal plane (depicted in blue). (Second
column) Cross-sectional (coronal) view from the T2 weighted images. The piston indentation can be seen, as well as the expansion of the phan- tom in the e1 −e2 directions under compression. Wave displacements
from the MRE imaging protocol can be seen in the e1 direction (third
column), in the e2 direction (fourth column) and in the e3 direction
(fifth column). It can be observed in the wave displacements, between
the three rows, that the wavelength increases under compression. The
wave images in the third, fourth and fifth columns have been cropped
around the phantom area, to exclude the surrounding noise tom in the e1 −e2 directions under compression. Wave displacements
from the MRE imaging protocol can be seen in the e1 direction (third
column), in the e2 direction (fourth column) and in the e3 direction
(fifth column). It can be observed in the wave displacements, between
the three rows, that the wavelength increases under compression. The
wave images in the third, fourth and fifth columns have been cropped
around the phantom area, to exclude the surrounding noise ments uc and reconstructs the complex stiffness modulus G∗
by assuming scaling due to large deformations into the wave displace-
ments gradient. Hence, instead of solving for G∗, a modified
form of the equations was developed, given below: (21) G∗= G∗I. −ρω2uc −∇· [M(G′ : ∇uc)]
−i∇· [(N(G′′ : ∇uc)] −∇· pc I = 0,
(22a)
∇· uc = 0,
(22b) (22a)
(22b) Thisisusuallyseparatedintothe(real)storagemodulus G′
and (imaginary) loss modulus G′′ (where G∗= G′ + iG′′). (22b) As explained in Sect. 2.1.2, the form of G∗presented in
Eq. 21 holds true in the absence of deformation and under
an isotropic standard material law, and the wave gradient
contribution becomes symmetric, as I : ∇uc = ∇uc+∇uT
c . 123 2.3.2 MRE data analysis The geometric data acquired were used to provide detailed
quantitative information of the geometrical configuration of
the material in reference and deformed states. Assuming that
the cuboid phantoms were compressed uniformly, then the
deformation gradient can be written as in Eq. 12, where λ
is the compression defined as the ratio of the compressed
and uncompressed heights. Hence, due to the design of the
MRE experiment, this form of the deformation gradient can
be employed with the stiffness moduli given by Eq. 10. MR acquisition consisted of two primary scans. First, a
geometric scan was acquired providing 55 cross-sectional
slices with a field of view (FOV) of 96×96 mm (resolution of
96×96 pixels and slice thickness of 1 mm). Scan parameters
wereadjustedtoenhancesignalofthePVAmaterial(TE/TR=
120.00 /5000.00 ms). The second scan was a MRE eXpresso Gradient Echo
sequence (Garteiser et al. 2013) providing 10 cross-sectional
slices with a FOV of 96 × 96 mm (resolution 96 × 96 pixels
and slice thickness of 2.0 mm). Driver frequency and motion
encoding gradients were set, by turn, at 120, 130 and 140 Hz,
and TE/TR at 6.91 /151.82 ms. The sequence encoded each
of the three displacement component direction—e1, e2, e3 From the recorded small amplitude displacements, two
reconstruction methods were used. Firstly, a divergence free
finite element reconstruction solving the set of Eq. 9 (Fovar-
gue et al. 2018a) was used to retrieve the stiffness of the PVA
phantom, merging the datasets acquired at 120, 130 and 140
Hz, for a better signal to noise ratio. This method takes as
input the material density ρ, frequency ω and wave displace- 12 3 A. Capilnasiu et al. Fig. 4 Images corresponding to the uncompressed (top row) and
compressed (middle and bottom rows) phantoms (here illustrated in
phantom 12). (First column) Phantom depicted at the different defor-
mation states. The uncompressed state is kept for reference. The piston
(grey bar) is indenting the phantom perpendicularly during the MRE
scan. Slices are acquired in the coronal plane (depicted in blue). (Second
column) Cross-sectional (coronal) view from the T2 weighted images
tom in the e1 −e2 directions under compression. Wave displac
from the MRE imaging protocol can be seen in the e1 directio
column), in the e2 direction (fourth column) and in the e3 d
(fifth column). 3.1 Nonlinear viscoelastic model for PVA With this understanding of the newly developed CR, the
PVA material model was parameterised, this time employing
the MRE data. The reference and second compression states
were used, and the left hand side of Eq. 22a was minimised
over all pixels. That is, the linear model parameters were
sought such that M and N were reconstructed as closely as
possible to unity for each pixel, over all pixels. The nonlinear
parameter α was fixed to be the one indicated by rheology
tests. The set of parameters thus obtained from the reference
and second compression states was tested against the first
compression state, for prediction value. The error was mea-
sured as the pixel-wise difference between the reconstructed
values M and unity in uncompressed and second compres-
sion case, normalised by the number of pixels, as The aim of the modelling process was to fit the viscoelas-
tic model 19 to the PVA data acquired in the rheological
setup. Hence, for each of the six phantoms used in the rhe-
ology experiment, the parameters were fit accounting for
all oscillatory and relaxation tests simultaneously. It will
be seen that the parameters cannot be uniquely identified,
which stems from the fact that the PVA has a small vis-
coelastic response, as it will be explained shortly. As such, a
model with a reduced number of parameters is sought, which
ensures unique parameter estimation. Initially, the 5-parameter model described by Eq. 19 was
fit to the data, considering α values between 0 and 1. Figure 5
(left) depicts the errors for each of the six phantoms used in
rheology. All phantoms yield a minimal error around an α
value of 0.03–0.09. The minimal error increases only slightly
at higher α values. The reason for the shallow changes in min-
imalerrorathighα valuesisthatthe2viscoelasticparameters
δ1 and δ2 become very small; hence, the data are fit mostly
with the elastic parameters C1 and C2. Due to the PVA mate-
rial having a small viscoelastic response, employing only
an elastic model is not highly detrimental to the model fit,
hence the small errors even at α = 1. The best parameter
fit for each phantom is summarised in Table 1. The errors
are small (less than ∼7%), which indicates that a good fit is
ensured for each test. An example of the model fit can be seen
in Fig. 6 (left). 2.3.2 MRE data analysis Hence, this method is suitable for reconstructing the stiffness
modulus when no deformation is employed, but will lead to
a biased result under deformation. Throughout the rest of
the paper, this method will be referred to as uncorrected
reconstruction (UR). where a solution is sought for M +i N. This second approach
will be referred to as the corrected reconstruction (CR). As previously mentioned, within the UR the wave gradi-
ent is contracted with the fourth-order identity tensor scaled
by the complex stiffness modulus, as (G∗I) : ∇uc. By
contrast, the CR allows the wave to be contracted with
two distinct fourth-order tensors, defined by the user. One
result is to be integrated with the real part and one with
the imaginary part of the complex stiffness modulus, as
M(G′ : ∇uc)+i N(G′′ : ∇uc), where M +i N is to be found. This is a novel way of analysing MRE data and is of utmost
importance, since we saw, in Eq. 10, that the real and imagi-
nary moduli G′ and G′′ are non-trivial and change differently
with deformation. These changes depend on metrics derived Secondly, the same reconstruction was adapted here, by
integrating the formal definition of the real and imaginary
stiffness moduli G′ and G′′ (Eq. 10) in order to account for
the bias in apparent stiffness introduced by the deformations. Conceptually, instead of relying solely on the wave displace-
ments and its gradient ∇uc, the reconstruction integrates the 123 123 Magnetic resonance elastography in nonlinear viscoelastic materials under load from the known deformation gradient F (B, IC, etc.), and,
for our PVA material, are scaled by the parameters determin-
ing the viscoelastic Eq. 19. The specific form can be found
in Appendix 2, which is applicable to the PVA material used
here. Thus, assuming that the viscoelastic model describes
the data well, then G′ and G′′ already incorporate the true
real and imaginary stiffness moduli. Hence, it is predicted
that M and N will be reconstructed as unity. bias. The deformation bias is shown in Sect. 3.2, by investi-
gating uniaxial compression in the MRE experimental setup. In Sect. 3.3 it is shown that, by incorporating the information
on rheology and deformation into the CR, the intrinsic stiff-
ness of the PVA material can be retrieved from the loaded
states of the phantoms. 3.1 Nonlinear viscoelastic model for PVA It can be observed that the compression level
and relaxation behaviour are properly captured, while micro-
oscillatory amplitudes are slightly underestimated. Notably, err =
n
k=1 (Mk −1)2
n
,
(23) (23) where n is the total number of pixels considered and subscript
k iterates over those pixels. 3 Results and discussion a large variability can be seen for all parameters with stan-
dard deviations around the mean spanning more than 100%. This can be explained by looking at the α parameter, which
is generally small (0.03–0.09). These values indicate that
the PVA has a very small viscoelastic response. Mathemat-
ically, if α = 0, then the linear parameters {C1, δ1} and
{C2, δ2} become pairwise redundant, since they scale the
same component. Hence, with a very low α, a coupling of the
parameters arises. For instance, it can be seen, in Table 1, This can be explained by looking at the α parameter, which
is generally small (0.03–0.09). These values indicate that
the PVA has a very small viscoelastic response. Mathemat-
ically, if α = 0, then the linear parameters {C1, δ1} and
{C2, δ2} become pairwise redundant, since they scale the
same component. Hence, with a very low α, a coupling of the
parameters arises. For instance, it can be seen, in Table 1, Fig. 6 Illustration of the model fit considering the 5-parameter model
or 3-parameter model in phantom 3. (Left) The model fit to the data
using all parameters: C1, C2, δ1, δ2 and α (with an error of 1.50%). (Right) The model fit to the data using a reduced number of parameters:
C2, δ1, and α (with an error of 1.78%). The parameter values can be
seen in Tables 1 and 3
Fig. 7 Contribution of each
parameter to the model (black)
compared to the data (red) (here,
in phantom 4). Each of the four
linear parameters (C1 (top left),
C2 (top right), δ1 (bottom left),
δ2 (bottom right)) was fit to the
data, while setting the other 3 to
be 0 (α was kept constant at
0.03). In each quadrant, the
traction is depicted along the
y-axis and the time along the
x-axis (waiting times between
tests not plotted for
convenience). The parameters
and errors can be seen in Table 2 (Right) The model fit to the data using a reduced number of parameters:
C2, δ1, and α (with an error of 1.78%). The parameter values can be
seen in Tables 1 and 3 Fig. 6 Illustration of the model fit considering the 5-parameter model
or 3-parameter model in phantom 3. (Left) The model fit to the data
using all parameters: C1, C2, δ1, δ2 and α (with an error of 1.50%). Fig. 3 Results and discussion The first essential step in the experimental work was tailor-
ing the viscoelastic law 19 to the PVA phantoms used in the
rheology testing. The results of this process are presented in
Sect. 3.1 and help us understand the rheological behaviour of
the PVA material used, which directly influences the loading Fig. 5 The minimum error (per Eq. 20) obtained by fitting the model
to the data for each phantom, sweeping over fixed values of α between
0.01 and 1. (Left) The error for the 5-parameter model. (Right) The
error for the 3-parameter model. The minimal error for each phantom,
obtained by employing the parameters in Table 1 (5-parameter model)
and Table 3 (3-parameter model), is enhanced error for the 3-parameter model. The minimal error for each phantom,
obtained by employing the parameters in Table 1 (5-parameter model)
and Table 3 (3-parameter model), is enhanced Fig. 5 The minimum error (per Eq. 20) obtained by fitting the model
to the data for each phantom, sweeping over fixed values of α between
0.01 and 1. (Left) The error for the 5-parameter model. (Right) The error for the 3-parameter model. The minimal error for each phantom,
obtained by employing the parameters in Table 1 (5-parameter model)
and Table 3 (3-parameter model), is enhanced Fig. 5 The minimum error (per Eq. 20) obtained by fitting the model
to the data for each phantom, sweeping over fixed values of α between
0.01 and 1. (Left) The error for the 5-parameter model. (Right) The 12 3 3 A. Capilnasiu et al. Table 1 Best fit parameters for each PVA phantom; the error, computed
as per Eq. 20, can be seen in the first column (in brackets)
Phantom
C1 (Pa)
C2 (Pa)
δ1 (Pa)
δ2 (Pa)
α
p1 (3.44%)
1545.66
0.18
195.76
149.40
0.06
p2 (5.69%)
1683.89
11.55
0.47
275.13
0.05
p3 (1.50%)
1606.03
4.50
1457.86
193.03
0.04
p4 (1.71%)
2491.84
57.71
628.60
79.40
0.09
p5 (1.01%)
647.09
174.31
2259.66
2.00
0.03
p6 (6.69%)
1746.13
0.04
0.00
272.97
0.05
Mean
1620.11
41.38
757.06
161.99
0.05
SD
537.34
62.71
839.25
98.87
0.019
a large variability can be seen for all parameters with stan-
dard deviations around the mean spanning more than 100%. This can be explained by looking at the α parameter, which
is generally small (0.03–0.09). These values indicate that
the PVA has a very small viscoelastic response. 3 Results and discussion Mathemat-
ically, if α = 0, then the linear parameters {C1, δ1} and
{C2, δ2} become pairwise redundant, since they scale the
same component. Hence, with a very low α, a coupling of the
parameters arises. For instance, it can be seen, in Table 1, Table 1 Best fit parameters for each PVA phantom; the error, computed
as per Eq. 20, can be seen in the first column (in brackets)
Phantom
C1 (Pa)
C2 (Pa)
δ1 (Pa)
δ2 (Pa)
α
p1 (3.44%)
1545.66
0.18
195.76
149.40
0.06
p2 (5.69%)
1683.89
11.55
0.47
275.13
0.05
p3 (1.50%)
1606.03
4.50
1457.86
193.03
0.04
p4 (1.71%)
2491.84
57.71
628.60
79.40
0.09
p5 (1.01%)
647.09
174.31
2259.66
2.00
0.03
p6 (6.69%)
1746.13
0.04
0.00
272.97
0.05
Mean
1620.11
41.38
757.06
161.99
0.05
SD
537.34
62.71
839.25
98.87
0.019 that the phantoms for which a high δ1 was yielded had a small
C1 and vice versa. Table 1 Best fit parameters for each PVA phantom; the error, computed
as per Eq. 20, can be seen in the first column (in brackets) Due to the small viscoelastic response of the PVA phan-
toms, parameters {C1, δ1} and {C2, δ2} tend to exhibit very
similar features. As such, in order to identify each param-
eter contribution to the model, α was set to 0.03 (the best
choice according to Fig. 5 (right)) and each of the linear
parameters was fit individually. The effects of each individ-
ual parameter on the model are illustrated in Fig. 7. When
considering only C1, the model component considered is Se1,
which is equivalent to the Neo-Hookean elastic law. This
parameter captures reasonably well the compression level of
each test, indicating that the PVA material used has a dom-
inant linearly elastic response. However, it lacks the ability
to capture the changes in oscillatory amplitudes—in the four
repetitions of the micro-oscillatory tests, the data indicates an
increased amplitude at higher compression levels, whereas
the model shows the same amplitude. The decrease in ampli-
tude seen in the macro-oscillatory test is a direct result of
the decrease in the actual displacement amplitude data, as
seen in Fig. 7. This amplitude behaviour is entirely expected
from the Neo-Hookean model, and the mismatch between a large variability can be seen for all parameters with stan-
dard deviations around the mean spanning more than 100%. 3 Results and discussion It is
stressed, once again, that the similarity occurs due to α being
very small. S = C2Se2 + δ1Dα
t Sv1 + Sp. (24) (24) The new set of parameters obtained for the 3-parameter
model can be seen in Table 3. It is notable that the error
increase is very small; thus, the new parametrisation does not
significantly deteriorate the quality of the fit. An example of
the parameterised 3-parameter model fit to the rheology data
can be seen in Fig. 6 (right). The error plot of the 3-parameter
model can be seen in Fig. 5 (right). Each phantom indicated a
best fit for a very small α (0.02 or 0.03). At low α values, the
errors are generally small, since the PVA is only slightly vis-
coelastic. However, at high α values, there is a sharp increase
in the minimal error, which eventually plateaus. This happens
when the contribution of the viscoelastic parameter becomes
insignificant (δ1 becomes very small, to counteract the effect
of α) and the only remaining parameter is thus C2. In this
case, as seen in Table 2 as well, the errors rise up to ∼30–
35%. In the low α regime, employing the 3-parameter model
increasestheerrorsonlyslightlycomparedtothe5-parameter
model, which is preferred due to reduced complexity. WheninvestigatingC2 ,thenonlinearityofthe Se2 compo-
nent becomes obvious—a linear increase in the compression
level (2mm between the micro-oscillatory tests) leads to a
nonlinear response in the traction force. Hence, this param-
eter alone provides an unsuitable fit to the data; however,
it adds value to the full model by being able to adjust for
the increased amplitude in the four micro-oscillation tests. As before, the viscoelastic counterpart δ2 captures the relax-
ation behaviour. Table 2 presents the best fit parameter for
each PVA phantom. By investigating Fig. 7, it is expected
that the C1 and δ1 parameters provide a better fit, and hence
yield smaller errors than the C2 and δ2 parameters. Having looked at each individual component, it is reason-
able to conclude that two out of the four linear parameters—
one elastic and one viscoelastic non-counterpart—are suffi-
cient for describing the PVA material behaviour. By keeping
either C1 or δ1, the compression level is ensured. By adding
either δ2 or C2, the amplitude fit can be improved for both
the micro- and macro-oscillations. 3 Results and discussion The relaxation is ensured
by considering either viscoelastic component—δ1 or δ2,
although from the individual parameter fit it looks like δ1
is more suitable for this aspect. 3 Results and discussion 20, can be seen below each parameter; each parameter was
fit considering the remaining three parameters to be 0
Phantom
C1 (Pa)
C2 (Pa)
δ1 (Pa)
δ2 (Pa)
α
p1
1835.42
685.33
2175.25
813.40
0.03
err (%)
11.69
53.97
9.80
58.12
p2
1933.73
884.40
2318.04
1031.81
0.03
err (%)
17.78
52.34
15.18
59.42
p3
3015.56
1190.96
3568.47
1433.38
0.03
err (%)
9.29
52.93
7.31
56.10
p4
3036.63
1072.38
3585.49
1258.29
0.03
err (%)
7.89
57.27
6.24
60.80
p5
2864.40
1132.68
3374.09
1346.91
0.03
err (%)
8.70
53.30
8.70
56.92
p6
1934.22
890.65
2363.49
922.69
0.03
err (%)
22.35
52.31
15.39
65.47
Mean
2436.66
976.06
2897.47
1134.41
NA
SD
590.75
189.85
677.24
248.31
NA Table 3 Best fit parameters for each phantom, restricting C1, δ2 = 0
Pa; the error, computed as per Eq. 20, can be seen in the first column,
in brackets mputed
er was
α
0.03
0.03
0.03
0.03
0.03
0.03
Table 3 Best fit parameters for each phantom, restricting C1, δ2 = 0
Pa; the error, computed as per Eq. 20, can be seen in the first column,
in brackets
Phantom
C2 (Pa)
δ1 (Pa)
α
p1 (3.95%)
118.94
1923.03
0.03
p2 (6.54%)
233.16
1900.25
0.03
p3 (1.78%)
169.10
3220.10
0.03
p4 (2.32%)
117.86
3143.31
0.02
p5 (1.28%)
176.42
2854.77
0.02
p6 (8.74%)
216.15
1973.74
0.03
Linear
Linear
Nonlin
Mean
171.94
2502.53
0.03
SD
43.71
581.34
0.005 These observations indicate that the best parameters to fit
are C2 and δ1, while setting C1 and δ2 to 0 Pa. Indeed, when
investigating all pairwise tests (i.e. fitting either {C1, δ1},
{C1, δ2}, {C2, δ1} or {C2, δ2}, with α = 0.03), the smallest
errors were obtained when considering C2 and δ1. Hence, the
initial model was reduced to 3 parameters—C2, δ1 and α-,
with δ1 modelling the Neo-Hookean viscoelastic component,
andC2 spanning the nonlinear behaviour. The simplified PK2
tensor that characterises the PVA material becomes the model and data indicates that the PVA displays nonlinear
characteristics. Lastly, no relaxation behaviour is displayed,
which is also expected due to the model component being
purely elastic. Conversely, the fractional derivative of the
Se1 component, scaled by δ1, is the viscoelastic counterpart
of the Neo-Hookean model. As such, investigating the value
of δ1 leads to similar observations as for C1, with the obvious
difference that it can capture the relaxation behaviour. 3 Results and discussion 6 Illustration of the model fit considering the 5-parameter model
or 3-parameter model in phantom 3. (Left) The model fit to the data
using all parameters: C1, C2, δ1, δ2 and α (with an error of 1.50%). (Right) The model fit to the data using a reduced number of parameters:
C2, δ1, and α (with an error of 1.78%). The parameter values can be
seen in Tables 1 and 3 Fig. 7 Contribution of each
parameter to the model (black)
compared to the data (red) (here,
in phantom 4). Each of the four
linear parameters (C1 (top left),
C2 (top right), δ1 (bottom left),
δ2 (bottom right)) was fit to the
data, while setting the other 3 to
be 0 (α was kept constant at
0.03). In each quadrant, the
traction is depicted along the
y-axis and the time along the
x-axis (waiting times between
tests not plotted for
convenience). The parameters
and errors can be seen in Table 2 Fig. 7 Contribution of each
parameter to the model (black)
compared to the data (red) (here,
in phantom 4). Each of the four
linear parameters (C1 (top left),
C2 (top right), δ1 (bottom left),
δ2 (bottom right)) was fit to the
data, while setting the other 3 to
be 0 (α was kept constant at
0.03). In each quadrant, the
traction is depicted along the
y-axis and the time along the
x-axis (waiting times between
tests not plotted for
convenience). The parameters
and errors can be seen in Table 2 123 12 Magnetic resonance elastography in nonlinear viscoelastic materials under load Table 2 Best fit parameter for each PVA phantom; the error, computed
as per Eq. 3.2 Loading bias in harmonic wave motion under
pure compression In this study, the PVA phantom material is described by the
moderately complex viscoelastic Eq. 24. Harmonic waves 12 3 A. Capilnasiu et al. Fig. 8 Stiffness maps G′ and corrected maps M corresponding to the
uncompressed (top row) and compressed (middle and bottom rows)
phantoms (here illustrated in phantom 12). (First column) Phantom
depicted at the different deformation states. The uncompressed state is
kept for reference. The piston (grey bar) is indenting the phantom per-
pendicularly during the MRE scan. Slices are acquired in the coronal
plane (depicted in blue). (Second column) Stiffness estimates using the
UR. The region of interest (ROI) of the apparent stiffness map G′ was
obtained by eroding the phantoms’ margins. (Third column) Estimates
of map M using the CR. The ROI of maps M was obtained by eroding
the phantoms’ margins Fig. 8 Stiffness maps G′ and corrected maps M corresponding to the
uncompressed (top row) and compressed (middle and bottom rows)
phantoms (here illustrated in phantom 12). (First column) Phantom
depicted at the different deformation states. The uncompressed state is
kept for reference. The piston (grey bar) is indenting the phantom per-
pendicularly during the MRE scan. Slices are acquired in the coronal
plane (depicted in blue). (Second column) Stiffness estimates using the
UR. The region of interest (ROI) of the apparent stiffness map G′ was
obtained by eroding the phantoms’ margins. (Third column) Estimates
of map M using the CR. The ROI of maps M was obtained by eroding
the phantoms’ margins Fig. 8 Stiffness maps G′ and corrected maps M corresponding to the
uncompressed (top row) and compressed (middle and bottom rows)
phantoms (here illustrated in phantom 12). (First column) Phantom
depicted at the different deformation states. The uncompressed state is
kept for reference. The piston (grey bar) is indenting the phantom per-
pendicularly during the MRE scan. Slices are acquired in the coronal plane (depicted in blue). (Second column) Stiffness estimates using the
UR. The region of interest (ROI) of the apparent stiffness map G′ was
obtained by eroding the phantoms’ margins. (Third column) Estimates
of map M using the CR. The ROI of maps M was obtained by eroding
the phantoms’ margins were designed to probe the PVA, as shown in Fig. 4 (columns
3–5). under compression, which could be a result of the complex
wave behaviour. 3.3 Intrinsic stiffness estimation Table 4 MRE minimisation parameters fixing α = 0.03; the error,
computed as per Eq. 23, can be seen in the first column, in brackets
Phantom
C2 (Pa)
δ1 (Pa)
α
p7 (15.85%)
93.68
2119.92
0.03
p8 (15.86%)
83.15
2085.28
0.03
p9 (14.80%)
88.36
1878.05
0.03
p10 (16.64%)
94.83
2126.26
0.03
p11 (17.08%)
103.43
2235.11
0.03
p12 (18.27%)
106.27
1945.07
0.03
p13 (11.15%)
246.54
1848.72
0.03
Linear
Linear
Nonlin
Mean
120.43
2019.75
NA
SD
56.96
139.60
NA Table 4 MRE minimisation parameters fixing α = 0.03; the error,
computed as per Eq. 23, can be seen in the first column, in brackets By understanding how loading biases stiffness estimation, it
is possible to estimate intrinsic stiffness values when using
CR. This requires knowledge of the deformation employed
and the material law. Since this study deals with pure
compression, the deformation was estimated using Eq. 12. Rheological testing of the PVA led to a 3-parameter material
law (Eq. 24). It was seen, in Sect. 3.1, that there is variation
in estimating the linear parameters C2 and δ1, whereas the
nonlinear parameter α is stable around 0.02–0.03. The opti-
misationperformedinrheologywasnotrepeatedforMRE,as
this was observed to lead to a parameter coupling between α
and δ1. Thus, α was fixed at 0.03, as indicated by the rheolog-
ical tests, and C2, δ1 were estimated as described in the data
analysis Sect. 2.3.2. The resulting parameters are presented
in Table 4. It is notable that the C2 parameter is generally
lower in MRE (Table 4) than in rheology (Table 3), with the
exception of phantom 13. This could be related to the degree
of relative compression achieved. In rheology, the maximum
relative compression is 28.5%. In MRE, however, relative
compressions reach up to 35%, with the exception of phan-
tom 13 (only 18%), as seen in Fig. 9. At lower deformation
levels, the viscoelastic component is less employed; thus, the
purely elastic part becomes more dominant. As C2 is scal-
ing the elastic component, its estimate is higher when less
uniaxial compression is achieved. Within each MRE phan-
tom, experimental imperfections lead to variability between
pixels. Looking at the apparent stiffness maps in Fig. 8,
excluding the boundary area (as shown in column 2), a small
degree of heterogeneity is observed, which accentuates under
higher compressions. One reason behind this is speculated
to lie in the crystallisation process during the F-T cycles of
PVA. 3.2 Loading bias in harmonic wave motion under
pure compression (Right)
Average of corrected maps M, obtained by employing CR (Eq. 22)
on the MRE data. The average value of the corrected maps M spans
between 0.96 and 1.11. The standard deviation for all phantoms in all
compression states cross the line at the ideal value M = 1 Fig. 9 Average of stiffness maps G′ and corrected maps M over all
pixels in a phantom (excluding boundaries), in all deformation states. (Left) Average of stiffness map G′ obtained using UR (Eq. 9) on the
MRE data. In undeformed state, when λ = 1, the average stiffness
of the phantoms lies within 4.5-5.3 kPa. With increased compression, Fig. 9 Average of stiffness maps G′ and corrected maps M over all
pixels in a phantom (excluding boundaries), in all deformation states. (Left) Average of stiffness map G′ obtained using UR (Eq. 9) on the
MRE data. In undeformed state, when λ = 1, the average stiffness
of the phantoms lies within 4.5-5.3 kPa. With increased compression, 3.2 Loading bias in harmonic wave motion under
pure compression Since the phantom PVA material becomes
apparently anisotropic under deformation, a shear wave (as
in Eq. 13) probing, locally, different directions would lead to
higher variability in the stiffness measurements. With this experimental design, when reconstructing with
UR the MRE phantom stiffness in the uncompressed and
two compression states, it is observed that the material
appears increasingly stiffer with increasing compression. This behaviour is observed in all seven phantoms, as shown
in Fig. 8 (column 2). There it can be seen that, in the uncom-
pressed case, the intrinsic stiffness is ∼4.5 kPa. At the first
compression level, the loading bias leads to an apparent stiff-
ness of ∼6 kPa, and at the second compression level to an
apparent stiffness of ∼7.5 kPa. A summary of the average
stiffness of every phantom in each deformation state can
be seen in Fig. 9. The statistics are based on a region
of interest (as seen in
Fig. 8) which excludes the phan-
tom’s margins, in order to avoid peculiar boundary effects. A nonlinear stiffening trend can be observed with increased
compression. The standard deviations increase as well, from
∼10 to ∼21%, suggesting a higher stiffness heterogeneity Recall that in Sect. 2.1.3 we discussed the motion of a
planar shear wave through a compressed body in order to gain
an intuition of the biasing effects of loading. It was seen that,
for an idealised planar wave (like in Eq. 15) moving through
a compressed Neo-Hookean body, the material appears to be
stiffer. Although the MRE experiment employed in this study
carries additional complexity compared to the idealised case
(particularly in the type of material used and wave structure),
the stiffening trend observed in PVA is in accordance with the
simplified theoretical outcomes just summarised. The same
stiffening effect was observed in an ultrasound study as well
(Gennisson et al. 2007), under similar conditions (idealised
planar wave and uniaxially compressed material). 3 123 Magnetic resonance elastography in nonlinear viscoelastic materials under load the phantoms appear to be stiffer, following a nonlinear trend. (Right)
Average of corrected maps M, obtained by employing CR (Eq. 22)
on the MRE data. The average value of the corrected maps M spans
between 0.96 and 1.11. The standard deviation for all phantoms in all
compression states cross the line at the ideal value M = 1 the phantoms appear to be stiffer, following a nonlinear trend. 3.4 Extension and impact in tissue This indicates
that the model, parameterised with the values presented in
Table 4, captures the bias induced by loading on the wave
motion. This shows that the parameters found can describe
the material at different deformation states and are suitable
for estimating the intrinsic stiffness of the PVA material. The most immediate extension of our approach could be
breast MRE. This imaging technique relies on the breast
being fixed in between two plates, which leads to tissue com-
pression. In the literature, the loading bias was observed in
cases where the breast was fixed tightly, compared to cases
where the breast was fixed more loosely (Sinkus et al. 2005a),
but was not accounted for. Undoing this bias using subject
specific deformation would help in correcting the observed
stiffness for both healthy and diseased areas. For instance,
provided that the risk of breast cancer is associated with tis-
sue stiffness (Boyd et al. 2014), accounting for the loading
bias would ensure that the risk assessment is not underes-
timated or overestimated. Clinically, this could improve the
prognostic value and disease management. Another extension of our study could be in the liver,
which is subjected to motion due to respiration. While this
results mainly in translational motion, parts of the liver can
be strained (Kang et al. 2012). This can be advantageous,
since some liver pathologies were shown to become more
apparent, compared to healthy tissue, under large strains (in
ex vivo) (Yeh et al. 2002). Measuring stiffness in strained
tissue could increase diagnostic accuracy, but would require
techniques to eliminate loading bias. Integrating rheology and MRE is a difficult problem to
tackle. While rheology can be employed to directly measure
a material’s stress-strain response, MRE relies on investi-
gating the wave propagation behaviour in order to estimate
material properties. Here, we tried to integrate the two meth-
ods by validating a viscoelastic model using rheological data
and then using it to derive the G′ and G′′ moduli to be used
within CR of MRE data. The model was shown to capture
the loading bias, thus proving the successful integration of
the methods. Furthermore, model parameterisation was done
separately for rheology and MRE, yet it is notable that the
standard deviations around the mean of the C2 and δ1 param-
eters are overlapping across the two experimental methods. 3.4 Extension and impact in tissue The theoretical framework presented in Sect. 2.1.2 showed
that the stiffness moduli are directly related to the material
law and deformation. The work done in phantoms supports
the theoretical concept. It was shown that, by incorporat-
ing the knowledge on material behaviour and deformation
into the MRE reconstruction, the intrinsic stiffness can be
estimated. This approach could be extended and applied in
tissues. As a first step, a biomechanical model describing
the tissue could be inferred and further adapted from animal
rheological work. Additionally, by employing registration
tools on anatomical images of an organ in undeformed and
deformed states, deformations can be quantified. Together, a
better characterisation of specific tissue rheology and large
deformation recordings using MRI could enable proper inter-
pretation of MRE data. The quantified average of map M for all phantoms in
all deformation states (uncompressed, first compression and
second compression) is shown in Fig. 9 (right). Same as for
the apparent stiffness G′, only a ROI was considered inside
each phantom, to avoid boundary effects. Depending on each
phantom’s height, a variable compression λ describes the first
or second compression. For instance, the data points on the
graph corresponding to λ between 0.75 and 0.8 depict the
first compression for taller phantoms and second compres-
sion for shorter phantoms. It is observed that the standard
deviations are sizeable (10–19%) and tend to be higher at
the larger compression levels. In the stiffness measurements,
this observation was attributed to the apparent anisotropy
effect and waves’ local probing directions. In the corrected
maps M, the apparent anisotropy effect is undone by incor-
porating the knowledge on the deformation gradient, given
by Eq. 12. Since the variance does not decrease in the M
maps compared to G′ maps, it suggests that the predicted
apparent anisotropy in compression is not the main cause of
the increasing standard deviations. Instead, it could be due to
experimental imperfections which are difficult to control. For
instance, the slight unevenness of the phantoms’ top or the
compression induced by the piston indentation are unavoid-
able, yet for simplicity their effects are not accounted for in
the deformation gradient, which employs an idealised form. Despite these, in all but one cases, the average of corrected
map M lies within 10% of the ideal value of 1. 3.3 Intrinsic stiffness estimation Assuming that the PVA freezes first at the mould edges
and then in the centre, this could affect the local structures
formed. Another reason could be that the top side of the phan- toms is not perfectly flat, as it was cut manually. As such,
the compression could have not been ideally uniform. Other
arguments could revolve around the compression that the pis-
ton is exerting on the phantom, but an analysis in this sense
is intricate and beyond the purpose of this study. Regardless
of the underlying reasons, the small local variations in each
phantom lead to the errors presented in Table 4, as slightly
different pixels are attempted to be fit to the same measure. Although the errors are higher than in rheology (Table 3),
the error metrics are not directly comparable. In rheology,
a transient behaviour is investigated, that is, a data point is
followed in time (Eq. 20), whereas in MRE a large spatial
sample size is examined—all the pixels across several slices
of each phantom, in uncompressed and second compressed
state (Eq. 23). The parameters obtained in
Table 4 were sought by
employing the CR on each phantom in uncompressed and
second compression states, as previously described. Sub-
sequently, they were used directly in reconstructing the
corrected maps M for the first compression case, corre- 12 3 A. Capilnasiu et al. spondingly for each phantom. The corrected maps M are
illustrated, in a phantom, in Fig. 8 (column 3). Since the
parameters are fit to best describe the data in uncompressed
and second compressed states, it was indeed expected that the
M map’s average for these cases is close to unity. Addition-
ally, the corrected map for the first compression case, whose
average also lies close to unity, demonstrates the prediction
value of the parameters. This, together with the undoing of the loading bias, demon-
strates the improvement that the newly developed CR brings
in analysing MRE data. Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of
interest. This study provides the means through which a material’s
intrinsic characteristics can be retrieved, despite it experienc-
ing loading. However, a reasonable quantification of intrinsic
material parameters relies on a good estimation of the defor-
mation state and on an appropriate constitutive material law. In more complex scenarios, like in vivo measurements, these
prerequisites are more challenging to attain. The deforma-
tion metrics would presumably need to rely on precise image
registration techniques. As medical image registration is a
greatly developed field, identification of suitable methods
is surely attainable. However, the material constitutive law
could be difficult to model, particularly in tissues which
exhibit anisotropy. Nonetheless, much work has been done
in this sense, as tissue models have been developed in many
forms—polynomial, exponential, logarithmic, power laws,
etc., and can be further improved and adapted. Undeniably,
a model with increased complexity would lead to convo- Open Access This article is distributed under the terms of the Creative
Commons Attribution 4.0 International License (http://creativecomm
ons.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. 3.5 Study limitations A limitation encountered during the analysis process of the
data presented in this study was the estimation of the viscous
modulus from MRE data. The rheological tests performed on
PVA phantoms identified a very low α, indicating that there
is little viscous response of the material. This is a conse-
quence of choosing a highly elastic material, with low wave
attenuation. As such, the characteristics of the viscous com-
ponent were difficult to identify under MRE testing, leading
to an overestimation of the reconstructed viscous modulus. This effect was already identified with the employed MRE
reconstruction (Fovargue et al. 2018a), where the overestima-
tion of the viscous modulus was observed in a purely elastic
phantom, particularly with increased noise. However, this
drawback is predicted to disappear when using in vivo data,
as tissue has a stronger viscous component. Nevertheless, in
this phantom study, the bias due to the spectrum of stiffness
prevented the characterisation of the full complex modulus,
thus shifting the focus only on the real stiffness modulus. 3.4 Extension and impact in tissue The heart is a particularly complex organ which presents
structural anisotropy, has dynamic stiffness, and undergoes
large motion during the cardiac cycle. The latter constitutes
one of the many challenges in assessing the unbiased stiffness
of the myocardium (Kolipaka et al. 2010, Glaser et al. 2012),
since the effects of deformation non-trivially interfere with
the intrinsic tissue properties. The framework presented in
this study would constitute a step forward in assessing the
tissue characteristics, by eliminating the deformation bias. 123 123 Magnetic resonance elastography in nonlinear viscoelastic materials under load Inordertofollowthispaper’sproposedpathwayofretriev-
ing intrinsic material parameters from MRE data, one would
have to consider Eq. 9 in the material frame as a starting
point. Since the moduli G′ and G′′ depend on constitutive
behaviour and deformation (e.g. Eq. 10), then knowledge on
the PK2 tensor and deformation gradient is necessary. The
material behaviour can be modelled by considering a con-
stitutive equation for the strain energy function W, like in
Eq. 17, from which tensor S can be derived, or by proposing
an adaption for S, e.g. Eq. 19. A formulation for the deforma-
tion gradient F is required, like the one presented in Eq. 12,
which needs to describe the unperturbed macro-deformation
that the body is undergoing. From these, a form of the moduli
G′ and G′′ can be inferred, which can be employed within
CR (Eq. 22) to eliminate the loading bias. luted G′ and G′′ moduli, yet they can be attained numerically
(by contrast with the analytic form presented here, for PVA,
in Appendix 2). Hence, despite an increased computational
cost, the use of a complex model would not be hindered. 4 Conclusions This paper presents a framework for integrating knowledge
on material constitutive behaviour and deformation into wave
dynamics in order to retrieve intrinsic material stiffness prop-
erties. First off, a theoretical foundation was established by
perturbing Cauchy’s equations of motion and transcribing
the resulting equations into material frame. This determined a
form of the stiffness moduli which was seen to depend on the
material model and deformation metrics. To support the the-
ory, experiments on PVA phantoms were employed. The PVA
material was tested in a rheological setup and its examined
behaviour was modelled using a viscoelastic law. The same
material was subjected to MRE experiments, where a known
deformation (uniaxial compression) was employed. It was
seen that, under the designed testing conditions, using stan-
dard MRE analysis, the material appeared to be stiffer with
increasing compression. Thus, the known material model and
deformation were integrated into the stiffness moduli and
subsequently into the MRE reconstruction. By doing this,
it was shown that intrinsic material stiffness parameters can
be estimated, thus undoing the loading bias observed using
standard analysis. Hence, this study provides a framework
demonstrated to work in phantoms, which can be adapted
and applied to MRE in more complex instances. Simplification to linearised elastic wave
equations Here we aim to simplify the total system of Eq. 8 by Pertur-
bation theory, expanding about the current state U. Starting by linearising the mass conservation equation,
using the matrix determinant multiplication rule (Golub and
Loan 1996), note that the first-order ε terms of the Jacobian 12 3 A. Capilnasiu et al. J ε may be clustered as3
we may focus on the deformation gradient expansion we may focus on the deformation gradient expansion J ε may be clustered as3 J(Fε) = J + Du[J][uε]
= J + Du[J(F)][uε]
= J + ∂J
∂F : Du[F][uε] + O(ε2)
= J + ∂J
∂F : ∇0uε + O(ε2). (25)
Fε = F +
∞
n=1
[∇F]n F
[: δF]n,
≈F + ∇0uε + O(ε2),
(31)
the hydrostatic part expansion Fε = F +
∞
n=1
[∇F]n F
[: δF]n,
≈F + ∇0uε + O(ε2),
(31) (31) (25)
the hydrostatic part expansion (25) Sε
p = Sp +
∞
n=1
[∇F]nSp
[: δF]n
+
∂n
∂Pn Sp
[: δP]n
≈
Sp + ∇F Sp : ∇0uε + pε J F−T
+ O(ε2),
(32) Since ∂J
∂F = J F−T (Bonet and Wood 2008), it follows that Since ∂J
∂F = J F−T (Bonet and Wood 2008), it follows that
Sε
p = Sp +
∞
n=1
[∇F]nSp
[: Since ∂J
∂F = J F−T (Bonet and Wood 2008), it follows that J(U + uε) ≈J + J F−T : ∇0uε. (26) (26) (32) This approximation, considered to hold strongly when trun-
cated to the first-order, reduces the mass balance in Eq. 8b
to This approximation, considered to hold strongly when trun-
cated to the first-order, reduces the mass balance in Eq. 8b
to the PK2 elastic part expansion the PK2 elastic part expansion J ε −1 = J(1 + F−T : ∇0uε) −1 = 0
on Ω0. (27)
Here note that J is assumed to satisfy the original balance
Sε
e = Se +
∞
n=1
[∇F]nSe
[: δF]n,
≈Se + ∇F Se : ∇0uε + O(ε2),
(33) J ε −1 = J(1 + F−T : ∇0uε) −1 = 0
on Ω0. (27) (27)
alance
Sε
e = Se +
∞
n=1
[∇F]nSe
[: δF]n,
≈Se + ∇F Se : ∇0uε + O(ε2),
(33) (27) (33) Here, note that J is assumed to satisfy the original balance
law in Eq. Simplification to linearised elastic wave
equations 3b, which leads Eq. 27 to become and the PK2 viscoelastic part expansion and the PK2 viscoelastic part expansion F−T : ∇0uε = 0. (28)
Dα
t Sε
v = Dα
t
Sv +
∞
[∇ F−T : ∇0uε = 0. (28)
Using Eqs 25 and 28 it is now clear that J ε = J +O(ε2) F−T : ∇0uε = 0. (28)
Using Eqs. 25 and 28, it is now clear that J ε = J +O(ε2),
which simplifies the momentum term in Eq. 8a to
2
Dα
t Sε
v = Dα
t
Sv +
∞
n=1
[∇F]n Sv
[: δF]n
≈Dα
t
Sv + ∇F Sv : ∇0uε
+ O(ε2)
≈∇F Sv : Dα
t
∇0uε
+ O(ε2), Dα
t Sε
v = Dα
t
Sv +
∞
n=1
[∇F]n Sv
[: δF]n
≈Dα
t
Sv + ∇F Sv : ∇0uε
+ O(ε2)
≈∇F Sv : Dα
t
∇0uε
+ O(ε2),
(3 F−T : ∇0uε = 0. (28) Using Eqs. 25 and 28, it is now clear that J ε = J +O(ε2),
which simplifies the momentum term in Eq. 8a to (34) ρ J ε∂ttUε = ρ J∂ttUε + O(ε2). (29) (29) where it is assumed that Sv and ∇F Sv are constant in time. Hence, the components of the stress term FεSε are given by To further reduce the momentum balance, we want to
expand the stress term FεSε in Eq. 8a using Taylor expan-
sion in (P, F). As a result, letting S =
Se(C(F)) +
Dα
t Sv(C(F)) + Sp(C(F), P), using ∇F to denote the
derivative with respect to F, i.e. FεSε ≈FS
+
F[∇F(Se + Sp)] + [I]S
: ∇0uε
+ F[∇F Sv] : Dα
t
∇0uε
+ pε J F−T
+ O(ε2),
(35) FεSε ≈FS +
F[∇F(Se + Sp)] + [I]S
: ∇0uε
+ F[∇F Sv] : Dα
t
∇0uε
+ pε J F−T
2 +
F[∇F(Se + Sp)] + [I]S
: ∇0uε
+ F[∇F Sv] : Dα
t
∇0uε
+ pε J F−T
+ O(ε2),
( (35) (∇F)i j = ∂/∂Fi j, (∇F)i j = ∂/∂Fi j, where the square bracketed terms denote fourth-order ten-
sors resulting from differentials of the PK2 stress tensor with
respect to F. For clarity, in indicial notation, and the deltas for the difference between the perturbed and
unperturbed states, i.e. Simplification to linearised elastic wave
equations 3a,
In indicial notation,
[F∇F(Se + (iω)αSv + Sp) + IS] : ∇0uc
i j
= Fik
∂(Se + (iω)αSv + Sp)kj
∂Fmn
∂(uc)m
∂xl
Fln
+ δimSnj
∂(uc)m
∂xl
Fln, In indicial notation, In indicial notation,
ρ
tt
0 (
)
+ ρ
tt
−∇0 ·
F∇F(Se + Sp) + IS
: ∇0uε
+ [F∇F Sv] : Dα
t
∇0uε
+ pε J F−T
= 0. (37)
Noting that the macro-deformation U satisfies Eq. 3a,
the first square bracketed term on the left hand side is zero. Hence, a set of linearised elastic wave equations in the ref-
erence frame can be formulated:
[F∇F(Se + (iω)αSv + Sp) + IS] : ∇0uc
i j
= Fik
∂(Se + (iω)αSv + Sp)kj
∂Fmn
∂(uc)m
∂xl
Fln
+ δimSnj
∂(uc)m
∂xl
Fln,
and
F−T : ∇0uc = ∂(uc)m
∂x
. (37) Noting that the macro-deformation U satisfies Eq. 3a,
the first square bracketed term on the left hand side is zero. Hence, a set of linearised elastic wave equations in the ref-
erence frame can be formulated: and F−T : ∇0uc = ∂(uc)m
∂xm
. ρ J∂ttuε −∇0 ·
F∇F(Se + Sp) + IS
: ∇0uε
+ [F∇F Sv] : Dα
t
∇0uε
+ pε J F−T
= 0. (38a)
F−T : ∇0uε = 0. (38b) Moreover, by noticing the property linking the divergence of
a tensor between the physical and reference domains (Gurtin
et al. 2010) (38a) (38b) F−T : ∇0uε = 0. J∇· H = ∇0 · [J H F−T ] , While the set of Eq. 38 deals generically with small scale
perturbations, in the case where the frequency is sufficiently
high or the time interval sufficiently long, harmonic waves
may be considered. Thus, inserting the wave form given by
Eq. 2 into the set of Eq. 38, and noting the Laplace transform
as Dα
t
eiωt
≈(iω)αeiωt ∀t >> 0, it follows that the divergence of the stress terms can be recast in terms of
the physical gradient operator ∇, i.e. the divergence of the stress terms can be recast in terms of
the physical gradient operator ∇, i.e. Simplification to linearised elastic wave
equations and the deltas for the difference between the perturbed and
unperturbed states, i.e. δF = Fε −F = ∇0uε,
δP = Pε −P = pε,
(30)
(FεSε)i j = FikSk δF = Fε −F = ∇0uε,
δP = Pε −P = pε,
(30)
(FεSε)i j = FikSkj + Fik
∂(Se + Sp)k
∂Fmn
+ Dα Fik
∂(Sv)kj ∂uε
m + δF = Fε −F = ∇0uε,
δP = Pε −P = pε,
(30)
3 Here, we denote by
(FεSε)i j = FikSkj + Fik
∂(Se + Sp)kj
∂Fmn
∂uε
m
∂Xn
+ Dα
t Fik
∂(Sv)kj
∂Fmn
∂uε
m
∂Xn
+ ∂uε
i
∂Xn
Snj
ε J F
1
O( 2) δF = Fε −F = ∇0uε,
δP = Pε −P = pε,
(30) (FεSε)i j = FikSkj + Fik
∂(Se + Sp)kj
∂Fmn
∂uε
m
∂Xn
+ Dα
t Fik
∂(Sv)kj
∂Fmn
∂uε
m
∂Xn
+ ∂uε
i
∂Xn
Snj
+ pε J F−1
ji + O(ε2). (36) (30)
f (x)
(FεSε)i j = FikSkj + Fik
∂(Se + Sp)kj
∂Fmn
∂uε
m
∂Xn
+ Dα
t Fik
∂(Sv)kj
∂Fmn
∂uε
m
∂Xn
+ ∂uε
i
∂Xn
Snj
+ pε J F−1
ji + O(ε2). (36) (30) 3 Here, we denote by + pε J F−1
ji + O(ε2). (36) (36) Dx[ f ][u] = lim
ε→0
f (x + εu) −f (x)
ε Thus, considering Eqs. 29 and 35, the conservation of
momentum can be split between macro-deformation (in
square brackets) and micro-deformation components, i.e. the directional derivative of f at a point x in the direction of increment
u. the directional derivative of f at a point x in the direction of increment
u. 3 Magnetic resonance elastography in nonlinear viscoelastic materials under load
ρ J∂ttU −∇0 · (FS)
+ ρ J∂ttuε
−∇0 ·
F∇F(Se + Sp) + IS
: ∇0uε
+ [F∇F Sv] : Dα
t
∇0uε
+ pε J F−T
= 0. (37)
Noting that the macro-deformation U satisfies Eq. Viscoelastic moduli estimates for PVA
material (5 + ∂Sp,ks
∂Fmn
+ ∂Fik
∂Fmn
Sks
Fln Fjs,
(48)
while the G′ and G′′ moduli become
G′ = 1
J
F
C1(∇F Se1) + C2(∇F Se2)
+ ∇S
+ IS
FT FT
= J
ωα sin
2
·
δ1 3 I C
∇uc + ∇uc
+ 8δ2
( B2 : ∇uc)
4 B2 + 2I −2I I C I
+
B∇uc B + ( B∇uc B)T + I I C
3
∇uc + ∇uT
c
· (I I C −3)
. (51) (48) while the G′ and G′′ moduli become
+ 8δ2
(B : ∇uc)
4B + 2I
2I IC I
+
B∇
B + ( B∇
B)T + I I C
∇
+ ∇
T while the G′ and G′′ moduli become G′ = 1
J
F
C1(∇F Se1) + C2(∇F Se2)
+ ∇F Sp
+ IS
FT FT
+ ωα
J cos
πα
2
F
δ1(∇F Se1)
+ δ2(∇F Se2)
FT FT ,
(49a)
G′′ = ωα
J sin
πα
2
F
δ1(∇F Se1)
+ δ2(∇F Se2)
FT FT . (49b)
Inthe
i.e. F
only
G′ : G′ = 1
J
F
C1(∇F Se1) + C2(∇F Se2)
+ ∇F Sp
+ IS
FT FT
+ ωα
J cos
πα
2
F
δ1(∇F Se1)
+ δ2(∇F Se2)
FT FT ,
(49a)
G′′ = ωα
J sin
πα
2
F
δ1(∇F Se1)
+ δ2(∇F Se2)
FT FT . (49b)
+
B∇uc B + (B∇uc B)
+
3
∇uc + ∇uc
· (I I C −3)
. (51)
Intheparticularcasewhenthereexistsnomacro-deformation,
i.e. F = I, it follows that the real scaling modulus retains
only the terms
G′ : ∇uc = 1
J
2C1
∇uc + ∇uT
c
−J P∇uT
c
+ 2δ1ωα cos
πα
2
∇uc + ∇uT
c
,
(52) (51) Intheparticularcasewhenthereexistsnomacro-deformation,
i.e. F = I, it follows that the real scaling modulus retains
only the terms (49a) G′ : ∇uc = 1
J
2C1
∇uc + ∇uT
c
−J P∇uT
c
+ 2δ1ωα cos
πα
2
∇uc + ∇uT
c
,
(52) (52) (49b) while the imaginary modulus reduces to The moduli G′ and G′′ are influencing the wave dynamics
∇uc, leading to an apparent wave dynamic. Simplification to linearised elastic wave
equations G′ = 1
J
F∇F Se + ωα cos
πα
2
F∇F Sv
+ F∇F Sp + IS
FT FT ,
(46a)
G′′ = ωα
J sin
πα
2
F∇F Sv FT FT . (46b) motion described in Sect. 2.1.1. After a cumbersome deriva-
tion and simplification process, the moduli defining the PVA
material used can be formulated as motion described in Sect. 2.1.1. After a cumbersome deriva-
tion and simplification process, the moduli defining the PVA
material used can be formulated as (46b) G′ : ∇uc
= 1
J
C1
−4
3 I( B : ∇uc) + 2
3 I C∇uT
c + 2∇uc B
+ 8C2
( B2 : ∇uc)
4 B2 + 4I −8
3 I I C I
+
B∇uc B + ( B∇uc B)T + ∇uc B2 + I I C
3 ∇uT
c
· (I I C −3)
−J P∇uT
c
+ ωα cos
πα
2
·
δ1
2
3 I C
∇uc + ∇uT
c
+ 8δ2
( B2 : ∇uc)
4 B2 + 2I −2I I C I
+
B∇uc B + ( B∇uc B)T + I I C
3
∇uc + ∇uT
c
· (I I C −3)
(50 G′ : ∇uc
= 1
J
C1
−4
3 I( B : ∇uc) + 2
3 I C∇uT
c + 2∇uc B
+ 8C2
( B2 : ∇uc)
4 B2 + 4I −8
3 I I C I
+
B∇uc B + ( B∇uc B)T + ∇uc B2 + I I C
3 ∇uT
c
· (I I C −3)
−J P∇uT
c G′ : ∇uc Simplification to linearised elastic wave
equations ∇0 ·
[F∇F(Se + (iω)αSv + Sp)FT
+ ISFT ] : ∇uc
= J∇· (C : ∇uc),
(42) (42) −ρ Jω2uc −∇0 ·
[F∇F(Se + (iω)αSv + Sp)
+IS] : ∇0uc + pc J F−T
= 0,
(39a)
F−T : ∇0uc = 0. (39b) where the fourth-order tensor C encompasses Ci jml = 1
J
Fik
∂(Se + (iω)αSv + Sp)ks
∂Fmn
+ ∂Fik
∂Fmn
Sks
Fln Fjs. (39a) Ci jml = 1
J
Fik
∂(Se + (iω)αSv + Sp)ks
∂Fmn (39b) + ∂Fik
∂Fmn
Sks
Fln Fjs. (43) (43) When comparing with real data, it is desirable to map
the momentum equations into the physical frame. Using the
property (Ogden 1997) that Combining Eqs. 40–42 and noting the property linking the
divergence of a scalar in the reference domain to the physical
frame (Gurtin et al. 2010) ∇y = (∇0 y)F−1, J∇w = ∇0 · [wJ F−T ] the gradients with respect to the reference frame can also be
transformed into the current domain. Hence, the terms in the
set of Eq. 39 become the equations analogous to the classical set of Eq. 3, may be
written as, −ρ Jω2uc −J∇·
pc I + C : ∇uc
= 0,
(44a)
∇· uc = 0, −ρ Jω2uc −J∇·
pc I + C : ∇uc
= 0,
(44a)
∇· uc = 0,
on Ω. (44b) [F∇F(Se + (iω)αSv + Sp) + IS] : ∇0uc
= [F∇F(Se + (iω)αSv + Sp) + IS] : [∇0uc F−1F]
= [F∇F(Se + (iω)αSv + Sp)FT + ISFT ] : ∇uc, (44a) ∇· uc = 0, (44b) on Ω. on Ω. (40) (40) Equation 44a can be written as and and −ρω2uc −∇· [(G′ + iG′′) : ∇uc + pc I] = 0,
(45a) (45a) F−T : ∇0uc = F−T : [∇0uc F−1F]
= I : ∇uc
= ∇· uc. F−T : ∇0uc = F−T : [∇0uc F−1F]
= I : ∇uc
= ∇· uc. with the understanding that the real and imaginary scaling
moduli G′ and G′′ are given by G′ = Re(C), G′′ = Im(C), with the understanding that the real and imaginary scaling
moduli G′ and G′′ are given by G′ = Re(C), G′′ = Im(C),
i.e. (41) 12 A. Capilnasiu et al. Viscoelastic moduli estimates for PVA
material In this section, the aim is to understand the effect of macrode-
formations on the wave behaviour in a viscoelastic material
described by Eq. 19. So far, general elastic and viscoelastic
stresses Se and Sv have been used. Recall that, for this work,
they were considered to comprise two parts each, based on a
Mooney–Rivlin type of law, as follows: + ωα cos
πα
2
·
δ1
2
3 I C
∇uc + ∇uT
c
+ 8δ2
( B2 : ∇uc)
4 B2 + 2I −2I I C I
+
B∇uc B + ( B∇uc B)T + I I C
3
∇uc + ∇uT
c
· (I I C −3)
( Se = C1Se1 + C2Se2,
Dα
t Sv = δ1Dα
t Se1 + δ2Dα
t Se2. Se = C1Se1 + C2Se2,
Dα
t Sv = δ1Dα
t Se1 + δ2Dα
t Se2. Dα
t Sv = δ1Dα
t Se1 + δ2Dα
t Se2. · (I I C −3)
(50) Hence, the fourth-order tensor C can be taken one step further
and written as Hence, the fourth-order tensor C can be taken one step further
and written as (50) Ci jml = 1
J
Fik
C1
∂Se1,ks
∂Fmn
+ C2
∂Se2,ks
∂Fmn
+ (iω)α
δ1
∂Sv1,ks
∂Fmn
+ δ2
∂Sv2,ks
∂Fmn
+ ∂Sp,ks
∂Fmn
+ ∂Fik
∂Fmn
Sks
Fln Fjs,
(48)
and
G′′ : ∇uc
= 1
J
ωα sin
πα
2
·
δ1
2
3 I C
∇uc + ∇uT
c
+ 8δ
( B2
∇
)
4 B2 + 2I
2I I I
and G′′ : ∇uc
= 1
J
ωα sin
πα
2
·
δ1
2
3 I C
∇uc + ∇uT
c
+ 8δ2
( B2 : ∇uc)
4 B2 + 2I −2I I C I
+
B∇uc B + ( B∇uc B)T + I I C
3
∇uc + ∇uT
c
· (I I C −3)
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Division of Biomedical Engineering and Imaging Sciences,
King’s College London, London, UK Affiliations Adela Capilnasiu1
· Myrianthi Hadjicharalambous1,2 · Daniel Fovargue1 · Dharmesh Patel4 · Ondrej Holub1 ·
Lynne Bilston5,6 · Hazel Screen4 · Ralph Sinkus1,7 · David Nordsletten1,3 Adela Capilnasiu1
· Myrianthi Hadjicharalambous1,2 · Daniel Fovargue1 · Dharmesh Patel4 · Ondrej Holub1 ·
Lynne Bilston5,6 · Hazel Screen4 · Ralph Sinkus1,7 · David Nordsletten1,3 4
Institute of Bioengineering, Queen Mary University of
London, London, UK 1
Division of Biomedical Engineering and Imaging Sciences,
King’s College London, London, UK 1
Division of Biomedical Engineering and Imaging Sciences,
King’s College London, London, UK 5
Prince of Wales Clinical School, University of New South
Wales, Sydney, Australia 2
Present Address: KIOS Research and Innovation Centre of
Excellence, University of Cyprus, Nicosia, Cyprus 2
Present Address: KIOS Research and Innovation Centre of
Excellence, University of Cyprus, Nicosia, Cyprus 6
Neuroscience Research Australia, Sydney, Australia 3
Department of Biomedical Engineering and Cardiac Surgery,
University of Michigan, Ann Arbor, USA 7
Inserm U1148, LVTS, University Paris Diderot, University
Paris 13, 75018 Paris, France 12 123
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Light emission despite doubly-forbidden radiative transitions in AlP/GaP quantum wells: Role of localized states
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Light emission despite doubly-forbidden radiative
transitions in AlP/GaP quantum wells: Role of localized
states
S. Bhuyan, R. Mondal, P. Khatua, M. Semtsiv, W. T. Masselink, Jean Léotin,
B. Pal, B. Bansal , R. Mondal, P. Khatua, M. Semtsiv, W. T. Masselink, Jean Léotin,
B. Pal, B. Bansal To cite this version: S. Bhuyan, R. Mondal, P. Khatua, M. Semtsiv, W. T. Masselink, et al.. Light emission despite
doubly-forbidden radiative transitions in AlP/GaP quantum wells: Role of localized states. Journal
of Applied Physics, 2013, 114, pp.163101. 10.1063/1.4825328. hal-00938395 Light emission despite doubly-forbidden radiative
transitions in AlP/GaP quantum wells: Role of localized
states
S. Bhuyan, R. Mondal, P. Khatua, M. Semtsiv, W. T. Masselink, Jean Léotin,
B. Pal, B. Bansal Distributed under a Creative Commons Attribution 4.0 International License I. INTRODUCTION disorder-related relaxation of the momentum selection rules
and carrier localization are responsible for unexpected bright
emission from these samples. Other ideas for improving light
emission include making the AlP quantum well (QW) only a
few atomic layers thin so that there may be large spillover of
the electron wave function outside the AlP QW and the C-X
valley mixing due to the abrupt heterointerface may lead to a
phonon-free pathway for light emission.7,8 Furthermore, for
the GaP/AlP/GaP type-II QWs, since the holes in GaP would
effectively be free, neighboring confinement systems to
confine the holes have also been tried.9–11 Finally, there was
also a report of insertion of an ultrathin AlP QW between
the GaAsP/GaP heterostucture to improve the radiative effi-
ciency of GaP.12 In most of these studies, a relatively effi-
cient photoluminescence (PL) has been observed which has
been tentatively attributed to carrier localization.4,11 But this
localization and its origin have never been clearly estab-
lished. It is hence surprising that apart from some very pre-
liminary work,13 there is no reported luminescence study
on more conventional, wider QWs of AlP/GaP. Without
the added complications of the above mentioned engineering
ideas, wider QWs are perhaps better suited to probe the
nature of localization. In this paper, we have studied
GaP/AlP/GaP multiple QW (MQW) samples of 3, 4, and
5 nm well widths. Through a systematic study of the PL in Despite the near lattice match, AlP/GaP is among the
least studied of the group III–V heterojunction pairs. This is
because both AlP and GaP have an indirect gap. To make
things bleaker for optoelectronics, the band alignment
between AlP and GaP is type-II; the X-valley electrons (C-
valley holes) see a lower potential in AlP (GaP) [Fig. 1]. As
a result, the overlap of electron and hole wave functions is
not expected to be optimal. Non-optimal overlap would lead
to poor radiative efficiency. Due to the expected poor emission efficiency, AlP-based
materials have been largely ignored. The little research
reported, mostly in 1990’s, has primarily focused on innova-
tive bandgap engineering ideas which may improve the poor
light emitting efficiency expected of these structures. Light emission despite doubly-forbidden radiative transitions in AlP/GaP
quantum wells: Role of localized states Sumi Bhuyan,1 Richarj Mondal,1 Pradip Khatua,1 Mykhaylo Semtsiv,2 W. T. Masselink,2
Jean Leotin,3 Bipul Pal,1 and Bhavtosh Bansal1,a)
1Indian Institute of Science Education and Research Kolkata, Mohanpur, Nadia 741252, West Bengal, India
2Department of Physics, Humboldt University Berlin, Newtonstrasse 15, D-12489 Berlin, Germany
3Laboratoire National des Champs Magnetiques Pulses, 143 avenue de Rangueil, 31400 Toulouse Cedex 4,
France The GaP/AlP/GaP heterostructure has an indirect gap both in real as well as momentum space,
making the first order radiative recombination doubly forbidden. Nevertheless, we have observed
relatively efficient emission from these structures. This paper comprehensively studies the origin of
this improved light emission through a detailed analysis of the photoluminescence (PL) spectra. Our
observations suggest that localized excitons within the acceptor states in GaP close to the
heterostructure interface are enough for efficient light emission in these structures, doing away with
the need for more complicated structures (superlattices or neighboring confinement structures). This
real space localization of holes, close to the interface, apart from increasing the wave function
overlap, also relaxes the delta-function momentum selection rule. Independent experimental evidence
for this assertion comes from (i) the PL spectrum at high excitation power where transitions from
both the localized as well as extended states are independently observed, (ii) the observation that
extended states emission has the expected band-bending-induced blue-shift with increase in excitation
power, whereas the localized states do not, (iii) observation of phonon replicas for PL from localized
states, and (iv) observation of persistent photoconductivity at low temperature. Finally, we propose a
simple analytical model that accounts for both the type-II nature as well as the indirect bandgap to
explain the improvement of radiative recombination efficiency with increased localization. The
experimental observations are reproduced within an order of magnitude. The model is very general
and it also provides a framework to study the optical properties of other such (type-II and/or
indirect gap) heterostructures. HAL Id: hal-00938395
https://hal.science/hal-00938395v1
Submitted on 21 Aug 2020 L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
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lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License a)Electronic mail: bhavtosh@iiserkol.ac.in II. EXPERIMENT AND OBSERVATIONS Multiple quantum wells composed of Si-doped AlP wells
and GaP barriers were grown using gas-source molecular-
beam epitaxy on unintentionally doped n-type GaP(001) sub-
strates with n 8 1015 cm3 at 300 K.14 Low temperature
(15 K) PL spectra were measured by nonresonantly exciting
the samples with a 405nm diode laser with the power inci-
dent on the sample varied between 20 lW and 20 mW. The
spectra were dispersed in a 0.5 m monochromator and were
recorded using a silicon charge coupled device with a spectral
resolution of 0.5 meV. GaP, 50 meV at the C point, and about 46 meV at L and X
valleys.18 Hence, the second lower energy peak is from a
phonon-assisted transition and the first peak is identified as
the no-phonon (NP) peak. Note that the wider QWs have rel-
atively weaker NP peaks. The actual transition energies for
NP peaks are shown in Fig. 2 (inset). The systematic blue-
shift of the spectra with the decreasing well width confirms
quantum confinement of carriers and hence our first impor-
tant conclusion is that at least the electrons participating in
PL are indeed from the AlP QWs. This rules out the trivial
defect recombination from GaP barrier layers alone. A simplified schematic band diagram of the AlP
QW sandwiched between GaP layers is shown in Fig. 1. The type-II band alignment makes the transition energy
( 2.01–2.05 eV) smaller than the energy gaps of both the
parent compounds. For simplicity, the strain-induced split-
ting of the three-fold degenerate X-valley of AlP is not
shown.15,16 It is important to note that there will be an elec-
trostatically produced band-bending.17 This will be espe-
cially important for the holes in the GaP layer. The valence
band holes should observe a carrier density-dependent con-
finement potential which may be approximated by a triangu-
lar well.17 It was perhaps not realized that this potential
makes the extra QWs created in the neighboring confinement
structures9–11 redundant. There will also be an accompany-
ing band bending in the AlP QW layer, but since this would
only add a small perturbation to the already present strong
QW confinement potential felt by electrons, the band bend-
ing inside the QW is not shown. Now onwards, we shall mostly be discussing the results
from the 3 nm MQW sample. I. INTRODUCTION Most
studies have been made on (AlP)m/(GaP)n superlattices,
where m and n are the number of monolayers.1–5 Like for the
Si/Ge system, zone folding is predicted to yield a direct gap
also in AlP/GaP for suitable m and n.1,6 Relatively, efficient
light emission has indeed been observed from these struc-
tures but conclusive evidence for direct transitions has never
been
found. It
is
now
generally
believed
that
the 1 FIG. 1. Schematic band diagram of GaP/AlP/GaP QWs under low optical
excitation. Note that the spatially-indirect optical transition between X-
valley electrons in AlP and holes in GaP occurs at energy smaller than the
bandgaps of both GaP and AlP. FIG. 2. Photoluminescence spectra from three AlP/GaP MWQ samples
measured at 15 K under very low excitation power (20 lW). The spectra
have been scaled such that the prominent no-phonon (NP) peak in different
samples has the same intensity and lies around zero energy. In each of the
three cases, the lower intensity (phonon-assisted) peak is about 45–50 meV
below the NP peak. Note that the intensity of the phonon peak relative to the
NP peak increases with increasing well width. (Inset) The actual energy
position of the NP peak as a function of the well width. The blue-shift in the
NP peak energy with decreasing well width ascertains that the electrons are
indeed those confined in the AlP QWs. FIG. 1. Schematic band diagram of GaP/AlP/GaP QWs under low optical
excitation. Note that the spatially-indirect optical transition between X-
valley electrons in AlP and holes in GaP occurs at energy smaller than the
bandgaps of both GaP and AlP. FIG. 2. Photoluminescence spectra from three AlP/GaP MWQ samples
measured at 15 K under very low excitation power (20 lW). The spectra
have been scaled such that the prominent no-phonon (NP) peak in different
samples has the same intensity and lies around zero energy. In each of the
three cases, the lower intensity (phonon-assisted) peak is about 45–50 meV
below the NP peak. Note that the intensity of the phonon peak relative to the
NP peak increases with increasing well width. (Inset) The actual energy
position of the NP peak as a function of the well width. The blue-shift in the
NP peak energy with decreasing well width ascertains that the electrons are
indeed those confined in the AlP QWs. I. INTRODUCTION these MQWs, we have attempted to elucidate the primary
mechanism responsible for the relatively efficient light
emission. II. EXPERIMENT AND OBSERVATIONS Temperature dependence of the PL spectra for 3 nm MQW sample
measured under 5 mW excitation power. Note that at higher temperature,
band-to-band recombination dominates over the acceptor-to-band transitions. FIG. 3. Photoluminescence spectra from 3 nm MQW sample measured at
15 K as a function of excitation power (20 lW20 mW). The peaks due to
NP acceptor-to-band [A-B(NP)], one LO phonon-assisted acceptor-to-band
[A-B(LO)], two LO phonon-assisted acceptor-to-band [A-B(2LO)], and NP
band-band [B-B(NP)] transitions are marked. The A-B transitions do not
show blue-shift, whereas the B-B(NP) transition is strongly blue-shifted
with increasing excitation power. The peak at 2.2 eV is not identified. was switched on and then abruptly switched off after a
steady state in the light-on condition was reached. Time-
resolved photoconductivity was inferred from the photore-
sistance measurement. Figure 6 shows the time evolution of
normalized photoconductivity from 3 nm MQW sample. The
decay of the photoconductivity rðtÞ after the abrupt switch-
ing off of light is replotted in Fig. 6 (inset). The experimental
data can be fitted20 to the Kohlrausch stretched exponential
form: rðtÞ ¼ rð0Þexpðt=sÞb with b ¼ 0:12 and with a very
large effective decay time s 3:5 107 s at 15 K. Hence,
we have observed persistent photoconductivity.20 was switched on and then abruptly switched off after a
steady state in the light-on condition was reached. Time-
resolved photoconductivity was inferred from the photore-
sistance measurement. Figure 6 shows the time evolution of
normalized photoconductivity from 3 nm MQW sample. The
decay of the photoconductivity rðtÞ after the abrupt switch-
ing off of light is replotted in Fig. 6 (inset). The experimental
data can be fitted20 to the Kohlrausch stretched exponential
form: rðtÞ ¼ rð0Þexpðt=sÞb with b ¼ 0:12 and with a very
large effective decay time s 3:5 107 s at 15 K. Hence,
we have observed persistent photoconductivity.20 of B-B peak plotted in Fig. 4 as a function of [power]1=3
nicely fits a straight line. This is well-known in type-II
systems19 and arises due to carrier induced band-bending
and resultant nearly triangular potential well (schematically
shown for the valance band in Fig. 1) whose steepness
increases with the excitation power leading to the [power]1=3
dependence of the blue-shift of PL peak.17 The temperature dependence of the PL from the same
3 nm MQW sample measured under 5 mW excitation power
is presented on a semi-logarithmic scale in Fig. 5. II. EXPERIMENT AND OBSERVATIONS However, note that the other two
samples (4 and 5nm MQWs) also showed nearly identical
results. Low temperature PL from the 3nm MQW sample with
the excitation power varied over three orders of magnitude is
plotted in Fig. 3 on a semilogarithmic scale. Phonon replicas of
the no-phonon peak due to longitudinal optical (LO) phonon-
assisted indirect transitions are seen at all excitation powers. Tentatively, we assign these peaks as A-B(NP) [acceptor-to-
band—NP transition], A-B(LO) [acceptor-to-band—one LO
phonon-assisted transition], and A-B(2LO) [acceptor-to-
band—two LO phonon-assisted transition], respectively. We
shall justify the assignment of acceptor-to-band transitions in
Sec. III. At higher powers, a second peak emerges at slightly
higher energy than the A–B(NP) peak. While the energy
positions of the first three peaks are independent of the exci-
tation power, the new peak, labeled as B–B(NP) [band-to-
band - NP transition] shows a blue-shift with power. Figure 2 shows PL spectra from three different AlP/GaP
MQW samples (well widths 3, 4, and 5 nm). All the spectra
have been scaled to the height of the strongest peak and the
abscissa shifted such that the strongest peak is centered at
zero energy. This clearly shows that all the three samples
have a second peak at approximately the same energy
(45 meV) below the first peak. This energy difference is
close to various longitudinal optical (LO) phonon energies in This is further analyzed in Fig. 4, where the power-
dependent PL data are re-plotted in the inset on a linear
scale. Here, the signal is normalized at the A-B(NP) peak for
a clear comparison. It is evident that the energy position of
the A–B peak is virtually constant with excitation power,
whereas the B–B peak shows a clear blue-shift. The energy 2 FIG. 3. Photoluminescence spectra from 3 nm MQW sample measured at
15 K as a function of excitation power (20 lW20 mW). The peaks due to
NP acceptor-to-band [A-B(NP)], one LO phonon-assisted acceptor-to-band
[A-B(LO)], two LO phonon-assisted acceptor-to-band [A-B(2LO)], and NP
band-band [B-B(NP)] transitions are marked. The A-B transitions do not
show blue-shift, whereas the B-B(NP) transition is strongly blue-shifted
with increasing excitation power. The peak at 2.2 eV is not identified. FIG. 5. Temperature dependence of the PL spectra for 3 nm MQW sample
measured under 5 mW excitation power. Note that at higher temperature,
band-to-band recombination dominates over the acceptor-to-band transitions. FIG. 5. A. Evidences for hole localization We shall now try to infer the nature of the light emitting
states from these observations. The first conclusion which
had already been made earlier on the basis of the shift in the
transition energy with well width [Fig. 2 (inset)] is that the
electron states are quantized within the QW. We also investigated time-resolved photoconductivity. The device was made with gold wires bonded on the sample
surface to indium contacts which were annealed to make
direct contact to the underlying QW layer. We measured
in-plane two-probe photoresistance when the sample main-
tained at 15 K was exposed to 532 nm laser light. The time
evolution of photoresistance was recorded as the light source Let us now pay attention to the hole states. Based on ex-
perimental observations stated above, we shall argue in the
following that at low excitation densities, the PL emission FIG. 4. Energy of the B-B(NP) peak in the 15 K PL spectra from 3 nm
MQW sample is plotted as a function of [power]1=3. The solid line is a linear
fit showing the expected [power]1=3 dependence of blue-shift. Inset: Power
dependent PL spectra normalized at the A-B(NP) peak at 2.05 eV. Note that
there is no energy shift in the A-B(NP) peak with power. FIG. 6. Time evolution of normalized photoconductivity under abrupt switch-
ing on and off of a green (532 nm) laser light exciting the MQW sample at
15 K. Inset: Decay of photoconductivity is fitted to Kohlrausch stretched expo-
nential: rðtÞ ¼ rð0Þexpðt=sÞb with b ¼ 0:12 and s 3:5 107 s. FIG. 6. Time evolution of normalized photoconductivity under abrupt switch-
ing on and off of a green (532 nm) laser light exciting the MQW sample at
15 K. Inset: Decay of photoconductivity is fitted to Kohlrausch stretched expo-
nential: rðtÞ ¼ rð0Þexpðt=sÞb with b ¼ 0:12 and s 3:5 107 s. FIG. 4. Energy of the B-B(NP) peak in the 15 K PL spectra from 3 nm
MQW sample is plotted as a function of [power]1=3. The solid line is a linear
fit showing the expected [power]1=3 dependence of blue-shift. Inset: Power
dependent PL spectra normalized at the A-B(NP) peak at 2.05 eV. Note that
there is no energy shift in the A-B(NP) peak with power. FIG. 4. II. EXPERIMENT AND OBSERVATIONS We
observe a rapid drop in intensity of the three lower energy
A-B peaks with increasing temperature and beyond 80 K,
only the high energy B-B peak can be clearly identified. A. Evidences for hole localization Even the electron
states in these narrow QW samples are localized within the
thin QW region. Stronger localization in narrower QWs leads
to
enhanced
no-phonon
transition
relative
to
the
phonon-assisted transitions compared with wider QWs as is
seen in Fig. 2. No blue-shift was observed for the A-B peaks in Fig. 4,
while
B-B
peak
suffered
a
characteristic
[power]1=3
blue-shift of type-II transition with increasing excitation
power. Absence of blue-shift of the A-B peak with excitation
power implies that corresponding states are too strongly
localized to be affected by the carrier induced band bending
and resultant triangular potential which quantizes the states
participating in B-B transitions. The temperature dependence
of PL (Fig. 5) where the strength of the B-B(NP) peak is
enhanced with respect to the bound exciton (A–B) peaks at
higher temperature also indicates the “ionization” of the
acceptor-bound localized hole states. Finally, the observation of persistent photoconductivity
(in the QW plane) is another independent indication of hole
localization. Optical excitation with 532 nm laser nonreso-
nantly creates electron-hole pairs mainly in the GaP layers. Though some of the photogenerated electrons can be depleted
due radiative and nonradiative recombination with holes in
GaP layers, a fraction of them would diffuse to the AlP QW
layer where the electrons see a lower potential. These QW
electrons will control the in-plane photoconductivity of the
sample. Depletion of these electrons due to recombination
with holes will lead to the decay of photoconductivity. Being
a type-II QW system, electron-hole wave function overlap is
not optimal for AlP/GaP QWs. Only the holes available near
the QW interface can recombine with QW electrons and this
cause the initial rapid decay of the photoconductivity in Fig. 6. With the electrons trapped in the QW, a biased diffusion of
holes from bulk GaP to the regions close to the QW interface
is expected due to the electrostatic attraction between electrons
and holes. If the holes are localized, this transport of holes
from the bulk GaP to the heterointerface will be an activated
process that is exponentially suppressed at low temperature. This will make the QW electrons available during a very long
period of time after their photocreation. Thus, localized holes
may lead to persistent photoconductivity. Further study of the
dependence of persistent photoconductivity on the sample
temperature and excitation laser wavelength may be useful for
a more conclusive evidence. A. Evidences for hole localization Energy of the B-B(NP) peak in the 15 K PL spectra from 3 nm
MQW sample is plotted as a function of [power]1=3. The solid line is a linear
fit showing the expected [power]1=3 dependence of blue-shift. Inset: Power
dependent PL spectra normalized at the A-B(NP) peak at 2.05 eV. Note that
there is no energy shift in the A-B(NP) peak with power. FIG. 6. Time evolution of normalized photoconductivity under abrupt switch-
ing on and off of a green (532 nm) laser light exciting the MQW sample at
15 K. Inset: Decay of photoconductivity is fitted to Kohlrausch stretched expo-
nential: rðtÞ ¼ rð0Þexpðt=sÞb with b ¼ 0:12 and s 3:5 107 s. 3 independently conclude that the A-B(NP) peak at 2.045 eV
is due to localized excitons. involves the localized hole states in GaP leading to the local-
ized hole-to-conduction band transition [A-B(NP)] in Fig. 3. Strong localization in real space leads to fuzziness in mo-
mentum owing to the position-momentum uncertainty princi-
ple. This facilitates electron-hole wave-function overlap in
momentum space and enhances optical transition probability
in indirect gap systems. This is schematically shown in
Fig. 7. To ensure nonzero wave-function overlap in real
space in the type-II systems, only the hole states localized
very close to the QW interfaces can participate in PL. Such
localization centers are much smaller in number than the
band states. At high excitation powers, these get saturated
and the band-to-band (B-B) emission also becomes visible. One may obtain a rough estimate of binding energy of these
localized states from the difference in the observed PL
energy of the B-B(NP) and A-B(NP) peaks. This is about
35 meV21 and is most likely related to a shallow acceptor
state. It is emphasized that unlike the type-I structures, here
the holes cannot be localized by the interface fluctuations
(there is no confinement potential for holes). The extent of
the bound hole wave function, d, can be estimated from the
uncertainty argument, d h=
ffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2mhEB
p
.22 With the heavy
hole mass in GaP mh ¼ 0:67m0, where m0 is the free electron
mass,
and
localization
energy
EB 35 meV,
we
get
d 1:2 nm, that is about two lattice constant. Such strongly
localized hole states would have a considerable spread in
their momentum states which would support phonon-free
recombination in this indirect gap system. A. Evidences for hole localization We have observed a large oscillator strength for the pho-
non satellites connected with the A-B transitions, whereas
there are no phonon satellites at all for the B-B peaks. While
in an indirect gap material, phonon-assisted transitions are
expected, these are usually extremely weak because the long
radiative recombination time associated with the second
order pathway has to compete with nonradiative processes. On the other hand, localization of excitons has an effect of
considerably enhancing oscillator strength of these satellites
due to the increase in the Huang-Rhys factor.23 Notably, the
absence of phonon satellites in the B-B(NP) peak leads us to B. Relaxation of momentum selection rule The additional
terms P2D and P1I signify the reduction of the overlap on
account of the type-II nature and the off-zone center position
of the electrons in the indirect gap AlP. Here, P1D; P2D, and P1I correspond to the terms in the three
parenthesis, respectively, in Eq. (4). We will only have the
first term P1D for a type-I direct gap QW. The additional
terms P2D and P1I signify the reduction of the overlap on
account of the type-II nature and the off-zone center position
of the electrons in the indirect gap AlP. wX
AlPðzÞ ¼ ðLp
ffiffiffiffiffiffi
2p
p
Þ1
2 exp½ðz z0Þ2=L2 ik0z:
(1) (1) The expðik0zÞ factor, with k0 ¼ p=0:545 nm1, takes care
of the fact that the electrons in AlP are not at the Brillouin
zone center and z0ð6¼ 0Þ accounts for the type-II nature of
the heterostructure since the center of the electron wave
function is displaced with respect to the center of the hole
wave function. The latter is assumed to be at the origin. The
envelope wave function of the acceptor-bound holes with a
localization length d in the z direction is taken as The expðik0zÞ factor, with k0 ¼ p=0:545 nm1, takes care
of the fact that the electrons in AlP are not at the Brillouin
zone center and z0ð6¼ 0Þ accounts for the type-II nature of
the heterostructure since the center of the electron wave
function is displaced with respect to the center of the hole
wave function. The latter is assumed to be at the origin. The
envelope wave function of the acceptor-bound holes with a
localization length d in the z direction is taken as g p
To make numerical estimate of these effects, we have
taken z0 ¼ L and L is estimated at 1.2 nm from the confine-
ment energy (33 meV) of the ground state electron in AlP
QW. Numerical evaluation of various terms in Eq. (5) is
plotted in Fig. 8 as a function of hole localization length. Dramatic enhancement in transition probabilities for indi-
rect gap (both type-II and type-I) transitions relative to that
of type-I direct gap transition is seen with decreasing hole
localization length. As expected, the relative transition
probability for type-II direct gap QW increases with
increasing hole localization length. B. Relaxation of momentum selection rule The localization length
for the acceptor-bound hole is estimated to be one-two lat-
tice constant (0.55–1.1 nm) in GaP. These positions are
marked with thick vertical lines in Fig. 8. In this case, an
enhancement of about 105–108 is achieved as compared
with the case for unbound holes. Compared with the direct
gap type-I QW, the transition probability for a type-II indi-
rect gap transition is still smaller by a factor of 102–105. Note that this calculation is rather approximate. The
assumption of Gaussian wave functions leads to an overlap
integral having exponential dependence on various parame-
ters, making the results in Fig. 8 extremely sensitive to the
parameter values. While having exponential wave functions
may be more realistic, the corresponding confinement
potentials are no longer simple to model. Harmonic confine-
ment was chosen for analytical simplicity. Experimentally,
the intensity of the PL signal from AlP/GaP was about two
orders of magnitude smaller than that measured from high
quality GaAs/AlGaAs QW. wC
GaPðzÞ ¼ ðdp
ffiffiffiffiffiffi
2p
p
Þ1
2 exp½z2=d2:
(2) (2) The transition probability (P) is proportional to the square of
the overlap integral and can be written as P
ð1
1
wX
AlP
ðzÞwC
GaPðzÞdz
2
(3) (3) or P ¼
A
2p L
d þ d
L
0
B
@
1
C
A
exp
2z2
0
L2 þ d2
exp
k2
0
2
1
L2 þ 1
d2
2
64
3
75
0
B
@
1
C
A: (4) FIG. 8. Numerically calculated transition probabilities for type-II direct
(P2D, dashed line), type-I indirect (P1I, dotted line), and type-II indirect
(P2D P1I, solid line) transitions relative to that of type-I direct transition as
a function of hole localization length. Vertical solid lines mark the localiza-
tion length of one and two lattice constants. Inset: Schematic of electron and
hole confinement potentials (assumed to be harmonic potentials) and the
ground state wave functions (Gaussians). B. Relaxation of momentum selection rule Let us now attempt a back-of-the-envelope estimate of
the extent of the relaxation of the momentum selection rule
due to the localization of holes. The electrons in AlP are at the
three equivalent (ignoring the lifted degeneracy) X-valleys,
where the wave vectors ðkx; ky; kzÞ are the three permutations
of (001). Furthermore, since the electron confinement in the
QW is along the z direction, if we consider only the (001)
X-valley electrons of AlP then we have direct transitions for
kx and ky, and we only need to worry about a mechanism for
the relaxation of the momentum conservation rule along the z
direction. The problem may thus be effectively treated in one
dimension, with only the wave functions along the growth
direction z being the important consideration. FIG. 7. Schematic representation of confined electron states in AlP QW and
localized hole states in GaP in (A) real and (B) momentum space. Spreads in
momentum for electron and hole states are related to the inverse of the cor-
responding localization lengths. Extended hole states would correspond to a
momentum d-function. FIG. 7. Schematic representation of confined electron states in AlP QW and
localized hole states in GaP in (A) real and (B) momentum space. Spreads in
momentum for electron and hole states are related to the inverse of the cor-
responding localization lengths. Extended hole states would correspond to a
momentum d-function. The X-valley electrons are QW bound states with a finite
momentum hk0 along one of the ~k directions depending on 4 Here, A is the constant of proportionality. The transition
probability neatly factorizes into three terms and may thus
be expressed as which of the three X-valleys they belong. For analytic sim-
plicity, let us assume harmonic confinement potentials for
electrons and holes resulting in Gaussian wave functions for
the ground state electrons and holes [Fig. 8 (inset)]. The
ground state wave function for X-valley electrons confined
with a confinement length L within the AlP QW thus has the
form P ¼ P1D P2D P1I:
(5) (5) Here, P1D; P2D, and P1I correspond to the terms in the three
parenthesis, respectively, in Eq. (4). We will only have the
first term P1D for a type-I direct gap QW. IV. SUMMARY We have shown that there can be relatively efficient
light emission from type-II indirect gap GaP/AlP/GaP
MQW structures. The observed efficiency of luminescence
has been attributed to the breakdown of the momentum
selection rule due to the localization of holes by acceptor
states close to the GaP/AlP interface. The hole localization
energy is estimated at 35 meV. Localization of holes is inde-
pendently established through (i) absence of shift of the low
energy no-phonon peak with excitation power, (ii) large in-
tensity of the phonon replica, (iii) temperature dependence
of the PL spectrum, and (iv) observation of persistent photo-
conductivity at low temperature. Furthermore, a true type-II
emission, as evidenced by the characteristic [power]1/3
dependence of the PL peak energy was also established at
higher excitation power. FIG. 8. Numerically calculated transition probabilities for type-II direct
(P2D, dashed line), type-I indirect (P1I, dotted line), and type-II indirect
(P2D P1I, solid line) transitions relative to that of type-I direct transition as
a function of hole localization length. Vertical solid lines mark the localiza-
tion length of one and two lattice constants. Inset: Schematic of electron and
hole confinement potentials (assumed to be harmonic potentials) and the
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Maintenance enteral electrolyte solutions for neonatal calves: sodium acetate and osmolarity effects
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Arquivo Brasileiro de Medicina Veterinária e Zootecnia/Arquivo brasileiro de medicina veterinária e zootecnia
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http://dx.doi.org/10.1590/1678-4162-11348 http://dx.doi.org/10.1590/1678-4162-11348 o
A.P. Lima
https://orcid.org/0000-0003-3490-5679
J.D. Ribeiro Filho
http://orcid.org/0000-0001-5223-9310
P.A.N. Ermita
http://orcid.org/0000-0001-5074-5754
L.C.F. Baptista Filho
https://orcid.org/0000-0002-4632-1569 M.F.B. Avanza
http://orcid.org/0000-0003-0064-8539
R.B. Viana
http://orcid.org/0000-0002-7540-585X
P.V.M. Santos
http://orcid.org/0000-0002-3340-7013
M.O. Silva
http://orcid.org/0000-0002-2951-1950
L.C. Monteiro
http://orcid.org/0000-0003-3391-5947
W.M.F. Dantas
http://orcid.org/0000-0003-4128-5623 Arq. Bras. Med. Vet. Zootec., v.72, n.1, p.18-24, 2020 ABSTRACT The use of hypotonic electrolytic solutions in enteral fluid therapy is still understudied in calves. The
objective of the present study was to evaluate the effects of maintenance enteral electrolytic solutions
with different concentrations of sodium acetate and different osmolarities in calves. For this, 18 Holstein
calves, six male and 12 female, 20 days old and weighing around 52kg, were used. The animals were
randomly divided into three groups and each group received one of the treatments. The three electrolytic
solutions contained the same components in different concentrations, resulting in a hyposmotic, an
isosmotic and a hyperosmotic solution. Each animal was maintained in enteral fluid therapy for 12 hours
with infusion rate of 15mL kg-1 h-1. Abdominal circumference, body weight, feces consistency, glucose
and plasma lactate, pH, pCO2, HCO-
3 and BE were measured at the following times: T0h, T6h, T12h and
T24h. The hyposmotic solution did not generate the onset of diarrhea, while the isosmotic and the
hyperosmotic did. Regardless of the dose used, acetate did not cause metabolic alkalosis in the evaluated
animals. The results suggest that the use of hyposmotic solution in diarrheic calves, dehydrated and
without metabolic acidosis, may be clinically important. Keywords: diarrhea, fluid therapy, hypotonic solution, volemia. Maintenance enteral electrolyte solutions for neonatal calves:
sodium acetate and osmolarity effects [Soluções eletrolíticas enterais de manutenção para bezerros neonatos: efeitos do
acetato de sódio e da osmolaridade]
h
M
h
L A.P. Lima1, J.D. Ribeiro Filho2, P.A.N. Ermita1, L.C.F. Baptista Filho3,
M.F.B. Avanza2, R.B. Viana4, P.V.M. Santos1*, M.O. Silva1,
L.C. Monteiro1, W.M.F. Dantas2 A.P. Lima1, J.D. Ribeiro Filho2, P.A.N. Ermita1, L.C.F. Baptista Filho3,
M.F.B. Avanza2, R.B. Viana4, P.V.M. Santos1*, M.O. Silva1,
L.C. Monteiro1, W.M.F. Dantas2 1Aluno de pós-graduação Universidade Federal de Viçosa ˗ Viçosa, MG
2Universidade Federal de Viçosa ˗ Viçosa, MG
3Universidade Federal Rural de Pernambuco ˗ Garanhuns, PE
4Universidade Federal Rural da Amazônia ˗ Belém, PA MATERIALS AND METHODS Six male and 12 female Holstein calves that were
an average of 20 days old and had a median body
weight of 52kg were used in this study. These
calves were clinically healthy with no history of
gastrointestinal disease, and underwent a clinical
and
laboratory
evaluation
prior
to
the
experiment. Calves remained in individual pens
(2×3m) and were provided with a wood shavings
bed, which was changed daily. They were fed 1L
of milk four times per day and were provided
with water ad libitum during the experimental
period. The study with enteral fluid therapy using
maintenance enteral electrolytic solutions is far
from being finished, as there is still little
information on their use, mainly in adult cattle. It
is believed that research with maintenance
enteral electrolytic solutions should emphasize
the optimal osmolarity these solutions must have. This is due the fact that hyperosmotic enteral
solutions in the intestinal tract may function as
osmotic laxatives, resulting in diarrhea in the
animals (McClure, 2001), while in isotonic
solutions these adverse effects are not observed. The use of hypotonic electrolyte solutions
associated with enteral fluid therapy in calves
remains understudied, despite studies in buffalo
calves (Ermita et al., 2016) and calves (Ribeiro
Filho et al., 2017). In horses this kind of solution
has been more researched. When compared with
isotonic solutions, they were effective in
expanding volemia without causing plasma
hyponatremia or a decrease in serum osmolarity,
providing a new option for enteral fluid therapy
(Ribeiro Filho et al., 2014). Animals were randomly distributed into three
equal-sized groups. Before starting treatments, a
nasorruminal tube was introduced and attached
to the halter of each animal. The probe was
connected to a coil tube, through which the
solution flowed from a 20L recipient positioned
above the animal’s head. Animals remained in
individual
stalls
and
received
electrolyte
solutions throughout the experimental period. All
of the electrolyte solutions contained the same
components but in different concentrations,
resulting in a hypoosmotic solution – HypoSol –
(200mOsmol L-1): 4g sodium chloride, 0.5g
potassium chloride, 1g sodium acetate, and 7.5g
dextrose per liter; an isosmotic solution - IsoSol,
(280mOsmol L-1): 5g sodium chloride, 1g
potassium chloride, 2g sodium acetate, and 10g
dextrose per liter; and a hyperosmotic solution -
HyperSol - (350mOsmol L-1): 6g sodium
chloride, 1g potassium chloride, 3g sodium
acetate, and 15g dextrose per liter. RESUMO O uso de soluções eletrolíticas hipotônicas na hidratação enteral ainda é pouco estudado em bezerros. O
objetivo do presente estudo foi avaliar os efeitos de soluções eletrolíticas enterais de manutenção com
diferentes concentrações de acetato de sódio e diferentes osmolaridades em bezerros. Para isso, foram
utilizados 18 bezerros, seis machos e 12 fêmeas, holandeses, com 20 dias de nascidos e pesando por volta
dos 52kg. Os animais foram divididos aleatoriamente em três grupos e cada grupo recebeu um dos
tratamentos. As três soluções eletrolíticas continham os mesmos componentes, mas em diferentes
concentrações, resultando em uma solução hiposmótica, uma isosmótica e uma hiperosmótica. Cada
animal foi mantido em hidratação enteral durante 12 horas com taxa de infusão de 15mL kg–1h–1. Foram
aferidos perímetro abdominal, peso corporal, consistência das fezes, glicose e lactato plasmático, pH,
pCO2, HCO-
3 e excesso de base nos seguintes tempos: T0h, T6h, T12h e T24h. A solução hiposmótica não
gerou aparecimento de diarreia, enquanto a isosmótica e a hiperosmótica geraram. Independentemente
da dose utilizada, o acetato não causou alcalose metabólica nos animais avaliados. Os resultados
sugerem que o uso da solução hiposmótica em bezerros diarreicos, desidratados e sem acidose
metabólica, pode ser clinicamente importante. Palavras-chave: diarreia, hidratação, solução hipotônica, volemia. Palavras-chave: diarreia, hidratação, solução hipotônica, volemia. Recebido em 15 de fevereiro de 2019
Aceito em 14 de junho de 2019
*Autor para correspondência (corresponding author)
E-mail: pvmorais@hotmail.com Recebido em 15 de fevereiro de 2019
Aceito em 14 de junho de 2019
*Autor para correspondência (corresponding author)
E-mail: pvmorais@hotmail.com Maintenance enteral… Arq. Bras. Med. Vet. Zootec., v.72, n.1, p.18-24, 2020 RESULTS AND DISCUSSION As expressed in Table 1, the animals that
received HypoSol treatment had no change in the
consistency of the feces at the end of the fluid
therapy phase (T12h), whereas in one (16.66%)
of the animals that received the IsoSol treatment,
diarrhea was detected. In the same period, two
animals (33.33%) of the HyperSol treatment
showed diarrhea. These results demonstrate that
the hypotonic solution (HypoSol) was absorbed
in greater quantity by the intestinal tract,
evidencing that this solution is an alternative for
the enteral fluid therapy of calves with diarrhea,
which is the main cause of dehydration and death
of neonatal calves. The isotonic solution, when
used in these patients, may increases the intensity
of
diarrhea. In
children,
oral
hypotonic
electrolyte solution reduces fecal production by
20%, vomiting episodes by 30% and intravenous
hydration use by 33%, it began to be used on a
global scale in 2005 (Oral…, 2006). Blood samples were collected in tubes with
sodium fluoride to obtain the plasm to measure
the glucose and lactate in plasma using an
automatic analyzer. The samples for blood gas
analysis were collected in 2mL disposable plastic
syringes that had previously been heparinized
with lithium heparin. The statistical program SAEG 9.1 (Sistema…,
2007) was used for data analysis. Data were
analyzed using repeated measures analysis of
variance (ANOVA), which evaluated the effects
of treatment and time, and the interaction Table 1. Lima et al. morning, after the last collection (T24h), they
returned to the pre-experiment diet. between these. When the analysis was significant
for one or more factor, Tukey’s test was used to
compare the mean values. Where the data did not
meet the assumptions of ANOVA, Kruskal-
Wallis test was applied. All analyses were
interpreted considering a significance level of
5% (P< 0.05). The abdominal circumference was measured by a
tape measure at a midpoint of the paralumbar
fossa for measurement in centimeters of the
abdominal contour, while body weight was
measured in bull scales. The feces were collected
from the rectal ampoule. Subsequently, they
were weighed, placed in aluminum trays, and
placed in a kiln at 60°C for dehydration. The
feces were then weighed daily until there were
no changes in their weight. The moisture content
of the feces was calculated by the formula:
Moisture (%) = [(fresh weight - dry weight) /
fresh weight] x 100. Arq. Bras. Med. Vet. Zootec., v.72, n.1, p.18-24, 2020 20
Arq. Bras. Med. Vet. Zootec., v.72, n.1, p.18-24, 2020 MATERIALS AND METHODS Each animal
received the appropriate solution for 12 hours at
a rate of 15mL kg–1 hr–1 via nasorruminal tube
(4mm diameter, 150cm long). During the fluid
therapy phase, animals remained fasted, both
food and water were withheld, and were kept
without movement restrictions. Dehydrated suckling calves usually present
metabolic acidosis and the addition of an
alkalizing agent in the electrolytic solution is
required (Bregadioli et al., 2017). Acetate is only
effective as an alkalizing agent after being
metabolized and cannot be used in cases of
severe metabolic acidosis. However, it does not
present the disadvantages of bicarbonate when
used in enteral solutions for neonatal calves, such
as alkalinization of the abomasal pH, which does
not allow milk to coagulate by interfering with
its digestion, leading to the fermentation of
undigested milk, lactic acidosis and worsening
metabolic acidosis (Naylor et al., 1990; Leal et
al., 2007; Constable et al., 2009). The aim of the present study was to evaluate the
effects of maintenance enteral electrolytic
solutions with different concentrations of sodium
acetate and different osmolarities administered in
a continuous flow through a small-caliber
nasorruminal tube over 12 hours on newborn
calves. It is hypothesized that the use of a
hypotonic electrolyte solution causes no change
in stool consistency and that the higher dose of
sodium acetate may lead to the appearance of
metabolic alkalosis. Abdominal circumference, body weight, stool
consistency, plasma glucose and lactate, pH,
pCO2, HCO-
3 and base excess (BE) were
measured
at
the
following
times:
T0h,
immediately prior to the initiation of treatment;
T6h, after 6 hours of treatment; T12h, after 12
hours of treatment (end of fluid therapy); and
T24h, 12 hours after the end of fluid therapy. After one hour of the end of fluid therapy
(T12h), the calves were fed 1L of milk, and
water was again provided ad libitum. In the next 19 Arq. Bras. Med. Vet. Zootec., v.72, n.1, p.18-24, 2020 Lima et al. 20
Arq. Bras. Med. Vet. Zootec., v.72, n.1, p.18-24, 20 RESULTS AND DISCUSSION Mean values and standard deviations of abdominal circumference (cm), weight (kg), plasma
glucose (mg/dL) and plasma lactate (mMol/L) in healthy neonatal calves treated with hypotonic enteric
electrolyte solutions (HypoSol), isotonic (IsoSol) and hypertonic (HyperSol) administered by continuous electrolyte solutions (HypoSol), isotonic (IsoSol) and hypertonic (HyperSol) administered by continuous
nasogastric tube for 12 hours
Treatment
T0h
T6h
T12h
T24h
Abdominal circumference (cm)
HypoSol
93.174.91
97.834.87
101.506.62
96.835.77
IsoSol
92.179.11
93.507.19
100.507.71
96.507.92
HyperSol
95.3312.5
101.5010.86
103.3312.26
95.506.73
Weight (kg)
HypoSol
56.6711.15
58.3411.34
60.0010.71
57.8310.14
IsoSol
51.5014.81
53.3414.68
54.3414.53
51.8414.49
HyperSol
55.6713.59
58.5013.92
60.5015.64
57.0013.53
Plasma glucose (mg/dL)
HypoSol
84.6711.48
72.839.15
73.337.06
101.1719.49
IsoSol
92.1714.59
83.8316.58
80.0013.55
92.8317.27
HyperSol
91.8317.86
105.8333.20
96.1717.97
103.8332.79
Plasma lactate (mMol/L)
HypoSol
0.960.38
1.070.73
1.150.36
1.150.30
IsoSol
1.140.43
1.050.41
1.060.40
1.190.49
HyperSol
1.00.43
1.080.36
1.090.52
1.060.41
(P< 0.05) by the Tukey test. Plasma glucose values (Table 2) did not show
differences between treatments and treatments
over time (P> 0.05). Similar results were
reported by Gomes et al. (2014), who analyzed
solutions containing glucose precursors and did
not observe variation in glycemia of treated
animals. Ribeiro Filho et al. (2014), using 15g/L
of dextrose in the elaboration of enteral
electrolytic solutions for horses, reported an
increase in glycemia during the fluid therapy
period. In cattle, Ribeiro Filho et al. (2011)
reported that when using an enteral electrolyte
solution containing 5g/L dextrose, the animals
did not show any difference in glycemia during
the treatment period. However, these authors
reported that the other solution used, containing a
precursor of glucose propylene glycol in the
volume of 15mL/L, promoted an increase of
plasma glucose during treatment and that this
persisted until 24 hours after the end of the fluid
therapy. In the HyperSol, a slight increase in glycemia
was observed during the experimental phase,
which persists until time T24h (Table 2). The
animals received electrolytic solution containing
15g/L of dextrose and 3g/L of sodium acetate,
which when oxidized, metabolizes into glucose
and carbon dioxide (Leal et al., 2007), which
caused this slight increase in glycemic levels. Plasma glucose concentrations did not show
changes between treatments and over time in
treatments (P> 0.05). In addition, their values
remained within the reference range for the
studied age (Table 2). The amount of glucose used in the electrolyte
solutions observed in the present assay was not
enough to promote a significant increase in the
glycemic rate in the animals. RESULTS AND DISCUSSION Frequency of fecal consistency in healthy neonatal calves treated with hypotonic (HypoSol
isotonic (IsoSol) and hypertonic (HyperSol) enteral electrolyte solutions administered by continuou g
Fecal Consistency
Time (h)
Dry
Well-formed pasty
Pasty
Diarrheal
Total
HypoSol
0h
0+ (0)++
83.33 (5)
16.67 (1)
0 (0)
25 (6)
6h
0 (0)
83.33 (5)
16.67 (1)
0 (0)
25 (6)
12h
0 (0)
33.33 (2)
66.67(4)
0 (0)
25 (6)
24h
0 (0)
83.33 (5)
16.67 (1)
0 (0)
25 (6)
Total
0 (0)
70.83 (17)
29.17 (7)
0 (0)
100 (24)
IsoSol
0h
0 (0)
83.33 (5)
16.67 (1)
0 (0)
25 (6)
6h
0 (0)
83.33 (5)
16.67 (1)
0 (0)
25 (6)
12h
0 (0)
66.67(4)
16.67 (1)
16.67 (1)
25 (6)
24h
0 (0)
83.33 (5)
0 (0)
16.67 (1)
25 (6)
Total
0 (0)
79.17 (19)
12.5 (3)
8.3 (2)
100 (24)
HyperSol
0h
0 (0)
83.33 (5)
16.67 (1)
0 (0)
25 (6)
6h
0 (0)
50 (3)
33.33 (2)
16.67 (1)
25 (6)
12h
0 (0)
16.67 (1)
50 (3)
33.33 (2)
25 (6)
24h
0 (0)
66.67(4)
33.33(2)
0 (0)
25 (6)
Total
0 (0)
54.17 (13)
33.33 (8)
12.5 (3)
100 (24)
+Frequency of fecal consistency (%); ++ Number of animals. 20 Maintenance enteral… body weight, but without difference (P> 0.05),
which can be attributed to the administration of
enteral
electrolyte
solutions. The
animals
received on average 8.2 liters of solution, about
15% of their body weight in 12 hours, which
may have generated this increase (Table 2). Similar results were reported in cattle by Ermita
et al (2018) and in goats by Atoji et al. (2012). In turn, diarrhea that appears during the use of
hypertonic
enteral
electrolyte
solutions
(HyperSol) was caused by the fact that the
electrolyte solution was not completely absorbed,
and consequently, the volume remaining in the
intestinal lumen caused softening of the stool. The animals of all treatments presented a slight
increase in the abdominal circumference and Table 2. Mean values and standard deviations of abdominal circumference (cm), weight (kg), plasma
glucose (mg/dL) and plasma lactate (mMol/L) in healthy neonatal calves treated with hypotonic enteric
electrolyte solutions (HypoSol), isotonic (IsoSol) and hypertonic (HyperSol) administered by continuous Table 2. Arq. Bras. Med. Vet. Zootec., v.72, n.1, p.18-24, 2020 Lima et al. As expressed in Table 2, no difference was
detected in mean plasma lactate values between
treatments and over time in treatments (P> 0.05). The sources of energy commonly used in enteral
electrolyte solutions, when too much, can lead to
the risk of allowing unabsorbed glucose to carry
over into the large intestine, where glucose may
be fermented to short-chain volatile fatty acids
and exacerbate fecal water loss as quoted by
Nouri and Constable (2006). The values verified
in the present study demonstrate that even the
amount
used
in
the
hypertonic
solution
(HyperSol), 15g/L dextrose, was not enough to
increase the lactate concentration in the blood
and to determine the appearance of adverse
effects. According to Nouri and Constable
(2006) the upper limit of glucose in an oral
electrolyte solution (OES) for a calf of 45kg-
body weight may be 1.0-3.6g/kg-body weight. possibility that the increase of carbohydrate in
the electrolytic solution causes an increase in
blood lactate concentration. As there was no
increase in its values (Table 2), this procedure
can be performed, though the possibility that the
increase of the energy source in the electrolytic
solution predisposes to the appearance of
acidosis should not be excluded. No changes were observed in mean blood pH
values between treatments (P> 0.05). In the
HyperSol treatment, at time T24h, there was a
variation (P< 0.05) of the mean values in relation
to the time T12h (Table 3). This increase in pH
occurred, possibly, due to the end of fluid
therapy and the return of feed. The same
behavior was observed in the solutions HypoSol
and IsoSol, although not significant, the return of
the mean values to basal concentrations (T0h)
occurred. Despite
the
difference,
values
remained within the reference range for neonatal
calves, from 7.32 to 7.40 (Lisbôa et al., 2002). It was suggested to increase the amount of
dextrose to provide a more expressive effect on
the glycemic rate of animals. However, there is a Table 3. RESULTS AND DISCUSSION This result indicates
that if there is a need to correct cases of
hypoglycemia, the amount of energy source
should be higher. 21 21 Arq. Bras. Med. Vet. Zootec., v.72, n.1, p.18-24, 2020 Arq. Bras. Med. Vet. Zootec., v.72, n.1, p.18-24, 2020 REFERENCES ATOJI,
K.H.;
RIBEIRO
FILHO,
J.D.;
MALAFAIA, P. Enteral fluid therapy through
nasogastric tube in rumen cannulated goats. Pesqui. Vet. Bras., v.32, p.1281-1284, 2012. BREGADIOLI,
G.C.;
PEREIRA,
P.F.V.;
FLAIBAN, K.K.M.C. et al. Enteral fluid therapy
in neonatal calves and features of commercially
available electrolyte solutions in Brazil. Ciênc. Rural, v.47, p.1-8, 2017. The reduction of BE during the fluid therapy
phase in IsoSol (T12h) and HyperSol (T6h)
treatments can be attributed to the composition
of referred electrolytic solutions (Table 3). Two
substances possibly influenced this result:
chloride and dextrose. Chloride participation was
discussed previously (bicarbonate concentration). The other possibility is related to the amount of
dextrose in the electrolytic solutions used,
because according to Zhang et al. (2003), the
carbohydrates are metabolized by bacteria in the
gastrointestinal tract, producing organic acids,
having an elevation of these in the plasma. Lactate isomers (L-lactate and D-lactate) are
some of the organic acids produced by bacterial
metabolism (Fall and Szerlip, 2005). D-lactate is
metabolized slowly in most mammals, causing a
serum accumulation of this organic acid,
reducing blood pH, bicarbonate
and BE
(Ewaschuk et al., 2005). CONSTABLE,
P.;
GRÜNBERG,
W.;
CARSTENSEN, L. Comparative effects of two
oral solutions on milk clotting, abomasal luminal
pH, and abomasal emptying rate in sucking
calves. J. Dairy Sci., v.92, p.296-312, 2009. ERMITA, P.A.N.; RIBEIRO FILHO, J.D.;
VIANA, R.B. et al. Enteral fluid therapy
administered in continuous flow by naso-ruminal
route
using
three
maintenance
electrolyte
solutions: effects on physiological biomarkers
and the hemogram of bovines. Ciênc. Rural,
v.48, p.1-7, 2018. ERMITA, P.A.N.; VIANA, R.B.; RIBEIRO
FILHO, J.D. et al. Effects of enteral fluid therapy
in continuous flow administered by nasogastric
tube in buffalo Calves. J. Buff. Sci., v.5, p.60-69,
2016. The increase in the values of BE occurred in the
three treatments at T24h, returning to values
similar to T0h, possibly due to the end of fluid
therapy and the return of feed, as occurred with
the pH (Table 3). It should be noted that the
amount of sodium acetate contained in the
treatment solutions, mainly HypoSol and IsoSol,
were not enough to cause an increase in the BE,
signaling that in calves with metabolic acidosis it
should be higher than that used in the present
test. EWASCHUCK,
J.B.;
NAYLON,
J.M.;
GORDON, A.Z. D-lactate in human and
ruminant metabolism. Critical review. J. Nut.,
v.135, p.1619-1625, 2005. FALL, P.J.; SZERLIP, H.M. Lactic acidosis:
from sour milk o septic shock. CONCLUSIONS means were within the normal range (48 to
60mmHg) reported by Lisbôa et al. (2002) in
healthy bovine calves and buffalo calves by Silva
et al. (2010). The HypoSol did not generate the onset of
diarrhea, while the IsoSol and HyperSol did. Regardless of dose used, acetate did not cause
metabolic alkalosis in the animals in the present
study. The results suggest that the use of the
HypoSol in diarrheal calves, dehydrated and
without metabolic acidosis may be clinically
important. Mean values of BE had no difference between
treatments (P> 0.05). In the evaluation over time,
a difference (P< 0.05) was detected in the groups
of electrolytic solutions IsoSol and HyperSol
(Table 3). In the IsoSol solution a reduction in
BE at the final moment of the fluid therapy
(T12h) was observed when compared to T0h
(before the period of fluid therapy) and T24h (12
hours after the end of the fluid therapy). In turn,
the animals hydrated with the HyperSol solution
presented a significant decrease in T6h in
relation to T24h. Arq. Bras. Med. Vet. Zootec., v.72, n.1, p.18-24, 2020 Lima et al. Mean values and standard deviations of blood pH, pCO2 (mmHg), cHCO-
3v (mMol/L) and cBasev
(mMol/L) in healthy neonatal calves treated with hypotonic (HypoSol), isotonic (IsoSol) and hypertonic
(HyperSol) enteral electrolyte solutions administered by continuous nasogastric tube for 12 hours
Treatment
T0h
T6h
T12h
T24h
Ph
HypoSol
7.380.019Aa
7.370.015Aa
7.350.031Aa
7.390.021Aa
IsoSol
7.350.046Aa
7.360.037Aa
7.320.046Aa
7.370.042Aa
HyperSol
7.370.016Aab
7.370.015Aab
7.360.018Ab
7.400.036Aa
pCO2 (mmHg)
HypoSol
48.332.63
48.832.40
50.502.17
49.201.92
IsoSol
52.334.46
48.504.37
50.331.97
50.673.78
HyperSol
51.04.15
50.332.34
52.672.34
50.02.61
cHCO-
3v (mMol/L)
HypoSol
27.721.18
26.731.25
26.602.88
28.702.25
IsoSol
30.683.29
28.303.13
28.853.45
30.482.94
HyperSol
27.903.53
25.603.08
24.932.91
27.482.51
cBasev (mMol/L)
HypoSol
3.221.06Aa
2.251.07Aa
1.622.87Aa
4.202.3Aa
IsoSol
4.31.47Aa
2.11.38Aab
1.041.34Ab
4.161.4Aa
HyperSol
3.711.81Aab
3.031.46Ab
3.810.5Aab
5.632.2Aa
Mean values followed by different capital letters in the same column or by different lowercase letters in the same row
differ from each other (P< 0.05) by the Tukey test. Table 3. Mean values and standard deviations of blood pH, pCO2 (mmHg), cHCO-
3v (mMol/L) and cBasev
(mMol/L) in healthy neonatal calves treated with hypotonic (HypoSol), isotonic (IsoSol) and hypertonic
(HyperSol) enteral electrolyte solutions administered by continuous nasogastric tube for 12 hours Table 3. Mean values and standard deviations of blood pH, pCO2 (mmHg), cHCO-
3v (mMol/L) and cBas
(mMol/L) in healthy neonatal calves treated with hypotonic (HypoSol), isotonic (IsoSol) and hyperton
(HyperSol) enteral electrolyte solutions administered by continuous nasogastric tube for 12 hours In the mean values of the partial pressure of
carbon dioxide, there were no differences (P>
0.05) between treatments and in the treatments
over time. Although not significant, a decrease of
the means during the fluid therapy period in the
IsoSol and HyperSol solutions can be observed
(Table 3). As with bicarbonate (Table 3) and base excess (BE), the slight reduction in pCO2
values was possibly due to the higher amount of
sugar and chloride in these solutions, which
would lead to an acidification that was
compensated by the respiratory buffer system,
causing a decrease of pCO2 values. However,
this variation had no clinical significance, as the 22 Arq. Bras. Med. Vet. Zootec., v.72, n.1, p.18-24, 2020 Maintenance enteral… Arq. Bras. Med. Vet. Zootec., v.72, n.1, p.18-24, 2020 REFERENCES Analytic reviews. J. Intensiv. Care Med., v.20, p.254-271, 2005. 23 Arq. Bras. Med. Vet. Zootec., v.72, n.1, p.18-24, 2020 Lima et al. RIBEIRO FILHO, J.D.; DANTAS, W.M.F.;
LIMA, A.P. et al. Enteral electrolyte solutions
with different osmolarities administered in a
continuous flow in newborn calves. Ciênc. Rural, v.47, 6p., 2017. GOMES, C.L.N.; RIBEIRO FILHO, J.D.;
FALEIROS, R.F. et al. Blood gas analysis, anion
gap, and strong ion difference in horses treated
with polyethylene glycol balanced solution (PEG
3350) or enteral and parenteral electrolyte
solutions. Ciênc. Rural, v.44, p.1086-1092, 2014. RIBEIRO FILHO, J.D.; GIMENES, A.M.;
FONSECA, E.F. et al. Enteral fluid therapy in
cattle: evaluation of isotonic electrolyte solutions
administered via nasogastric tube on continuous
flow. Ciênc. Rural, v.41, p.285-290, 2011. LEAL, M.L.R.; MARUTA, C.A.; ORTOLANI,
E.L. Uso de bicarbonato e lactato-L para
correção da acidose metabólica sistêmica em
bovinos com acidose láctica ruminal aguda. Arq. Bras. Med. Vet. Zootec., v.59, p.971-976, 2007. RIBEIRO FILHO, J.D.; PESSIN, A.E.; ATOJI,
K. et al. Enteral fluid therapy: biochemical
profile of horses treated with hypotonic enteral
electrolyte solutions associated with energy
sources. J. Equine Vet. Sci., v.34, p.759-764,
2014. LISBÔA, J.A.N.; BENESI, F.J.; LEAL, M.L.R.;
TEIXEIRA, C.M.C. Efeito da idade sobre o
equilíbrio ácido-básico de bezerras sadias no
primeiro mês de vida. Braz. J. Vet. Res. Anim. Sci., v.39, p.136-142, 2002. SILVA, D.G.; SILVA, P.R.L.; FAGLIARI, J.J. Hemograma e perfil bioquímico, inclusive
hemogasométrico,
de
bezerros
infectados
experimentalmente com Salmonella Dublin. Arq. Bras. Med. Vet. Zootec., v.62, p.251-257, 2010. McCLURE, J.T. Oral fluid therapy for treatment
of neonatal diarrhoea in calves. Vet. J., v.162,
p.87-89, 2001. NAYLOR,
J.M.;
RODRIGUEZ,
M.I.;
SKILNICK, P. A comparison of three oral
solutions in the treatment of diarrheic calves. Can. Vet. J., v.31, p.753-760, 1990. SISTEMA para análises estatísticas - SAEG. Versão 9.1. Viçosa: UFV. Fundação Arthur
Bernardes, 2007. 301p. NOURI, M.; CONSTABLE, P.D. Comparison of
two oral electrolyte solutions and route of
administration on the abomasal emptying rate of
Holstein‐Friesian calves. J. Vet. Inter. Med.,
v.20, p.620-626, 2006. ZHANG, D.L.; JIANG, Z.W.; JIANG, J. et al. D-lactic acidosis secondary to short bowel
syndrome. Postgrad. Med. J., v.79, p.110-112,
2003. ORAL rehydration salts: production of the new ORAL rehydration salts: production of the new
ORS. Geneva:
Bulletin
of
Word
Health
Organization and UNICEF, 2006. 89p. ORS. Geneva:
Bulletin
of
Word
Health
Organization and UNICEF, 2006. 89p. 24
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Development of Strategies for SNP Detection in RNA-Seq Data: Application to Lymphoblastoid Cell Lines and Evaluation Using 1000 Genomes Data
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Supplementary Figure 1: Number of SNPs per method in RNA-seq data. This figure displays the number of SNPs called
for each of the 3 samples using 4 different methods. The proportion of heterozygous (grey) and homozygous (black) SNP calls
is also displayed. Details of the numbers of SNPs called are listed in supplementary table S2a-c. Supplementary Figure 1: Number of SNPs per method in RNA-seq data. This figure displays the number of SNPs called
for each of the 3 samples using 4 different methods. The proportion of heterozygous (grey) and homozygous (black) SNP calls
is also displayed. Details of the numbers of SNPs called are listed in supplementary table S2a-c.
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Access to artemisinin-based combination therapy (ACT) and quinine in malaria holoendemic regions of western Kenya
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Malaria journal
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RESEARCH Open Access Watsierah and Ouma Malaria Journal 2014, 13:290
http://www.malariajournal.com/content/13/1/290 © 2014 Watsierah and Ouma; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the
Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public
Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this
article, unless otherwise stated. Access to artemisinin-based combination therapy
(ACT) and quinine in malaria holoendemic
regions of western Kenya Carren A Watsierah1 and Collins Ouma2* Carren A Watsierah1 and Collins Ouma2* Abstract Background: Artemisinin-based combination therapy (ACT) has been adopted as the most effective treatment
against malaria in many endemic countries like Kenya while quinine has remained the second line. The objective of
the current study was to assess access to Kenya’s policy recommended anti-malarials, ACT and quinine in the public,
private and not-for-profit drug outlets in western Kenya. Methods: A cross-sectional survey using purposive sampling of 288 outlets (126 public, 96 private, 66 not-for-profit)
was conducted in western Kenya in two regions with varying Plasmodium falciparum endemicities. Information on
access (availability, price, affordability) on ACT and quinine was collected using the WHO and Healthcare Associated
Infection (HAI) standardized methodologies for availability, prices and affordability of drugs. From a Ministry of
Health database, the following were included in the analyses: one (1) main public hospital, followed by random
selection of five hospitals under this main facility. Eight other public outlets under each of the hospitals were
selected, to a total of 96. Matching number of private outlets (n = 96), all (66) not-for-profit outlets and additional 30
public health facilities were sampled to get the required sample size of 288. Results: More public 111 (88.1%) and not-for-profit 27 (40.9%) outlets stocked subsidized ACT (artemether-lumefantrine,
AL). Other artemisinin-based combinations were widely available for both children 93 (96.9%) and adults 82
(85.0%) in private outlets. Frequent stock-outs were in public in 106 (84%), reporting three times or more stock-outs
in three months. Subsidized ACT (AL) was sold at median price of USD 0.94 and 0.75 in private and not-for-profit outlets
respectively. The costs was higher than recommended price of USD 0.5 and requiring up to 0.20-0.25 days of disposable
income for households in lowest economic status. Conclusion: There is low availability of subsidized ACT (AL) and higher frequency of stock-outs in government
facilities, while private sector sells AL at higher prices, thus making it less affordable to many households. These
factors determine the adherence to the dosing schedules during the treatment course and thus the evaluation of
the subsidy policy, its implementation and role in malaria burden in this region is compulsory. * Correspondence: collinouma@yahoo.com
2Department of Biomedical Sciences and Technology, Maseno University,
Private Bag, Maseno, Kenya
Full list of author information is available at the end of the article Background malarial drugs, such as chloroquine and sulphadoxine-
pyrimethamine (SP) [2]. Kenya adopted the new malaria
policy in 2004, which recommends the use of arte-
mether 20 mg-lumefantrine 120 mg (AL) as the first-
line drug for treatment of uncomplicated malaria [3],
while quinine is preferred for complicated and severe
malaria. Malaria still poses a great burden in many parts of
Africa despite the availability of many interventions
focused on preventive and therapeutic approaches [1]. Artemisinin-based combination therapy (ACT) has been
adopted as the most effective treatment option against
malaria in many countries following the widespread
malarial parasite resistance to more affordable anti- The World Health Organization (WHO) however,
stresses the fact that many strategies to increase access
to effective anti-malarial drugs have been put in place,
but in the year 2009, less than 15% of children under-
five years of age received ACT when they presented with * Correspondence: collinouma@yahoo.com
2Department of Biomedical Sciences and Technology, Maseno University,
Private Bag, Maseno, Kenya
Full list of author information is available at the end of the article Page 2 of 7 Watsierah and Ouma Malaria Journal 2014, 13:290
http://www.malariajournal.com/content/13/1/290 Watsierah and Ouma Malaria Journal 2014, 13:290
http://www.malariajournal.com/content/13/1/290 fever in 11 out of 13 African countries [4]. In addition,
most mortality in children who are able to attend a
health facility with adequate staff and supplies occurs
within 24 hours after admission [5]. Consequently, it is
evident that most mortality occurring in such set-ups
are as a result of delay in home management with effect-
ive drugs, thus underscoring the importance of early
treatment for preventing such mortalities [5]. It is, there-
fore, critical to improve access to recommended drugs. One way of approaching this problem is to ensure access
to treatment using effective anti-malarials like ACT and
quinine which could be given promptly even at primary
health care facilities as highlighted by WHO, to ensure
access and minimize parasite resistance [5]. subsidized prices at any given time, especially in malaria
endemic areas, with high levels (≥40%) of malaria preva-
lence throughout the year remains unknown [11]. Fre-
quent stock-outs have been reported in a few studies as
leading to the use of several inappropriate anti-malarials
in the households [15-18]. Study design and study area A cross-sectional study was conducted between February
and May 2012 in Nyanza Province in western Kenya. Two regions, endemic for Plasmodium falciparum trans-
mission, but with different levels of risk for malaria in
the province, were targeted: the lowlands of the Province
(Kisumu, Siaya and Bondo regions) around Lake Victoria
with a holoendemic and stable P. falciparum transmis-
sion (altitude 0–1,300 metres) and Kisii highlands (Kisii,
Gucha and Nyamira regions), with an epidemic transmis-
sion (>1,300-1,750 metres) [21]. To enhance ACT policy implementation and further
proper management of malaria, Kenya government has
scaled up case management strategies as follows: 1) all
public health institutions offer ACT free-of-charge;
2) introducing cheaper artemether-lumefantrine (AL)
to be sold at Kenyan shillings 40 (USD 0.5) in retail drug
outlets [11]; and 3) children who are below 13 years of age
are treated for free in public health facilities. Despite the
efforts to improve access, ACT remains registered as
prescription-only drug recommended for formal sector
providers [12] and, in 2010, the treatment policy was uni-
versally changed to include the use of ACT for only
laboratory-confirmed malaria cases [13,14]. The two regu-
lations hence limit access of households to these drugs
further causing delay in treatment for more than 24 hours. However, access to ACT at the point-of-care and at the Malaria in the region accounts for 40% of out-patient
visits and 40% of hospital in-patient admissions, with be-
tween 10–15 paediatric cases of severe malaria often com-
plicated with anaemia and malnutrition, on a daily basis
[22]. Malaria transmission occurs all year round, peaking
in the rainy season months of April and May. The high-
lands areas suffer epidemic levels, where temperature in-
creases and rainfall variation impacts on vector breeding
and malaria transmission with some areas experiencing a
prevalence of up to 20% [23]. Background Availability of a particular drug
in the market is an important factor in determining which
anti-malarials are bought for use [19] of which, in addition
to high cost of anti-malarial, are important contributing
factors to lack of access [20]. Furthermore, two years fol-
lowing the Kenyan government rolling-out the subsidy
policy on ACT, namely artemether 20 mg-lumefantrine
120 mg (AL) as the first-line treatment, and quinine still
being the preferred treatment of severe malaria, no survey
has been carried out to date to evaluate availability of anti-
malarials in malaria-prone regions. Most of the studies
reporting ACT availability before or immediately after the
roll-out were based on country-wide surveys and results
were not attributed to fit the differences in regional preva-
lence of malaria. Even with the efforts to improve the case management
of malaria, the success of policy implementation and ef-
fectiveness is measured by the availability of the recom-
mended drugs at the point of care [6]. Many patients in
Africa use private sector as their primary source of med-
icines, with 50% of febrile cases reported to be treated in
this sector [7], despite the fact that the manufacturer’s
selling prices and final patient prices range from 56% to
358%, making treatment unaffordable [8]. On average,
households in many malaria endemic countries spend
up to 90% of their household expenditure on medicines,
portraying high anti-malarial pricing as an important
contributing factor to the lack of access to ACT [9]. Fur-
thermore, a recent study [10] demonstrated that there
were huge increases in quality-assured ACT (QAACT)
availability (25.8—51.9% points), and market share (15.9—
40.3% points), driven mainly by changes in the private
for-profit sector. The same study observed that large
falls in median price for QAACTs per adult equivalent
dose was seen in the private for-profit sector in six pi-
lots, ranging from US$1.28 to $4.82. The recent findings
provide additional evidence that anti-malarial pricing is
a critical factor in access to ACT in sub-Saharan African
countries. It is on this background that the current study assessed
access to Kenya’s policy recommended anti-malarials,
ACT and quinine in the public, private and not-for-profit
drug outlets using the WHO and Healthcare Associated
Infection (HAI) standardized methodologies for availabil-
ity, prices and affordability of drugs. Selection of outlets and households Three-stage sampling approach was used to select drug
outlets. For each region, a list of public medical facilities
was compiled using a database obtained from the Kenyan Page 3 of 7 Watsierah and Ouma Malaria Journal 2014, 13:290
http://www.malariajournal.com/content/13/1/290 Watsierah and Ouma Malaria Journal 2014, 13:290
http://www.malariajournal.com/content/13/1/290 Ministry of Health [24]. First, from each of the two se-
lected study areas, the main public hospital was selected,
and then five hospitals under the main facility were ran-
domly sampled. Lastly, eight other public outlets under
each of the hospitals were selected, to a total of 96. Match-
ing number of private outlets was randomly selected. All
(66) not-for-profit outlets and additional 30 public health
facilities were sampled to get the required sample size of
288. For every outlet sampled, one household served by
the outlet was sampled. access (price, availability and affordability) was established. For the availability analysis, different dose strengths of
the same drug were combined to calculate the overall
availability of that particular anti-malarial on the day of
the survey. The median prices of anti-malarials were calculated
for each anti-malarial category identified. For compari-
son, the median price ratios (MPR) of the ACT median
price to the current government recommended price for
a single treatment course with AL was computed in pri-
vate and not-for-profit outlets at USD 0.5 (KES 40). For
quinine, the MPR of private to not-for-profit outlets was
calculated, since there was no government-subsidized
price at the moment. Availability of packs per weight group of subsidized ACT,
other forms of ACT and quinine tablet formulation Availability of packs per weight group of subsidized ACT,
other forms of ACT and quinine tablet formulation Data collection Data was collected through drug auditing and interviews
by enumerators who visited the outlets as in pairs. Focus
was on tablet anti-malarials only – typically, since this is
the most common formulation. Prior to data collection,
enumerators who had nursing qualification were trained
on the use of the tools prior to pilot testing in nine of
each outlet type with regular supervision. Drug outlets
were recruited for the survey. In the outlets, providers
who consented to participate were asked two screening
questions to determine whether (1) the outlet had stocked
ACT or quinine, within the previous three months, and
(2) if ACT or quinine were available on the day of the sur-
vey. All providers answering “yes” to at least one of these
questions gave information about the ACT and quinine
after informed consent was obtained from them. In outlets
with more than one attendant, the provider who was
serving the clients at the time of the survey provided
the information. Affordability was expressed as the number of days
households with lowest daily disposable income level at
KES 150,USD 1.875 [16] would need to work in order to
pay for the full adult dose of treatment course with AL,
other artemisinin-based combinations and quinine avail-
able in the outlet. Chi-square analyses were used for
proportionality. Statistical significance was assessed at a
p ≤0.05. Availability of anti-malarials All outlets stocked more than one category of anti-
malarial. More public 111 (88.1%) and not-for-profit out-
lets 27 (40.9%) surveyed stocked AL, the recommended
first-line treatment for uncomplicated malaria. However,
less than a third of the private outlets 27 (28.1%) had
stocked AL (Table 1). Other forms of ACT were stocked
mostly by private outlets 87 (90.6%) and less by not-for
profit 8 (12%), while public outlets did not stock any
other ACT. All outlets stocked both first-line subsidized
ACT (AL) and second-line (quinine) anti-malarials in
varied proportions (Table 1). The information collected in the drug outlets included
brand name, amount in stock and the frequency of stock
out in the last three months, retail price, and dosage
form as well as information on packs per weight group. The price of anti-malarials was originally collected in
Kenya Shillings (KES), and then later converted to US
dollars (USD) for international standardization in costs. Data management and statistical analysis The subsidized ACT was available in all the four weight-
specific presentations in less than half of the total outlets
88 (30.6%), while quinine was available in 137 (47.6%) Data collected was checked in the field and at the end of
each day cleaned to ensure completeness, consistency,
credibility and eligibility. Information captured in audit
sheets and interview guide on availability, affordability
and prices of anti-malarials as well as their use in house-
holds was coded and entered into Statistical Package for
Social Sciences (SPSS) version 19. Analysis adopted the
WHO and Healthcare Associated Infection (HAI) stan-
dardized methodologies for availability, prices and af-
fordability of drugs. Data from the outlets were grouped
into three categories and analyzed separately into: 1)
public outlets, 2) private outlets and 3) not-for-profit
outlets. Furthermore, anti-malarials were stratified as the
government subsidized ACT (AL), other formulations of
ACT and quinine. Using the mean and median, drug Table 1 Availability of subsidized ACT (AL), other forms
of ACT and quinine in the three outlet types
Outlet type
Public
n = 126 (%)
Private
n = 96 (%)
Not-for-profit
n = 66 (%)
Availability of anti-malarial
drugs
Subsidized ACT (AL)
111 (88.1)
27 (28.1)
27 (40.9)
Other ACT
0 (0)
87 (90.6)
8 (12.0)
Quinine
59 (46.8)
68 (70.8)
36 (54.5)
ACT, artemisinin-based combination therapy, AL, artemether-lumefantrine. Table 1 Availability of subsidized ACT (AL), other form
of ACT and quinine in the three outlet types Table 1 Availability of subsidized ACT (AL), other forms Watsierah and Ouma Malaria Journal 2014, 13:290
http://www.malariajournal.com/content/13/1/290 Page 4 of 7 outlets. Other forms of ACT were widely available for
both children 93 (96.9%) and adults 82 (85.0%) in private
outlets. The availability varied with the outlet type as
shown in Table 2. Availability of packs for different weight
groups was also evaluated for AL in the three outlet types. The six tablet pack was in stock in more public outlets 47
(37.3%) than in private 21 (21.9%) and not-for profit 16
(24.2%) outlets (Table 2). The same trend was observed
for the twelve AL tablet packs, however, the eighteen pack
was in stock in public 66 (52.4%) and not-for-profit 33
(50.0) than in private 12 (12.5%) outlets. Apparently, a
higher proportion of quinine tablet formulation was
available in private 46 (47.9%) than in public 44 (34.9%)
and not-for-profit 28 (42.4%) outlets (Table 2). median prices were computed. Data management and statistical analysis The overall median prices
for AL, other forms of ACT and quinine were USD 0.94
(R = 0.63-1.25), USD 5.63 (R = 1.88-8.13) and USD 1.25
(R = 0.75-1.25), respectively. The median price for all
anti-malarials categories was much higher in private
outlets than in not-for-profit outlets, as shown in Table 3. Other forms of ACT were more highly priced than the
subsidized ACT and quinine. The median price ratio (MPR) for other forms of ACT
was computed using the government subsidized price of
ACT of USD 0.5. The private outlets had higher MPR
for other forms of ACT compared to their not-for-profit
counterparts (Table 3). Although AL was available in all
the outlet types, the government recommended price of
USD 0.5 did not apply and AL was sold at median price
of USD 0.94 (R = 0.63-1.25) and USD 0.75 (R = 0.63-1.0)
in private and not-for-profit outlets, respectively. Other
forms of ACT were on average 12 times higher in price
than AL in private outlets and five times higher in not-
for-profit outlets (Table 3). Discussion The current study was designed to assess access to Kenya’s
policy recommended anti-malarials, ACT and quinine in
the public, private and not-for-profit drug outlets using
the WHO and Healthcare Associated Infection (HAI)
standardized methodologies for availability, prices and
affordability of drugs. The findings of the current survey
revealed that the government-recommended anti-malarials
were available in all outlet types with public outlets Affordability of anti-malarials All the anti-malarials were more affordable in not-for-
profit outlets than private outlets. The quinine pur-
chased within not-for-profit outlets was most affordable
to household with lowest disposable income levels
(Table 4). Other forms of ACT were least affordable and
would cost up to 3.20 days of disposable income in pri-
vate outlets. The decision for restocking a particular anti-malarial
was mainly mediated by the recommendation by the
government in 158 (54.7%) outlets followed by consumer
demand in 73 (25.5%) outlets. The government recom-
mendation guided decision for restocking in both public
in 125 (99.5%) and not-for-profit in 51 (76.7%) outlets
while private outlets restocked depending on most prof-
itable in 57 (59.2%) and consumer demand in 25 (26.3%)
among other reasons. Stock-out of anti-malarials Stock-out in the past three months was defined as ab-
sence of drugs from stock for at least seven consecutive
days [25]. The stock-out rates in public outlets was most
frequent in the last three months with 106 (84.0%) out-
lets reporting three times or more stock-outs. On the
other hand, private sector suffered stock-outs once in
three months in 70 (73.0%) outlets while in not-for-
profit outlets, there was no stock-out in 57 (87.0%) in
the past three months. The price of anti-malarials Anti-malarials in the public outlets were provided for free
and were not included in the price analysis. Prices of anti-
malarials with the same generic names were recorded and Table 2 Availability of packs per weight group of subsidized ACT (AL)
Outlet type
Public n = 126 (%)
Private n = 96 (%)
Not-for-profit n = 66 (%)
All the four AL weight specific packs
53 (42.1)
23 (23.9)
12 (18.2)
AL 6 tablet pack for 5-14 kg
47 (37.3)
21 (21.9)
16 (24.2)
AL 12 tablet pack for15-24 kg
62 (49.2)
25 (26.0)
12 (18.1)
AL 18 tablet pack for 25-34 kg
66 (52.4)
12 (12.5)
33 (50.0)
AL 24 tablet pack for ≥35 kg
111 (88.1)
42 (43.8)
46 (69.7)
Other forms of ACT for children
0
93 (96.9)
8 (12.1)
Other forms of ACT for adults
0
82 (85.0)
9 (13.6)
Quinine (tablet formulation only)
44 (34.9)
46 (47.9)
28 (42.4)
ACT, Artemisinin-based Combination Therapy, AL, Artemether-Lumefantrine. Table 2 Availability of packs per weight group of subsidized ACT (AL) Watsierah and Ouma Malaria Journal 2014, 13:290
http://www.malariajournal.com/content/13/1/290 Page 5 of 7 Table 3 Median prices and Median Price Ratios of anti-malarials being sold in private and not-for-profit outlets
Outlet type
Anti-malarials
Private
Not-for-profit
Median price (range) USD
Median price ratio
Median price (range) USD
Median price ratio
AL*
0.94 (0.63-1.25)
1.88
0.75 (0.63-1.00)
1.5
Other forms of ACT**
6.0 (1.88-10.63)
12
2.5 (1.88-3.13)
5
Quinine*
1.0 (0.75-1.25)
1.33
0.75 (0.63-1.13)
1
USD, United States Dollars; ACT, artemisinin-based combination therapy; AL, artemether-lumefantrine. *Median price ratio was calculated using median price of quinine in not-for-profit as reference since there is no government subsidized price. **Median price ratio was calculated as anti-malarial median price to the government recommended price of subsidized ACT (AL) (KES 40, USD 0.5). D, United States Dollars; ACT, artemisinin-based combination therapy; AL, artemether-lumefantrine. edian price ratio was calculated using median price of quinine in not-for-profit as reference since there is no government subsidized p
Median price ratio was calculated as anti-malarial median price to the government recommended price of subsidized ACT (AL) (KES 40, study carried out in Uganda showed that patients’ adher-
ence was high when they received AL blister packs with
a weight-specific number of tablets and including pictor-
ial instructions on how to use it [28]. The price of anti-malarials This observation
was also recorded in a study in the coastal region of
Kenya, where simplicity of drug regimen was associated
with increased treatment adherence [29]. providing only the two policy recommended anti-
malarials; subsidized ACT (AL) and quinine. The pri-
vate outlets on the other hand had stocked various
other Artemisinin-based combinations in addition to
the policy recommended anti-malarials. Stocking other
forms of ACT in private outlets could be due to the fact
that selling subsidized ACT would be unprofitable. Other studies in the region similar to the current study
site (in terms of malaria endemicity) showed that pri-
vate sectors prefer stocking drugs which can retail at
competitive prices [26]. This is a matter of concern
given that many households in the current study bought
drugs from private outlets, where anti-malarials were
sold at higher prices. The current study findings, consistent with previous
ones [20] observed that lack of drugs in the formal sector
contributes to people buying drugs from non-formal out-
lets. In such informal set-ups, the quality of drugs is less
controlled and information on dose is not often provided. Yet in another study, about a third of individuals who
sought care from public health facilities did not get drugs
from the hospital pharmacy because they were out of
stock [30]. The high stock-out rates questions the logic be-
hind WHO’s call to treat malaria cases within 24 hours. Further questions are also raised on the recommendation
to treat only laboratory-confirmed malaria cases, as well
as confining ACT to formal sector providers, since these
drugs are inaccessible especially in the rural areas, a part
poorly covered by these facilities. Unless the communities
are involved in the implementation of these guidelines, the
current trend in malaria treatment in this region might
start leaning towards the negative. Low availability of all the four weight-specific packs of
AL is of more concern, and worse still, the low availabil-
ity of the six-tablet Pack meant for the treatment of
under-fives, which is the most vulnerable group due to
malaria infections. Coupled with the higher frequency of
stock-outs recorded in this study, the calculation of
appropriate regimen may not be guaranteed when the
right package size is lacking, especially with low know-
ledge of providers on AL regimen in the private outlets
as observed in a previous study in this region [27]. Recommendations in a recent multi-country independent evaluation survey
in which the median prices ranged from USD 0.58 to USD
1.96 [31]. Conversely, the price of first-line treatment in
the current study is higher than the price of first-line treat-
ment in Burundi which was USD 0.16 in 2011 [26]. The study is in support of programmes that can support
the subsidy policy and should be recommends that the
programmes should be extended to include other anti-
malarials to make them affordable in all sector outlets. In addition, frequent stock-outs of the required anti-
malarials for different weight groups calls for more em-
phasis on the implementation of malaria treatment
policy. It is highly advised that a creation of private-
public partnership in the procurement and distribution
of recommended anti-malaria drugs is the goal to make
them available for the customers in all the outlets. The
factors that influence availability should be tackled accord-
ing to regional context. These should include monitoring
net sales/consumption rates through inventory, time
frame estimates and physical stock recording, especially
according to seasonality of illness and differences in dis-
trict health needs. Internal agreement to transfer anti-
malarials from one sector outlet to another and from
one region to another depending on the disease burden
and consumption rate is proposed. Higher prices than recommended in private outlets in-
dicate lack of regulations and price control. This lack of
control affects public health in the society, especially the
most vulnerable groups to malaria-related morbidity and
mortality due to poverty [32]. However, the price differ-
ences between the private and not-for-profit outlets for
both subsidized ACT and quinine need to be reconsid-
ered. Given the 3.2-fold price differences of other forms
of ACT in the private outlets as compared to the not-
for-profit outlets, there is need to lower these prices in
the private outlets to make them comparable to those in
other outlets. The reasons for reconciling and regulating
prices may range from the fact that many households
bought anti-malarials from private outlets due to fre-
quent stock-outs in the public outlets. Secondly, the
price is a vital factor in determining how drugs are used
in the household. Thirdly, the private outlets are more
accessible in terms of distance, as it has been previously
demonstrated in a study in this region [16]. The price of anti-malarials For
example, giving four packets of the six-tablet pack for
treatment of malaria in patients who need a 24-tablet
pack or splitting the blister pack into 12-tablet pack for
a child weighing 9 kg might result into uncertainty in
the use of the drug as per its recommended regimen. Simplified packaging of anti-malarials has been found to
improve the use of these drugs. For example, a previous The prices of subsidized ACT remained lower than
other anti-malarials in all the outlets. The lower median
price in not-for-profit outlets was as a result of this sector
being supplied with subsidized ACT by the government. In this sector, there was a full adherence to the subsidy
policy as much as the anti-malarials were not free as in
the public outlets. The not-for-profit outlets had add-
itional service charge, further raising the price of the
drugs. Lower prices of subsidized ACT are highly com-
mendable, although it is not affordable to people who are
of lowest economic levels. For instance, buying a full
course of subsidized ACT would cost USD 0.94 in private
and USD 0.75 in not-for-profit outlets, thus requiring up to
0.5 and 0.4 days of disposable household income, respect-
ively. These observations from private outlets are consistent
with previous data obtained from Kenya, Madagascar and
Tanzania while the observed prices in the current study are
lower than for Ghana, Zanzibar, Niger, Nigeria and Uganda Table 4 Number of days of disposable income to afford
anti-malarials
Outlet type
Private
Not-for-profit
Anti-malarials
AL
0.50
0.40
Other forms of ACT
3.20
1.34
Quinine
0.54
0.40
AL, artemether-lumefantrine; ACT, artemisinin-based combination therapy. Affordability was expressed as median price (numerator) against lowest daily
disposable income level (at KES 150, USD 1.875 = denominator). Table 4 Number of days of disposable income to afford
anti-malarials Watsierah and Ouma Malaria Journal 2014, 13:290
http://www.malariajournal.com/content/13/1/290 Watsierah and Ouma Malaria Journal 2014, 13:290
http://www.malariajournal.com/content/13/1/290 Page 6 of 7 Page 6 of 7 Recommendations Considering
the three reasons which show evidence of the role played
by private sector in providing treatment for patients in
western Kenya, it is important to effectively increase the
affordability of other artemisinin-based combinations in
these outlets. This strategy would further increase adher-
ence to treatment regimen since the affected population
would be able to afford the full course of anti-malarials. The end results would be among other reasons, prevent-
ing the irrational use of drugs, hence slowing down the
development of resistance by malarial parasite. The ob-
servations in the current study fully support the WHO
echoes that when health systems are not able to provide
ACT at little or no cost, consumers may have to pur-
chase them in the private sector [4]. The government needs to implement price regulatory
policies to protect the most vulnerable groups from ex-
ploitation by establishing effective surveillance mecha-
nisms. The prices should also be regulated to ensure
uniformity in pricing of the same product in all sector
outlets. Finally, appropriate health promotion programmes
on the importance of anti-malarial adherence can be made
available to the community. This could be done, for ex-
ample, through pamphlets in medical facilities, regular
public service announcements and mass media includ-
ing use of television, radio and video shows or through
a more community-focused method. Following a switch
in national drug policy to ACT, behaviour change com-
munication is recommended during which all outlets
staff should be trained in unbiased manner on the
current malaria policy to improve drug use in the com-
munity. In areas where such interventions have been
performed, then further scaling up of these programmes
is a necessity. Authors’ contribution Authors contribution
CAW designed, carried out the survey studies in the drug outlets and
households. CAW and CO performed the statistical analysis and participated
in drafting the manuscript. All authors read and approved the final
manuscript. Conclusion
l b l Availability of anti-malarials (ACT and quinine) still re-
mains a challenge in the outlets in malaria endemic regions
of western Kenya. First-line therapy for uncomplicated
malaria, the subsidized ACT, was more available in public
and not-for-profit facilities although with frequent stock-
outs reported in public outlets. The prices remains high in
the private outlets despite the fact that more households
get their drugs from this sector hence making affordability
a challenge. Private-sector marketing of ACT has resulted
into use of sub-optimal doses because of partial sales of
course of treatment packages. Collectively, availability,
price and affordability of the drugs affect adherence to the
dosing schedules during the treatment course. Competing interests
Th
i
i Competing interests
There is no competing interest from any of the authors of the manuscript
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Cite this article as: Watsierah and Ouma: Access to artemisinin-based
combination therapy (ACT) and quinine in malaria holoendemic regions
of western Kenya. Malaria Journal 2014 13:290. analysis of outlet survey data. Lancet 2012, 380:1916–1926. 11. DOMC: Ministry of Health (MoH), Kenya malaria worksheet. 2011. www.kemri.org 2011, 25th Feb. 2011 at 1620 hours. 12. Amin AA, Zurovac D, Kangwana BB, Greenfield J, Otieno DN, Akhwale W,
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Kenya: Ministry of Public Health and Sanitation, Division of Malaria Control;
2009. Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit Submit your next manuscript to BioMed Central
and take full advantage of: Author details
1 20. Chuma J, Musimbi J, Okungu V, Goodman C, Molyneux C: Reducing user
fees for primary health care in Kenya: policy on paper or policy in
practice? Int J Equity Health 2009, 8:15. 1Department of Public Health, Maseno University, Private Bag, Maseno,
Kenya. 2Department of Biomedical Sciences and Technology, Maseno
University, Private Bag, Maseno, Kenya. 21. Kenya Malaria Fact Sheet: Malaria in Kenya at a Glance. 1600 HRS,
www.kemri.org. Received: 6 April 2014 Accepted: 22 July 2014
Published: 28 July 2014 Received: 6 April 2014 Accepted: 22 July 2014
Published: 28 July 2014 doi:10.1186/1475-2875-13-290
Cite this article as: Watsierah and Ouma: Access to artemisinin-based
combination therapy (ACT) and quinine in malaria holoendemic regions
of western Kenya. Malaria Journal 2014 13:290. Submit your next manuscript to BioMed Central
and take full advantage of: 14. MoPHS: National Guidelines for Diagnosis, Treatment and Prevention of
Malaria for Health Workers. Nairobi, Kenya: Ministry of Public Health and
Sanitation, Division of Malaria Control; 2010. • Convenient online submission 15. KNBS: Kenya Demographic and Health Survey 2008–09. Kenya National Bureau
of Statistics (KNBS) and ICF Macro., 2010. Calverton, Maryland: KNBS and ICF
Macro; 2010. • Thorough peer review 16. Watsierah CA, Jura WG, Oyugi H, Abong’o B, Ouma C: Factors determining
anti-malarial drug use in a peri-urban population from malaria
holoendemic region of western Kenya. Malar J 2010, 9:295. 17. Kangwana BB, Njogu J, Wasunna B, Kedenge SV, Memusi DN, Goodman CA,
Zurovac D, Snow RW: Malaria drug shortages in Kenya: a major failure to
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80:737–738. Submit your manuscript at
www.biomedcentral.com/submit
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English
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Association of Maximum Temperature With Sedentary Time in Older British Men
|
Journal of physical activity & health
| 2,017
|
cc-by
| 4,779
|
Claudio Sartini, Richard W Morris, Peter H Whincup, S Goya Wannamethee, Sarah Ash,
Lucy Lennon, and Barbara J Jefferis Background: Sedentary behavior is very common in older adults and a risk factor for mortality. Understanding determinants
of sedentary behavior may help in defining strategies aimed to reduce the time spent sedentary. The degree of difference in
sedentary time attributable to varying temperatures has not been yet estimated in older men. Methods: Men aged 71 to 91
years participating in an established UK population-based cohort study were invited to wear an Actigraph GT3X accelerometer
for 1 week in 2010–12. Outcome was sedentary time (<1.5 Metabolic Equivalent of Task) in minutes per day. Associations
between daily outdoor maximum temperature and accelerometer-measured sedentary time were estimated using multilevel
models. Results: 43% (1361/3137) of invited men participated in the study and provided adequate data. Men spent on average
615 minutes in sedentary time per day (72% of the total accelerometer-wear time). After adjusting for covariates, men spent 26
minutes more per day (P < .001) in sedentary time when temperatures were in the lowest (–3.5; 9.2°C) versus highest quintile
(19.1; 29.5°C). Conclusions: Sedentary time in older adults is highest at lowest temperatures, typically recorded in winter. Findings are relevant for guidelines: interventions may consider targeting older men in winter providing recommendations for
minimizing sedentariness on daily basis. Keywords: sedentary behavior, older adults, weather, epidemiology, accelerometry A standard definition of sedentary behavior has not yet been
established, although contemporary researchers agree that seden-
tary behavior is not simply a lack of physical activity.1 Sedentary
behavior can be defined as the time spent in activities engendering
less than 1.5 Metabolic Equivalent of Task (METs).2 In recent
years, there have been an increasing number of studies which have
reported associations between prolonged sedentary behavior and
health outcomes, such as mortality and cardiovascular disease,
which have been independent of physical activity levels.3 Therefore,
understanding determinants of sedentary behavior may help in
defining strategies aimed to reduce the time spent sedentary. This
is particularly important in older adults, who are known as the most
sedentary of all age-groups.4 physical activity,5,6 although an association with sedentary time
was not investigated. We would intuitively expect sedentary time
to be higher at lower temperatures, as occur during the winter
season. However, the degree of difference in sedentary time
attributable to varying outdoor temperatures has not been esti-
mated in previous studies of older adults. Methods © 2017 The Authors. Published by Human Kinetics, Inc. This is an Open
Access article distributed under the terms of the Creative Commons Attribu-
tion License CC BY 4.0, which permits unrestricted noncommercial and
commercial use, distribution, and reproduction in any medium, provided
the original work is properly cited, the new use includes a link to the
license, and any changes are indicated. See https://creativecommons.org/
licenses/by/4.0/. This license does not cover any third-party material that
may appear with permission in the article. Claudio Sartini, Richard W Morris, Peter H Whincup, S Goya Wannamethee, Sarah Ash,
Lucy Lennon, and Barbara J Jefferis Outdoor temperature
has been overlooked in sedentary behavior guidelines,7 and as
determinant of sedentary time.8,9 To our knowledge an association
of temperature with sedentary time in older adults has not been
previously documented. Considering the gaps in knowledge of previous research, we
have therefore investigated how sedentary time (<1.5 METs) varies
according to outdoor maximum temperature in a large UK popula-
tion based cohort study of community-dwelling older men. A few previous studies in older adults have demonstrated that
low outdoor temperatures were associated with less time spent in ORIGINAL RESEARCH ORIGINAL RESEARCH Journal of Physical Activity and Health, 2017, 14, 265 -269
https://doi.org/10.1123/jpah.2016-0468 Association of Maximum Temperature With Sedentary Time
in Older British Men Claudio Sartini, Richard W Morris, Peter H Whincup, S Goya Wannamethee, Sarah Ash,
Lucy Lennon, and Barbara J Jefferis Measurements and Data Analysis Repeated measures of physical activity levels per each participant
were recorded over the course of 1 week by using accelerometers. Methods for accelerometer-data extraction and processing were
previously described in detail,11 and added here as supplementary
material (see Online Supplementary Material, Appendix S1). In brief, the number of minutes per day in spent in sedentary
behavior, light physical activity (LIPA) and moderate-to-vigor-
ous physical activity (MVPA) was derived and categorized using
count-based intensity threshold values of counts per minute
(CPM) developed for older adults, as in previous studies; the
cut-points used were <100, 100 to 1040, >1040 CPM for seden-
tary time (<1.5 METs), time spent in LIPA (<1.5 to 2.9 METs)
and MVPA (≥3 METs) respectively.4,12,13 Number of steps per
day was also recorded as a measure of overall physical activity. Then, maximum temperatures were linked to the accelerometer
data for each day the men wore the device. Daily temperatures
(maximum and minimum), hours of sunshine, and relative
humidity were provided by the UK Meteorological (MET)
Office (see Online Supplementary Material, Appendix S1). Maximum temperature was used as the main exposure variable
and divided into quintiles. Quintiles were chosen as temperatures
in the lowest quintile (1Q, –3.5°C; 9.2°C) were representative
of the typical UK winter, while temperatures in highest quintile
(5Q: 19.1°C; 29.5°C) were representative of the typical UK
summer.14 The main outcome investigated was sedentary time
measured in minutes per day. In preliminary analysis, the cor-
relations between sedentary time and other PA variables (steps,
LIPA, and MVPA) were calculated. Linear multilevel models
(level 1 was the date of wear and level 2 was the individual)
with random intercept only were used to estimate associations
between quintiles of maximum temperature and sedentary time. Quintiles of maximum temperature were derived counting every
day each participant wore an accelerometer. The highest quintile
of maximum temperature (5th quintile, 5Q) was chosen as refer-
ence quintile, and the results were reported as mean difference
in sedentary time between the reference vs lower quintiles. As in
one previous study,11 the model was adjusted for measurement
variables [accelerometer-wear time, wear day order (first day
of wear, second, etc), day of the week, age, social class, Body
Mass Index (BMI), chronic conditions, mobility limitations,
geriatric depression scale, vision problems, smoking status, and
day length (a proxy for season)]. Descriptive Statistics The median for maximum temperature in the lowest quintile was
6.3°C (between -3.5°C and 9.2°C) and in highest quintiles was
20.8°C (between 19.1°C and 29.5°C). In descriptive plots, unadjusted
sedentary time was highest when temperatures were in the lowest
quintiles, and then decreased at higher temperatures (Figure 1). Participants The British Regional Heart Study (BRHS) is a prospective cohort
of men recruited from a single local primary care center in 24 Brit-
ish towns in 1978–80.10 In 2010–2012, the surviving participants
resident in the United Kingdom (UK), then aged 71 to 91, were
invited to attend a further physical examination and to participate
in a study of objectively measured physical activity, on which the
analyses presented here are based. Men who met the inclusion
criteria (not living in a residential home and not being on wheel-
chair) were included. Participants completed a log diary (detailing
when the accelerometer was worn) and a comprehensive health
status questionnaire. The participants’ individual characteristics
and questionnaire data were already described elsewhere.11 The
National Research Ethics Service Committee for London provided Sartini, Morris, Wannamethee, Ash, Lennon, and Jefferis are with the Dept
of Primary Care & Population Health; Sartini and Jefferis are also with
the UCL Physical Activity Research Group (PARG); University College
London, UK. Morris is also with the School of Social and Community
Medicine, University of Bristol, Bristol, UK. Whincup is with the Popula-
tion Health Research Institute, St George’s University of London, London,
UK. Sartini (c.sartini@ucl.ac.uk) is corresponding author. 265 Sartini et al 266 ethical approval. Participants provided informed written consent
to the investigation, which was performed in accordance with the
Declaration of Helsinki. Associations of minimum temperature, hours of sunshine, and
relative humidity (continuous variables) with sedentary time were
also estimated.i A further investigation was performed to corroborate findings
from previous studies which made use of different physical activ-
ity outcomes, rather than sedentary time. Therefore, associations
of temperatures (maximum and minimum), hours of sunshine, and
relative humidity with daily (i) number of steps, (ii) minutes spent
in LIPA, and (iii) minutes spent in MVPA were estimated.i Associations Between Maximum Temperatures
and Sedentary Time The adjusted associations from multilevel models between quintiles
of maximum temperature and sedentary time are shown in Table 1. In summary, lower temperatures were associated with more time
spent in sedentary behavior (P < .001). In particular, men spent
26 minutes more per day (95% CI 19–33) in sedentary time when
temperatures were in the lowest compared with the highest quintile
(Table 1). When analyzing maximum temperature as continuous
variable, a negative linear association with sedentary time was
observed: a decrease in 1 SD (5.8°C) in maximum temperature was
associated with an increase of 11 minutes per day (95% CI 8–13)
in sedentary time (P < .001). The adjustment for day length did
not alter the magnitude of these associations; day length was not
significantly associated with sedentary time (P = .212). Measurements and Data Analysis The adjustment for day length
was made to check whether there was confounding between
temperature and a different seasonal term (collinearity was not
observed as the Variance Inflation Factor (VIF) score was less
than 1.5). As sensitivity analysis, a linear model was performed
using maximum temperature as a continuous variable instead
of the quintiles. We also performed stratified analysis by excluding men who
were depressed or/and with mobility limitations. All analyses were
carried out using STATA/SE 1315 and MLwiN Version 2.30.16 Results 1455 (46%) surviving men participated and met the inclusion cri-
teria. 1361 men (43.4%) had data on all covariates (complete case
analysis) and they had same mean age (78.5 years, SD = 4.6) and
BMI (26.7, SD = 3.3) in comparison with 1455 men who met the
inclusion criteria. The 1361 men with complete data were used in
the final analysis: men had a mean of 6.5 (SD = 1.2) valid days of
accelerometer wear; they wore the accelerometer for 855 minutes
per day (SD = 93) and took on average 4872 steps per day (SD =
2767). The average sedentary time per day was 615 minutes (SD
= 83), corresponding to 72% of the total accelerometer wear time;
time spent in LIPA and MVPA was 198 minutes (SD = 65) and 39
minutes (SD = 32) respectively. The correlations between daily
sedentary time with steps, LIPA, and MVPA were –0.46, –0.54,
and –0.47 respectively (all P-values < 0.001). Overall Findings The analysis of maximum temperature subdivided in quintiles
offered a simple and intuitive interpretation of the results: during
a typical winter day (temperature in the lowest quintile) older men
spent 26 minutes more per day in sedentary time in comparison
with a typical summer day (temperatures in the highest quintiles). Perception of cold may particularly inhibit older individuals from
spending time outdoors. Apart from the discomfort and need to
wear suitable clothing, there may be a fear of falling due to ice. Consequently, older adults may prefer replacing some incidental
light physical activity outdoors (eg, a gentle walk for pleasure) with
sedentary behaviors indoors, such as television watching.17 Figure 1 — Raw data (n = 1361). Plots depicting relationship between
sedentary time (mean, 95% CI), and quintiles (Q) of maximum temperature. Note. Quintiles of maximum temperature were derived counting every day
each participant wore an accelerometer (median, minimum and maximum):
1Q: 6.3 (–3.5, 9.2); 2Q: 11.0 (9.3, 13.0); 3Q: 15.3 (13.1, 16.5); 4Q: 17.9
(16.6, 19.0); 5Q: 20.8 (19.1, 29.5). P-value for the difference between the
quintiles was P < .001. Table 1 Adjusted Associations Between Quintiles (Q)
of Maximum Temperature and Sedentary Time
(n = 1361)a
Quintiles of maximum temperature (°C)
Mean difference (95%
CI) in sedentary time
(minutes per day)
5Q (19.1; 29.5)
Reference
4Q (16.6; 19.0)
+7 (3; 11)
3Q (13.1; 16.5)
+14 (10; 19)
2Q (9.3; 13.0)
+21 (15; 27)
1Q (–3.5; 9.2)
+26 (19; 33)
a Multilevel regression models (level 1 = date, level 2= individual) adjusted for age,
social class, BMI, chronic conditions, mobility limitations, geriatric depression
scale, vision problems, smoking status, daily wear time, day of the week, wear day
order, and day length. Note. P-value for trend < 0.001. We focused our investigation on maximum temperature as pri-
mary determinant as it is more accurate than other meteorological
factors due to a lower spatial variability.18 However, in subsidiary
analysis we also demonstrated that less hours of sunshine and higher
relative humidity, typical elements of the winter season in UK, were
also associated with an increase in sedentary time. To our knowledge
these findings are novel and not previously reported. Overall Findings Literature in
this field is sparse; 1 small study of 46 adults demonstrated that
accelerometer-measured sedentary time is higher in winter than
summer, although the participants were about 40 years younger than
our population.19 The majority of the studies investigated children
or adolescents, which are known to have a different life-style in
comparison with older adults.20 Table 1 Adjusted Associations Between Quintiles (Q)
of Maximum Temperature and Sedentary Time
(n = 1361)a Table 1 Adjusted Associations Between Quintiles (Q)
of Maximum Temperature and Sedentary Time
(n = 1361)a Subsidiary Analyses A decrease of 1 SD (5.8°C) in maximum temperature was associ-
ated with a decrease of 2 (95% CI 1–3) breaks in sedentary time
per day, and an increase of 0.2 (95% CI 0.1–0.3) daily number of
longer sedentary bouts (≥30 minutes). No association was found
between maximum temperature and daily number of shorter sed-
entary bouts (<30 minutes). For completeness of information, we investigated associations of
maximum temperature with different outcomes related to sedentary
behavior: total number of sedentary breaks per day, daily number
of sedentary bouts of <30 minutes, and daily number of sedentary
bouts of ≥30 minutes. JPAH Vol. 14, No. 4, 2017 Temperature and Sedentary Time in Older Men 267 Figure 1 — Raw data (n = 1361). Plots depicting relationship between
sedentary time (mean, 95% CI), and quintiles (Q) of maximum temperature. Note. Quintiles of maximum temperature were derived counting every day
each participant wore an accelerometer (median, minimum and maximum):
1Q: 6.3 (–3.5, 9.2); 2Q: 11.0 (9.3, 13.0); 3Q: 15.3 (13.1, 16.5); 4Q: 17.9
(16.6, 19.0); 5Q: 20.8 (19.1, 29.5). P-value for the difference between the
quintiles was P < .001. with variations of time spent in LIPA, MVPA, and steps per day,
although the magnitude of associations was smaller. Association
of minimum temperature with physical activity was not significant
(Online Supplementary Material, Appendix S1, Table 1).i In stratified analysis, the magnitude of associations between
temperature and sedentary time were not materially affected by
excluding men who were depressed or/and with mobility limitations
(Online Supplementary Material, Appendix S1, Table 2). Discussion In this large study of older British men, outdoor maximum tem-
perature was associated with accelerometer-measured sedentary
time: a decrease in maximum temperatures was associated with an
increase in sedentary time after controlling for potential confound-
ing variables (measurement variables, individual characteristics,
and day length). Strengths and Limitation This study used data from the BRHS, which is a large scale popula-
tion-based cohort of older men, rather than an institutionalized older
population. The magnitude of associations between temperature and
sedentary time were not materially affected by excluding men who
were depressed or/and with mobility limitations. Thanks to acceler-
ometers it was possible to overcome problems of recall error, which
is known to be more common in older individuals.21 Therefore, an
objective measure is more accurate and recommended, considering
the proportion of time older adults spent in sedentary behaviors.22
Moreover, we corroborated previous findings which have investi-
gated accelerometer-measured physical activity outcomes: as in
earlier studies we showed that low maximum temperatures, fewer
hours of sunshine, and higher relative humidity were associated with
fewer steps per day, and less time spent in LIPA and MVPA.5,6 We
also demonstrated that the association of maximum temperature with Variations in hours of sunshine and relative humidity were
associated with variations in sedentary time. On the other hand,
association of minimum temperature with sedentary time was not
significant (Online Supplementary Material, Appendix S1, Table 1). Maximum temperature was also strongly associated with
other physical activity outcomes: a decrease of 1 SD in maximum
temperature was associated with –7 minutes in LIPA per day (95%
CI –9 to –5), –4 minutes in MVPA per day (95% CI –5 to –2), and
–323 steps per day (95% CI –428 to –218). Similarly, variations
of hours of sunshine and relative humidity were also associated Variations in hours of sunshine and relative humidity were
associated with variations in sedentary time. On the other hand,
association of minimum temperature with sedentary time was not
significant (Online Supplementary Material, Appendix S1, Table 1). Maximum temperature was also strongly associated with
other physical activity outcomes: a decrease of 1 SD in maximum
temperature was associated with –7 minutes in LIPA per day (95%
CI –9 to –5), –4 minutes in MVPA per day (95% CI –5 to –2), and
–323 steps per day (95% CI –428 to –218). Similarly, variations
of hours of sunshine and relative humidity were also associated JPAH Vol. 14, No. Strengths and Limitation 4, 2017 Sartini et al 268 The temperature-related variation in sedentary time observed
in this study could be relevant to the temperature-related variation
in mortality risk.32 It is plausible that persisting low temperatures
in winter (primary determinant) may be a contributing factor
which increases the sedentary time, as well as other risk factors
levels (eg, inflammatory markers, such as C-Reactive Protein and
Interleukin-633) contributing to the excess of winter mortality.34 We
estimated an increase of 26 minutes in sedentary time at lower versus
higher temperatures. According to previous studies in older adults,
replacing 30 minutes of sedentary time with light physical activity
was independently associated with a significant reduction in mortal-
ity risk (HR = 0.80).35 However, future investigations are needed to
establish how temperature-related variations in sedentary time may
contribute to the temperature-related variations in mortality risk. physical activity was strongest in comparison with associations of
sunshine duration and humidity with physical activity. Our findings
suggested that maximum temperature is the most important predictor
of physical activity in the UK. However, earlier studies which took
place in Germany, Scotland and Japan had identified a range of dif-
ferent meteorological factors as being the most important, such as
global radiation,23 day length and diurnal minimum temperature,5
rainfall and mean temperature.24,25 However, we would expect
that, as in our results, radiation and other temperature variables are
positively correlated with maximum temperature. The study have some limitations: men who did not accept our
invitation to participate in the study were about 2 years older and
had higher BMI measured 10 years earlier; implying that overall
physical activity (eg, total number of steps) might be lower in the
general population. Our study is also limited by studying almost
exclusively white European older men, who would be expected to
spend more time in sedentary behavior, compared with younger
individuals.4 Moreover, our results may not be generalizable to
women, or to other ethnic groups.26i Acknowledgments The British Regional Heart study is supported by a British Heart Founda-
tion program grant (RG/08/013/25942). This research was supported by
an NIHR Post-Doctoral Fellowship (2010–03–023) awarded to BJ and
by British Heart Foundation grants (PG/13/41/30304 and PG09/024). The funders had no role in the design, methods, subject recruitment, data
collections, analysis or preparation of the paper. The views expressed in
this publication are those of the author(s) and not necessarily those of the
Funders. The authors thank the United Kingdom Meteorological Office
for providing the weather data. Also, during the study period maximum temperatures never
reached levels above 30°C. At those high temperatures, more typi-
cal of warmer climate zones than the UK, sedentary time may start
to increase. During heat waves local authorities tend to alert older
individuals, who are usually asked to remain indoors in the heat of
the day, to get some rest and sit when necessary, and not engage in
strenuous activities. Conclusions In this study of older adults, we demonstrated that sedentary time
increased at lower maximum temperatures. These findings are
relevant for guidelines: interventions may consider targeting older
adults in winter, when temperatures are lower, providing recom-
mendations for minimizing sedentariness on a daily basis. We defined sedentary behavior based solely on intensity, rather
than intensity and posture (more widely used), as this study did not
aim to investigate the “type” of sedentary behaviors (eg, sitting at
a computer, lying on the couch, driving, etc). However, the activity
monitors we used provide useful estimates of sedentary time, as
they have minimal bias in comparison with other devices able to
detect intensity, position and posture.27 The importance of position
and posture is widely recognized and future studies could further
investigate the particular types of sedentary behaviors (eg, watching
TV) carried out during the lowest peaks of activity. Implications 1. Gibbs BB, Hergenroeder AL, Katzmarzyk PT, Lee IM, Jakicic JM. Definition, measurement, and health risks associated with sedentary
behavior. Med Sci Sports Exerc. 2015;47(6):1295–1300. PubMed
doi:10.1249/MSS.0000000000000517 The results may have important implications for guidelines. The
UK recommendations suggest that older adults should aim to mini-
mize the time they spend being sedentary each day.8 Our findings
provided more justification for minimizing sedentary behaviors
particularly at low temperatures, a typical element of the winter
season. Replacing some of the time spent in sedentary behaviors
into more active behaviors may have beneficial effects on health. However, to find ways to reduce sedentariness is challenging, as in
modern life opportunities for sedentary behaviors are everywhere. To date, findings from the ProActive65+ trial suggested that older
adults with poor self-rated health, higher BMI and history of smok-
ing are more likely to reduce the sedentary time from an exercise
intervention.28 On the other hand, it is likely that interventions target-
ing individuals’ psychological and environmental barriers (beliefs,
feelings, and perspectives on participations in physical activity) may
be a valid alternative for replacing sedentary time with more active
behaviors.29,30 Providing recommendations for simple do-it-yourself
exercises (eg, standing up or walking while watching TV, toe rises,
calf and chest stretching) could be helpful. In older individuals,
simple targets can make the reduction in sedentary behavior easier
to achieve and relevant on a daily basis.31 Also, providing physically
and economically accessible indoor opportunities for promoting
more active behaviors during winter should be encouraged. 2. Pate RR, O’Neill JR, Lobelo F. The evolving definition of “sedentary”. Exerc Sport Sci Rev. 2008;36(4):173–178. PubMed doi:10.1097/ JES.0b013e3181877d1a Downloaded by on 06/05/17, Volume 14, Article Number 4 13. Freedson PS, Melanson E, Sirard J. Calibration of the Computer
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Development and Validation of the Behavioral Tendencies Questionnaire
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Development and Validation of the
Behavioral Tendencies Questionnaire
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Van Dam, Nicholas T.; Brown, Anna; Mole, Tom B.; Davis, Jake H.;
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http://hdl.handle.net/20.500.14038/39886 RESEARCH ARTICLE Nicholas T. Van Dam1,2, Anna Brown3, Tom B. Mole4, Jake H. Davis5, Willoughby
B. Britton6, Judson A. Brewer7,8* 1 Department of Psychiatry, Icahn School of Medicine at Mount Sinai, New York, New York, United States of
America, 2 Nathan S. Kline Institute for Psychiatric Research, Orangeburg, New York, United States of
America, 3 School of Psychology, University of Kent, Canterbury, United Kingdom, 4 Department of
Psychiatry, University of Cambridge, Cambridge, United Kingdom, 5 Graduate Center, City University of
New York, New York, New York, United States of America, 6 Department of Behavioral and Social Sciences,
Brown University Medical School, Providence, Rhode Island, United States of America, 7 Departments of
Medicine and Psychiatry, University of Massachusetts Medical School, Worcester, Massachusetts, United
States of America, 8 Department of Psychiatry, Yale University School of Medicine, New Haven,
Connecticut, United States of America Abstract This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited. * judson.brewer@umassmed.edu Development and Validation of the
Behavioral Tendencies Questionnaire Nicholas T. Van Dam1,2, Anna Brown3, Tom B. Mole4, Jake H. Davis5, Willoughby
B. Britton6, Judson A. Brewer7,8* Abstract At a fundamental level, taxonomy of behavior and behavioral tendencies can be described
in terms of approach, avoid, or equivocate (i.e., neither approach nor avoid). While there are
numerous theories of personality, temperament, and character, few seem to take advan-
tage of parsimonious taxonomy. The present study sought to implement this taxonomy by
creating a questionnaire based on a categorization of behavioral temperaments/tendencies
first identified in Buddhist accounts over fifteen hundred years ago. Items were developed
using historical and contemporary texts of the behavioral temperaments, described as
“Greedy/Faithful”, “Aversive/Discerning”, and “Deluded/Speculative”. To both maintain
this categorical typology and benefit from the advantageous properties of forced-choice
response format (e.g., reduction of response biases), binary pairwise preferences for items
were modeled using Latent Class Analysis (LCA). One sample (n1 = 394) was used to esti-
mate the item parameters, and the second sample (n2 = 504) was used to classify the partic-
ipants using the established parameters and cross-validate the classification against
multiple other measures. The cross-validated measure exhibited good nomothetic span
(construct-consistent relationships with related measures) that seemed to corroborate the
ideas present in the original Buddhist source documents. The final 13-block questionnaire
created from the best performing items (the Behavioral Tendencies Questionnaire or BTQ)
is a psychometrically valid questionnaire that is historically consistent, based in behavioral
tendencies, and promises practical and clinical utility particularly in settings that teach and
study meditation practices such as Mindfulness Based Stress Reduction (MBSR). OPEN ACCESS
Citation: Van Dam NT, Brown A, Mole TB, Davis JH,
Britton WB, Brewer JA (2015) Development and
Validation of the Behavioral Tendencies
Questionnaire. PLoS ONE 10(11): e0140867. doi:10.1371/journal.pone.0140867
Editor: Martin Voracek, University of Vienna,
AUSTRIA
Received: February 13, 2014
Accepted: October 1, 2015
Published: November 4, 2015
Copyright: © 2015 Van Dam et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited. OPEN ACCESS
Citation: Van Dam NT, Brown A, Mole TB, Davis JH,
Britton WB, Brewer JA (2015) Development and
Validation of the Behavioral Tendencies
Questionnaire. PLoS ONE 10(11): e0140867. doi:10.1371/journal.pone.0140867
Editor: Martin Voracek, University of Vienna,
AUSTRIA
Received: February 13, 2014
Accepted: October 1, 2015
Published: November 4, 2015
Copyright: © 2015 Van Dam et al. Introduction Even at the level of a single-celled organism, behavior must necessarily fall into one of three
possible categories: move towards (approach), move away (withdraw) or neither (no response). These three basic options cannot be reduced further and therefore would seem to represent the
most parsimonious description of behavioral tendencies. Human beings share an evolutionary
heritage, which has disposed us for certain common proclivities [1]. Variations on these gen-
eral proclivities, along with differences in our adaptations to considerably more recent chal-
lenges (in evolutionary terms) have led to relatively stable (both across individuals and
cultures), and largely behavioral, inter-individual differences, identified as dispositional traits
[2]. While these tendencies may vary considerably across individuals and cultures and may
comprise complicated constructs such as personality (e.g., [3]), temperament, (e.g., [4]), and
character (e.g., [5]), at a basic level, these constructs would seem to build upon a tripartite
behavioral categorization of approach, avoid, and neither approach nor avoid (i.e., inaction). g
The focus on these basic behavioral tendencies is not meant to undermine the laudable
efforts at characterizing human personality, as many theories have resulted in impressive
results. For example, relatively recent work has shown that the Big 5 (Openness, Conscien-
tiousness, Extraversion, Agreeableness, and Neuroticism), the most widely used categories of
personality [3], show individual relationships to regional neuroanatomical volume [6] and
intrinsic functional connectivity patterns of the brain [7]. However, the Big 5 may be character-
ized by two meta-traits, “Stability” and “Plasticity” [8], which may loosely relate to avoidance
tendencies (stability—comprised of agreeableness, conscientiousness, and the inverse of neu-
roticism) and approach tendencies (plasticity—comprised of extraversion and openness). One attempt to consider temperament/personality as tendencies for approach/avoidance
was based in part on Gray’s neuropsychological model of anxiety [9]. Accordingly, Carver and
White [10] developed a measure of the behavioral inhibition system (BIS) and the behavioral
activation system (BAS), to which the fight-flight-freeze system (FFFS) has been appended in
some conceptualizations [11]. While this early attempt to match personality to basic neurobe-
havioral tendencies is noteworthy, it is not without limitations. The original BIS/BAS [10]
contained 13 items across 3 subscales related to appetitive motives (Drive, Fun Seeking, and
Reward Responsiveness), but only 7 items on one subscale for avoidance. OPEN ACCESS Citation: Van Dam NT, Brown A, Mole TB, Davis JH,
Britton WB, Brewer JA (2015) Development and
Validation of the Behavioral Tendencies
Questionnaire. PLoS ONE 10(11): e0140867. doi:10.1371/journal.pone.0140867 Citation: Van Dam NT, Brown A, Mole TB, Davis JH,
Britton WB, Brewer JA (2015) Development and
Validation of the Behavioral Tendencies
Questionnaire. PLoS ONE 10(11): e0140867. doi:10.1371/journal.pone.0140867 Funding: This research was supported by the Kent
Albert research fund at Yale University. 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. 1 / 21 PLOS ONE | DOI:10.1371/journal.pone.0140867
November 4, 2015 Behavioral Tendencies PLOS ONE | DOI:10.1371/journal.pone.0140867
November 4, 2015 Introduction Further, the authors
equated avoidance with behavioral inhibition (inaction) and fear, rather than the full range of
behaviors commonly associated with the construct, including antipathy, repugnance, repul-
sion, dislike, hostility, anger, fear, and avoidance. Accordingly, the BIS items may reflect more
fearful and neurotic tendencies than truly aversive ones, as reflected in strong correlations of
the scale to negative affect, temperament, and harm avoidance [10]. Thus, while the BIS/BAS
does reflect components of approach and avoidance, it may fail to capture essential pieces of
avoidance. Further, it does not differentiate avoidance/aversion from the third fundamental
option of inaction. In addition to lacking basis in what could be considered the most parsimonious behavioral
taxonomy (i.e., approach, avoidance, equivocation), most theories of temperament, personality,
and character (e.g., [3–5, 12, 13]) are largely ‘Western’ in nature [3], potentially neglecting the
cultural basis of meaning-making that forms the cornerstone of individual autobiographical
representations [2]. Thus while existing models may meet aspects of criteria for one proposed
evaluative system for testing personality theory (i.e., compatibility and predictive power); they
may lack critical aspects of these criteria with regards to cross-cultural validity and parsimony
[14, 15]. Further, because of their relatively recent conceptualization, current personality tax-
onomies have limited historical evidence to demonstrate their utility as a sustainably practical
and cross-generational taxonomy tool. PLOS ONE | DOI:10.1371/journal.pone.0140867
November 4, 2015 2 / 21 Behavioral Tendencies Buddhist Approach to Temperament Unlike most contemporary theories of personality, which are predominantly Western, and less
than a century old [16], a Buddhist theory of temperament [17], outlined in the Visuddhi-
magga, a 5th century commentary on the early teachings of Theravada Buddhist doctrine, offers
a non-western approach and one that has been in evidence for over fifteen hundred years
(since at least the 5th Century C.E.). Although age itself does not necessarily translate to better
classification, the consistent use of such a system over time and cultures (see e.g., [17–19]) sug-
gests the possibility of cross-generational validity. Further, the traditional text from which this
typology is drawn characterizes the system not only as predictive of numerous aspects of
behavior (private and public), but also, primarily, as a means to prescribe lifestyle advice. These
lifestyles are described as maximally effective for encouraging the cultivation of skillful habits
and offsetting unskillful habits [19, 20], a practice that continues among modern-day practi-
tioners of various types of meditation practices [19], as well as yoga and alternative medical
practices among others [18, 21]. This classification system is still in use and has been adapted
by different cultures across different contexts. Specifically, the successful translation of mind-
fulness practices described in the Visuddhimagga (“The Path of Purification”) to modern evi-
dence-based interventions suggests that other concepts from the same textual sources may also
be amenable to empirical evaluation. PLOS ONE | DOI:10.1371/journal.pone.0140867
November 4, 2015 The Buddhist Behavioral Temperaments For example, the greedy and faithful types,
both manifest the same basic tendency, but the former in an unskillful way that leads to the
perpetuation of and increase in suffering, whereas the latter manifests a skillful way that leads
to the reduction of suffering ([17], p. 101–102). Each of these tendencies manifest in habitual
ways, but importantly, also can be altered ([17], p. 102–3). 1. Greedy/Faithful temperament (Approach Oriented). A style of seeking pleasant,
attractive, optimistic, beautiful, and enjoyable things and experiences characterizes the Greedy/
Faithful temperament. In general, individuals of this type tend to be passionate, extroverted,
spontaneous, and seek out novel opportunities and experiences. With an attentional bias
towards the positive, these individuals tend to overlook faults or dangers. They are biased away
from seeing their own faults, for instance, and thus may tend toward personal vanity. Equally,
such positive biases can lead to a neglect of possible negative consequences (e.g. ‘selling’ an idea
without responsibly mentioning possible downsides). On the other hand, this trait can manifest
in positive ways, inclining individuals to be generous with their trust and with material gifts,
and to take visionary leaps of faith necessary to move good works forward, saying ‘yes!’ when
more detail-oriented individuals might consider all the ways in which things could go wrong. Greed has also been characterized as unwholesome craving or desire. Predominantly greedy
individuals could be characterized by always wanting more. “It desires fulfillment through
pleasures, finding what it likes in the world of the senses. From liking, it can move quickly to
craving, passion and sensuality” ([19], p. 137). If greed is a destructive and unskillful emotion
motivating the pursuit of sensual objects, the corresponding quality of “faith”, here, is a motiva-
tion to pursue what is skillful and virtuous, “. . .greed does not give up what is harmful, while
faith does not give up what is beneficial” ([17], p.102). As Gethin [22] puts it, “. . .Buddhist
texts understand faith. . .not so much as a matter of intellectual assent to certain propositions
about the world. . .[but]as a state of trust, confidence, affection, and devotion. . .” (p. 167). Thus while benevolence or generosity might be seen as the more precise opposite of greed, here
benevolence is only one example of a virtuous quality that is motivated by faith. The Buddhist Behavioral Temperaments Buddhist theory posits that human beings react to pleasant experience by craving for its contin-
uation, to unpleasant experience by craving for it to stop, and to neutral experience by ignoring
it. Due to innate and learned dispositions, individuals are biased towards particular types of
behavioral responses to stimuli. The Visuddhimagga refers to such temperaments using an
Indic term, cariya, that translates literally as a ‘manner of going about’, or ‘manner of behavior’. This text presents its classification of behavioral types as a useful tool in tailoring interventions
to individuals’ temperaments to achieve maximal benefit, specifying particular types of hous-
ing, clothing, and food, for instance, in addition to particular meditation techniques [17]. This
may be one of the first descriptions of “personalized medicine”–matching an individual with a
treatment based on the individual’s personal characteristics to improve outcomes. The Buddhist classification of behavioral types considers numerous influences on how a
person thinks, behaves, and feels, as well as basic organizational principles of the individual as
an organism; the approach delineates these various influences in both external and internal fac-
tors. External signs include such factors as style of dress, postures and manner of walking, man-
ner of eating, sleeping and waking, working style, and style of interacting with others. Internal
signs mostly focus on the frequency of certain states of mind (e.g. anger, pride, etc.; more akin
to many Western personality theories) [17, 19, 20]. The temperaments are characterized by three fundamental motivations (similar to those
described above), greed (a pulling towards), aversion (a pushing away), and equivocation or
confusion (characterized by neither pulling nor pushing but instead allowing the mind to wan-
der either to disengage or escape from unsatisfactory conditions and/or to entertain tangential
positive experiences related to satisfactory conditions). These three motivations correspond to
three broad types of temperaments, which function as prototypes rather than exhaustive classi-
fiers. In introducing the temperaments, the Visuddhimagga actually lists six types: greedy,
faithful, aversive, discriminating, deluded, and speculative. However, the text suggests that the
six types can be understood as forming three pairs of opposing properties corresponding to
approach, avoid, and equivocate: 1) Greedy/Faithful type, the 2) Aversive/Discerning type, and
the 3) Deluded/Speculative type. The two poles of each generally represent the skillful and PLOS ONE | DOI:10.1371/journal.pone.0140867
November 4, 2015 3 / 21 Behavioral Tendencies unskillful aspects of each of these character types. PLOS ONE | DOI:10.1371/journal.pone.0140867
November 4, 2015 Behavioral Tendencies Table 1. Features of three Buddhist temperaments described in the Visuddhimagga by different characteristics. TEMPERAMENT
Greedy/Faithful
Aversive/Discerning
Deluded/Speculative
COGNITIVE
FEATURES
attentional bias
positive; towards pleasant, overlooks
faults/dangers
negative; towards unpleasant, overlooks
pleasant/virtues
inattention or attentional bias away
from present conditions towards
distraction/mind-wandering
skillful mind states
trust; generosity; motivation to
cultivate virtuous qualities
discernment; conscientiousness; prudence
equanimity; creativity
unskillful mind
states
deceit, vanity, pride, self-
centeredness, jealousy, avarice, and
addiction
judgment, criticism, hostility, hatred, prejudice
and ill-will, and aggression
negligence, confusion, doubt,
paralyzing indecisiveness,
speculation and inaction
BEHAVIORAL
FEATURES
posture/movement
graciously, with an elegant and
springy step
stiffly and unevenly, with tension and tightness
hesitantly with a perplexed gait/
shuffle
clothing style
aesthetically arranged; neither too
loose nor to tight
tightly
loosely; disheveled
food preferences
rich and sweet
sour and rough
no preference
eating habit
unhurriedly
hurriedly without savoring it
messily
sleep habit
comfortably
"with a scowl"
limbs sprawling or facedown
morning awakening
slowly
as if annoyed
with a "huh?"
social interactions
(meet new people)
seize on trivial virtues and discount
genuine faults; avoid conflict, even
by dishonesty
come across as distant, tired, or bored; leave
quickly as if anxious to go
copy what others are saying:
confused, not knowing what to do or
how to act
response to novel
environments
notice whatever is pleasing, fixate
upon it; leave pleasant
circumstances slowly and with
regret. notice whatever is wrong; fixate on difficulties;
seize upon any slightly unpleasant object; notice
trivial faults and discount virtues
initially unaffected by pleasing or
unpleasing features because
unnoticed until others point out
doi 10 1371/jo rnal pone 0140867 t001 doi:10.1371/journal.pone.0140867.t001 aversive types to judgment, criticism, hostility, hatred, prejudice, ill will, and aggression, all of
which can lead to conflict and unpopularity. “The aversive temperament is constructed around
judgment and rejection of experience. . .It is critical, quickly displeased, quarrelsome and dis-
paraging of many things. Its quality of aversion can give rise to states of anger, vindictiveness,
haughtiness, hatred, cruelty, aggression and struggle to control. . .” ([19], p. 174). Skillful and
unskillful aspects are both recognized in the source text, which notes that whereas aversion is
an unskillful and unwholesome motivation to find fault, discernment finds fault with those
conditions that are not beneficial, in particular, unwholesome states of mind [17]. For addi-
tional discussion of the characteristics of the aversive/discerning type, especially behavioral
descriptors, see Table 1. The Buddhist Behavioral Temperaments While affable,
and often well liked, individuals of the greedy/faithful type often have difficulty with discipline,
restraint, and commitment, and can become vain, self-centered, and unreliable while looking
for the “best offer”. They are vulnerable to overindulging and addictions, sexual infidelity, and
deception [17, 19, 20]. For additional discussion of the characteristics of the greedy/faithful
type, especially behavioral descriptors, see Table 1. In suggesting how to tailor a therapeutic intervention to persons of the greedy type, the
Visuddhimagga advises providing conditions that counteract the tendency to grasp after sen-
sual pleasures. Thus it is suggested that providing luxurious housing, clothing, and food is not
beneficial for one who is predominantly greedy, and moreover that it is helpful to have per-
sonal interactions that discourage pride and vanity ([17], p. 107–8). For similar reasons, medi-
tative contemplation of the body is especially suggested as a meditation object for those of a
greedy type, including contemplation of bodily anatomy such as organs and fluids ([17],
p. 113). This seems to be intended to counter attachment to the body, others’ as well as one’s
own, and aimed ultimately at fostering a more balanced mental attitude. 2. Aversive/Discerning temperament (Avoidance Oriented). In contrast to the greedy
type, the aversive type seeks to avoid or remove what is unpleasant, and therefore has an atten-
tional bias towards faults, imperfections, burdens, and threats. Aversive types thrive in intellec-
tual pursuits that require high levels of discernment, accuracy, and precision, and are often
perceived as having high levels of competency or understanding. Where the greedy/faithful
type is good at making the visionary leaps of faith necessary to move good works forward, aver-
sive/discerning types excel at making detailed contingency plans to anticipate the many ways
in which plans could go wrong. On a more negative note, however, these same qualities incline PLOS ONE | DOI:10.1371/journal.pone.0140867
November 4, 2015 4 / 21 PLOS ONE | DOI:10.1371/journal.pone.0140867
November 4, 2015 In suggesting how to tailor a therapeutic intervention to persons of this type, the Visuddhi-
magga advises providing much more sensually pleasing conditions than those suggested for the
greedy type. This text suggests aesthetically pleasing living quarters, with pleasing decorations,
flowers, clothes, and perfumes, fine light clothing, as well as food that is inviting and “superior
in every way” ([17], p. 108). The conditions suggested thus seem to be aimed at counteracting
the tendency to aversion and of pushing away. Similarly, the development of loving kindness,
compassion, sympathetic joy, and equanimity are especially recommended as meditation prac-
tices for one of an aversive temperament ([17], p. 114). 3. Deluded/Speculative temperament (Equivocate). While the greedy/faithful and aver-
sive/discerning types are characterized by their strong motivational tendency of either grasping 5 / 21 PLOS ONE | DOI:10.1371/journal.pone.0140867
November 4, 2015 Behavioral Tendencies or pushing away, the deluded/speculative type is distinguished by the absence of a strong moti-
vational tendency. It is characterized by a lack of awareness of present conditions, whether
pleasant, unpleasant, or neutral. As a result, deluded/speculative types tend not to have strong
immediate reactions and not to hold fixed opinions. On the positive side, this allows for crea-
tivity, being open to many possible options, and ‘thinking outside the box’. On the negative
side, the deluded type can seem lost, constantly scattered, and prone to following the opinions
of others because of uncertainty. Without an agenda, they can be laidback and easy-going, crea-
tive and “out of the box” pioneers. However, the lack of clarity, goals, or direction can lead to
confusion, doubt, paralyzing indecisiveness, speculation, and inaction. “They seek to establish
ease by ignoring what is happening or through dullness or inaction. The deluded temperament
gives rise to perplexity and worry, doubt, negligence, scattered thoughts, anxiety and agitation.”
([19], p. 175). For additional discussion of the confused/speculative type, especially behavioral
descriptors, see Table 1. PLOS ONE | DOI:10.1371/journal.pone.0140867
November 4, 2015 Current Study The current study aimed to develop a historically accurate and contemporarily applicable tem-
perament questionnaire based on both traditional and contemporary texts, originating from
the Visuddhimagga [17]. To counteract the effects of response styles and biases that can be par-
ticularly detrimental in cross-cultural research [23], we utilized a forced-choice response for-
mat, whereby items representing behaviors characteristic of the three types were compared. The psychometric properties of the questionnaire and specifically its ability to discriminate
between the types were tested in two separate samples via Latent Class Analysis of choices
made between pairs of items. Similar to Thurstonian Item Response Theory (IRT) modeling
[24–27], this approach uses dummy-coded outcomes of rankings in each block of items for
analysis. However, the measurement model assumes that categorical (latent classes) rather
than continuous (latent traits) variables underlie the responses. To examine construct validity,
we probed the relationship of the classes identified by this questionnaire to the predominant
contemporary personality typology (i.e., the Big 5), as well as to approach/avoidance tendencies
and behaviors, interpersonal tendencies, and decision-making styles. Measures Behavioral Tendencies Questionnaire (BTQ). An interdisciplinary team (comprising
contemplative Buddhist scholars and/or practitioners, philosophers, clinical, social, personal-
ity, and other psychologists, as well as neuroscientists and psychiatrists) was formed to discuss
questionnaire development. Appropriate source documents were first identified to develop a
comprehensive representation of each of the different temperament types. After becoming
familiar with the various descriptions of each of the temperament types, thematic analysis was
performed and preliminary summaries of the temperaments were created using contemporary
and classic texts (e.g., [17, 19]). Source documents consistently listed eight behavioral contexts
(i.e. sleeping, waking, social interactions, etc.; see Table 1 for details). Individuals from the
team drafted items for each behavioral category for each temperament type and consensus
building was used to compile questions upon which the group agreed. The initial items were
piloted on a small group for content consistency. After the initial pilot, a set of 43 blocks of three items remained, each containing a stem
phrase and three different possible responses, corresponding to each of the three temperament
types. The response format for the BTQ was multidimensional forced choice, wherein partici-
pants were instructed to rank response options (i.e., 1st, 2nd, 3rd) based on how well each option
characterized them for a given stem. Participants were instructed to rank the response option
that was most like them as 1st and the option that was least like them as 3rd in each block. Big Five Aspect Scales. The BFAS [12] is a 100-item measure of the ‘Big 5’ personality
characteristics (openness, conscientiousness, extraversion, agreeableness, and neuroticism). Each ‘Big 5’ characteristic is comprised of two aspects: Openness = Intellect and Openness;
Conscientiousness = Industriousness and Orderliness; Extraversion = Enthusiasm and Asser-
tiveness; Agreeableness = Compassion and Politeness; Neuroticism = Withdrawal and Volatil-
ity. Each item is rated on a 1 (strongly disagree) to 5 (strongly agree) Likert-type scale. Past
studies have revealed good psychometric properties and relationships to individual differences
in brain structure [6, 12]. Participants A small sample of participants were recruited from the communities of Providence, RI and
New Haven, CT (n = 36). All other participants (n = 859) were recruited through Amazon. com’s mechanical turk (MTURK) system. The MTURK works by creating a Human Intelli-
gence Task (HIT), which are completed by an experimenter-selected number of potential par-
ticipants from a pool of 500,000+ workers that use MTURK (see www.mturk.com). The
experimenter creates an account to upload funds and create HITs. Reimbursement rate for a
specified HIT is determined by the experimenter, often based on current norms among the
MTURK for comparable amounts of time required to complete a HIT (see e.g., [28] for discus-
sion of varying pay rates and its relatively small impact on the quality of data). Previous data
analysis with MTURK has shown excellent internal consistency and good reliability, including
high test-retest reliability [28]. Additionally, recent work has shown that data acquired via
MTURK is not only equivalent to data collected in person, but also tends to provide more
diverse demographics [29]. Data were collected in two waves. Data from the first wave were
collected from a total of 394 individuals, 36 locally, and 354 from MTURK. Data from the sec-
ond wave were collected from a total of 502 participants, all via MTURK. All procedures were PLOS ONE | DOI:10.1371/journal.pone.0140867
November 4, 2015 6 / 21 Behavioral Tendencies determined to be exempt from institutional review board review due to the completely anony-
mous nature of the data collection. determined to be exempt from institutional review board review due to the completely anony-
mous nature of the data collection. Measures The index is an over-
all sum of absolute differences between pre-identified pairs of highly correlated items [31]. In
the present sample, internal consistency was as follows: Inattention and Memory Problems, α
= .840; Hyperactivity and Restlessness, α = .697; Impulsivity and Emotional Lability, α = .814;
Problems with Self-Concept, α = .907. assesses four factor-derived subscales (Inattention and Memory Problems, Hyperactivity and
Restlessness, Impulsivity and Emotional Lability, and Problems with Self-Concept), in addition
to providing an inconsistency index. The inconsistency index consists of 16 items that are
rated on a Likert-type scale ranging from 0 (not at all, never) to 3 (very much, frequently). The
absolute difference for response pairs is summed and a cut-score is used. The index is an over-
all sum of absolute differences between pre-identified pairs of highly correlated items [31]. In
the present sample, internal consistency was as follows: Inattention and Memory Problems, α
= .840; Hyperactivity and Restlessness, α = .697; Impulsivity and Emotional Lability, α = .814;
Problems with Self-Concept, α = .907. Coping Responses Inventory. The CRI [32] is a 48-item questionnaire designed to assess
approach and avoidance coping strategies in response to dealing with problems. The scale
consists of 24 items that assess approach coping (subscales include logical analysis, positive
reappraisal, seeking guidance and support, and problem solving) and 24 items that assess
avoidance coping (subscales include cognitive avoidance, acceptance/resignation, seeking alter-
native rewards, and emotional discharge). Individual items are rated on a 1 (not at all) to 4
(fairly often) Likert-type response scale. The CRI has shown good predictive validity of psycho-
logical problems (esp. problem drinking) [33]. In the present sample, internal consistency was
as follows: Approach coping, α = .890 (Logical analysis, α = .701; Positive reappraisal, α = .846;
Seeking guidance and support, α = .637; Problem solving, α = .766); Avoidance coping, α =
.850 (Cognitive avoidance, α = .890; Acceptance or resignation, α = .818; Seeking alternative
rewards, α = .704; Emotional discharge, α = .514). Life Orientation Test—Revised. The LOT-R [34] is a 6-item questionnaire (10 items if
filler items are included) designed to measure optimism/pessimism. Three of the six items
directly assess optimism and three directly assess pessimism. All items are scored so that higher
scores indicate greater optimism. Individual items are rated on a 1 (I agree a lot) to 5 (I disagree
a lot) Likert-type scale. Measures In the present study, internal consistency was Cronbach’s α = .902. Melbourne Decision Making Questionnaire. The MDMQ [35] is a 22-item questionnaire
designed to assess coping patterns associated with the conflict theory of decision making [36]. Items are rated on a 0 (not true for me) to 2 (true for me), 3-point Likert-type response scale. Factor analysis supported a 4-factor structure comprised of the constructs of Buck-Passing
(i.e., encouraging others to make difficult decisions), Hypervigilance (i.e., a hurried, anxious
style of decision making), Procrastination (i.e., delaying/avoiding making decisions), and Vigi-
lance (i.e., an exhaustive search style of decision making). The MDMQ has shown good psy-
chometric properties across international samples [35]. In the present sample, internal
consistency was as follows: Buck Passing, α = .891; Hypervigilance, α = .785; Procrastination α
= .877; Vigilance α = .805. Trust Inventory. The TI [37] is a 40-item measure of trust in others, generally (20 items)
and romantic partners, specifically (20 items). Items are rated on a 1 (strongly disagree) to 4
(strongly agree), 4-point Likert-type rating scale. The scale has exhibited generally good psy-
chometric properties [37]. Only the general trust items were administered in the present study. In the present study, internal consistency for the general subscale was, α = .708. Measures In the present sample, internal consistency for Big 5 (and domains)
was as follows: Neuroticism, α = .906 (Withdrawal, α = .799; Volatility, α = .898); Agreeable-
ness, α = .889 (Compassion, α = .897; Politeness, α = .776); Conscientiousness, α = .869
(Industriousness, α = .847; Orderliness, α = .811); Extraversion, α = .830 (Enthusiasm, α =
.609; Assertiveness, α = .857); Openness, α = .869 (Openness, α = .839; Intellect, α = .814). Behavioral Inhibition/Behavioral Activation Scales. The BIS/BAS [10] is a 20-item ques-
tionnaire designed to assess behavioral approach and avoidance tendencies. The behavioral
approach system (BAS) is assessed across 13 items, while the behavioral inhibition system
(BIS) is assessed across 7 items. Participants rate each item on a 4-point Likert-type response
scale ranging from 1 (very true for me) to 4 (very false for me). In order to facilitate easy inter-
pretation alongside other measures, we reverse-scored the scale such that higher mean scores
indicate higher approach or inhibition. The BIS/BAS has shown generally good psychometric
properties, with a 4-factor structure (BIS + 3 BAS subscales), and reliability/consistency values
mostly > .7 in both nonclinical [10] and clinical samples [30]. In the present sample, internal
consistency was as follows: BAS, α = .860 (Drive, α = .844; Fun Seeking, α = .776; Reward
Responsiveness, α = .779); BIS, α = .854. Conner’s Adult ADHD Rating Scales. The CAARS:S [31] is a 26-item, short-form assess-
ment tool of Attention Deficit Hyperactivity Disorder symptoms. The items are rated on a 0
(not at all, never) to 3 (very much, very frequently), Likert-type response scale. The CAARS:S Conner’s Adult ADHD Rating Scales. The CAARS:S [31] is a 26-item, short-form assess-
ment tool of Attention Deficit Hyperactivity Disorder symptoms. The items are rated on a 0
(not at all, never) to 3 (very much, very frequently), Likert-type response scale. The CAARS:S 7 / 21 PLOS ONE | DOI:10.1371/journal.pone.0140867
November 4, 2015 Behavioral Tendencies assesses four factor-derived subscales (Inattention and Memory Problems, Hyperactivity and
Restlessness, Impulsivity and Emotional Lability, and Problems with Self-Concept), in addition
to providing an inconsistency index. The inconsistency index consists of 16 items that are
rated on a Likert-type scale ranging from 0 (not at all, never) to 3 (very much, frequently). The
absolute difference for response pairs is summed and a cut-score is used. Modeling Forced-Choice Data A forced-choice response format has several benefits, including elimination of response biases
acting uniformly across all items such as extreme responding and central tendency responding,
and acquiescence [38], as well as minimization of exaggerated emotional coherence in assess-
ments of different qualities (halo effect) [39]. Additionally, forced-choice formats eliminate the
need for a rating scale, which assumes that all participants interpret that response option 8 / 21 PLOS ONE | DOI:10.1371/journal.pone.0140867
November 4, 2015 Behavioral Tendencies categories in the same way—often a problematic assumption (see e.g., [25]). However, forced-
ranking items present unique challenges to data analysis. The primary problem is that if one
considers means or sums of the rankings (e.g., 1, 2, 3), the values will be the same for each
block (i.e., M = 2, ∑= 6), regardless of the actual ranking. The resulting data has been called
ordinal ipsative data (e.g., [40]) and requires complex analytic approaches (e.g., [41, 42]), with
difficult to interpret results. Fortunately, a solution to modeling multidimensional force-choice questionnaire data has
recently been proposed [24–27]. This method, identified as Thurstonian Item Response Theory
(IRT) allows for the recovery of latent trait structures underlying a response set [24–27]. Thus,
for each ranking block of three items, all pairwise comparisons of items (i.e., A vs. B, A vs. C, B
vs. C) are considered. Each pairwise comparison (say, A vs. B) is coded {A, B} = 1 if item A
was preferred to B, and {A, B} = 0 otherwise. Hence, the three comparisons/contrasts (binary
dummy variables) fully describe any given rank order of three items. For example, if a respon-
dent assigns the rank orders A = 2, B = 1, and C = 3, the dummy coding will be {A, B} = 0, {A,
C} = 1, {B, C} = 1. Because questionnaire items are usually designed to indicate some underly-
ing trait, each pairwise comparison indicates two traits. Here we assume that items within a
block indicate different traits. Thus, comparisons {A, B} and {A, C} provide information about
the latent trait associated with A, comparisons {A, B} and {B, C} provide information about the
latent trait associated with B, and comparisons {A, C} and {B, C} provide information about
the latent trait associated with C. Detailed information about the development of these models
and their implementation can be found in previous publications [24–27]. PLOS ONE | DOI:10.1371/journal.pone.0140867
November 4, 2015 Results Demographics and Data Screening Modeling Forced-Choice Data However, the questionnaire in the present study was designed not to indicate latent traits
(continua along which participants may be placed), but latent classes (nominal types into
which participants may be placed). These types (greedy, aversive and deluded) were hypothe-
sized to underlie the observed preferences; hence, the measurement model suitable for the
questionnaire is not an IRT model, but a Latent Class Analysis (LCA) model. This model is log-
ically straightforward: each binary dummy variable resulting from pairwise comparison of two
items is underlain by a categorical latent variable (latent class). Specifically, outcomes resulting
from comparisons between Greedy and Aversive items (the first and the second items in each
BTQ block, see S1 Appendix) are underlain by a latent categorical variable taking two possible
values, or two classes—Greedy and Aversive. Those respondents who belong to the Greedy
class, should tend to prefer Greedy items, hence their response to all item-pairs of this kind
should be primarily {iG, iA} = 1. Conversely, respondents belonging to the Aversive class,
should demonstrate primarily responses {iG, iA} = 0. The other two types of item comparisons
are modeled in the same way. Thus, to explain the patterns of choices between items indicating
Greedy and Deluded (first and third items in each block), a latent categorical variable with two
levels is needed. Finally, choices between Aversive and Deluded items (second and third items
in each block) are explained by a latent categorical variable with two levels. In this study, we tested the three separate LCA models described above, which have an
advantage of simplicity at the initial stages of developing and testing the questionnaire. Condi-
tional on the latent classes, the pairwise responses within each model are independent; hence,
all assumptions of a LCA measurement model are met to explore the optimal number of classes
needed to describe the variability in responses, and assess the performance of individual items
in measuring the intended classes. Due to local dependencies occurring between pairwise com-
parisons involving the same item (for example, {A, B} and {A, C}), and associated modeling
challenges, we do not test all pairwise comparisons simultaneously in this study. The LCA analyses were performed in MPLUS v7.2 [43], using the maximum likelihood esti-
mator (robust). All other analyses were conducted in SPSS v20. PLOS ONE | DOI:10.1371/journal.pone.0140867
November 4, 2015 9 / 21 Behavioral Tendencies Demographics and Data Screening Demographics. Demographic details are provided in Table 2. Demographic comparisons. Within sample 1, there was no age difference between partic-
ipants recruited locally and those recruited via Amazon.com’s Mechnical Turk, t(382) = 1.40, p
= .16. Similarly, there was no difference in gender distribution, χ2
(1) = 0.12, p > .5, racial/ethnic
background, χ2
(5) = 2.74, p > .5, marital status, χ2
(5) = 0.80, p > .5, or household income, χ2
(8)
= 10.40, p = .24. There were, however, significant differences in educational level, χ2
(4) = 21.89,
p < .001, and occupational status, χ2
(5) = 18.21, p < .01. The local sample had higher rates of
completed graduate/professional training (36.1% vs. 12.6%), college/university completion
(52.8% vs. 43.0%), but lower rates of partial college (11.1% vs. 41.1%). The local sample had
substantially lower rates of unemployment (0.0% vs. 19.3%) and part-time employment
(5.6% vs. 21.5%), but higher rates of full-time employment (63.9% vs. 36.6%) and not in labor
force (27.8% vs. 20.7%). The rates of inconsistent responding, based on item pairs from the
BFAS, were no different between groups, suggesting combination of groups would not be
inappropriate. Missing data. Among sample 1, there were 33 (8.6%) participants who completed less
than 95% of all questionnaire items. These participants were excluded from further analyses. No participants from sample 2 had any missing data. Behavioral Tendencies Table 2. Group Demographics. Sample 1 (n = 394a)
Sample 2 (n = 504)
M (SD)
M (SD)
t
Age
33.1 (12.9)
36.5 (12.6)
4.16***
% (n)
% (n)
χ2
Gender
4.47*
% Female
63.7 (244)
58.2 (293)
Race/Ethnicity
12.12*
% White
83.5 (320)
76.6 (386)
% Asian
6.1 (23)
7.0 (35)
% African American
3.3 (13)
8.3 (42)
% Hispanic/Latino
2.5 (9)
3.9 (20)
% Multi-racial
3.6 (14)
3.3 (17)
% Other
1.0 (4)
0.9 (4)
Educational Attainment
32.80***
% Graduate/Professional
14.7 (56)
11.1 (56)
% College/University
43.9 (168)
41.1 (207)
% Some College
38.3 (147)
33.5 (169)
% High School
2.5 (10)
12.0 (65)
% < 7 years
0.5 (2)
1.3 (7)
Work Status
30.09***
% Full-time employed
39.1 (150)
49.0 (247)
% Part-time employed
20.1 (77)
24.9 (126)
% Unemployed > 1 month
15.5 (59)
13.1 (66)
% Unemployed < 1 month
2.0 (8)
1.1 (6)
Never employed
2.0 (8)
2.4 (12)
Not in work force
21.3 (81)
9.4 (47)
a Data missing for n = 11. Representative sample is n = 383. ***p < .001,
*p < .05. d i 10 1371/j
l
0140867 t002 Table 2. Group Demographics. doi:10.1371/journal.pone.0140867.t002 Aversive versus deluded. Dummy coded binary contrasts between the 2nd and 3rd item in
each block were explored fitting a sequence of LCA models. From Table 3 it can be seen that
the 2-class solution was again the best. The two classes were clearly interpretable as the Aver-
sive (45.2% estimated prevalence) and Deluded types (54.8% prevalence). Exploring the
between-class odds ratios, three blocks showed large effect sizes, 11 blocks showed medium to
large effect sizes, and 14 small to medium. Fifteen blocks discriminated poorly with effect sizes
less than 1.5 (small). Exploratory LCA Analyses in Sample 1 Data The dummy coded binary contrasts in each model were explored fitting a sequence of LCA
models with 1, 2, 3 etc. classes in sample 1, which contained responses to the full BTQ (43
blocks). Bayesian Information Criterion (BIC) was used to determine the best number of clas-
ses [44]. Exploring the between-class odds ratios of preferring the first option in each item-pair
to the second option in calibration sample (sample 1) helped determine well performing items
(items that discriminate well between the two classes) and flag poorly performing items. To
this end, we used the recommended effect sizes for odds ratios (small = 1.5, medium = 3.5 and
large = 9 [45]). Greedy versus aversive. Dummy coded binary contrasts between the 1st and the 2nd item
in each block were explored fitting a sequence of LCA models with 1, 2, 3 etc. classes. Table 3
reports the BIC and the entropy (index of classification quality) for each of the competing
models. It can be seen that the 2-class solution was the best. The two classes were very clearly
interpretable—the conditional probabilities of preferring the first item to the second in each
pair were always higher in the first class. Hence, the first class represented the Greedy type
(57% estimated prevalence), and the second represented Aversive (43% prevalence). Exploring
the between-class odds ratios, two blocks showed large effect sizes, 12 blocks showed medium
to large effect sizes, and 21 small to medium. Eight blocks discriminated poorly with effect
sizes less than 1.5 (small). Greedy versus deluded. Dummy coded binary contrasts between the 1st and 3rd item in
each block were explored fitting a sequence of LCA models. From Table 3 it can be seen that
the 2-class solution was the best. The two classes were clearly interpretable as the Greedy
type (58.2% estimated prevalence), and the Deluded type (41.8% prevalence). Exploring the
between-class odds ratios, there were no large effects, 13 blocks showed medium to large effect
sizes and 19 small to medium. Eleven blocks discriminated poorly with effect sizes less than 1.5
(small). 10 / 21 PLOS ONE | DOI:10.1371/journal.pone.0140867
November 4, 2015 Behavioral Tendencies Table 3. Goodness of fit of competing LCA models for Behavioral Tendencies Questionnaire (original 43-block version). Sample 1 (n = 394)
Model
# Classes
# parameters
Loglikelihood
BIC
Entropy
G vs. A
1
43
-11025.12
22307.21
2
87
-10633.16
21786.27
.83
3
131
-10541.48
21865.86
.83
4
175
-10454.94
21955.74
.82
G vs. D
1
43
-11047.06
22351.10
2
87
-10746.22
22012.38
.75
3
131
-10638.07
22059.04
.82
4
175
-10550.41
22146.69
.80
A vs. D
1
43
-10664.59
21586.17
2
87
-10371.46
21262.87
.77
3
131
-10277.75
21338.41
.76
4
175
-10188.06
21421.98
.81
Note: G = Greedy, A = Aversive, D = Deluded; BIC = Bayesian Information Criterion. doi:10.1371/journal.pone.0140867.t003 ness of fit of competing LCA models for Behavioral Tendencies Questionnaire (original 43-block version). Table 3. Goodness of fit of competing LCA models for Behavioral Tendencies Questio Note: G = Greedy, A = Aversive, D = Deluded; BIC = Bayesian Information Criterion. 1. the probability of being Greedy from the model contrasting Greedy and Aversive, PGA(G),
and the complementary probability of being Aversive PGA(A) = 1 –PGA(G); 2. the probability of being Greedy from the model contrasting Greedy and Deluded, PGD(G),
and the complementary probability of being Deluded PGD(D) = 1 –PGD(G); and 3. the probability of being Aversive from the model contrasting Aversive and Deluded,
PAD(A), and the complementary probability of being Deluded PAD(D) = 1 –PAD(A). Thus, membership in each temperament class was based on the evidence from two separate
models. Having these multiple evidences was beneficial since it allowed determining not only
primary temperament/tendency type, but also secondary type. For instance, if an individual has
the probabilities PGA(G)>PGA(A), PGD(G)>PGD(D) and PAD(A)>PAD(D), it must follow that
his/her primary type is Greedy (since it was the most likely type compared to the alternatives),
and his/her secondary type is Aversive. Given 3 classifications with 2 classes each, theoretically
there should be 23 = 8 perturbations of the probability inequalities. However, two perturbations
out of eight are not admissible since they represent intransitive inequalities: (1) PGA(G)>
PGA(A), PGD(G)<PGD(D) and PAD(A)>PAD(D) and (2) PGA(G)<PGA(A), PGD(G)>PGD(D)
and PAD(A)<PAD(D). The remaining six inequalities are transitive and result in the following
primary and secondary type combinations: GA, GD, AG, AD, DG, and DA. Considering only
the primary type classification, out of all valid cases from sample 2, 42% were classified as the
Greedy type, 32.6% as the Aversive type and 25.4% as the Deluded type. The primary type attri-
butions were then used to validate the BTQ against other measures used in this study. Classification of Participants in Sample 2 After the item-pair parameters (i.e. thresholds) conditional on latent class membership have
been established using sample 1, these parameters were used to calculate the conditional proba-
bilities of belonging to latent classes for sample 2. Because sample 2 was administered a reduced
version of the BTQ (with 29 blocks), only parameters for the available blocks were used. This
calculation led to obtaining for each participant: 11 / 21 PLOS ONE | DOI:10.1371/journal.pone.0140867
November 4, 2015 1. the probability of being Greedy from the model contrasting Greedy and Aversive, PGA(G),
and the complementary probability of being Aversive PGA(A) = 1 –PGA(G); Behavioral Tendencies Table 4. Comparison of Groups in Sample 1 on Big Five Aspects and Traits. Greedy
Aversive
Deluded
GvA
GvD
AvD
M (SD)
M (SD)
M (SD)
p
Post-Hoc
Cohen’s d
Big Five
Aspect
Neuroticism
2.69 (0.63)
2.81 (0.77)
2.99 (0.75)
.002
D>G
-0.17
-0.43
-0.24
Withdrawal
2.75 (0.68)
2.86 (0.80)
3.09 (0.83)
.001
D>G
-0.15
-0.45
-0.28
Volatility
2.62 (0.70)
2.76 (0.84)
2.89 (0.84)
.022
D>G
-0.18
-0.35
-0.15
Agreeableness
3.85 (0.61)
3.77 (0.52)
3.74 (0.56)
.215
-
0.14
0.19
0.06
Compassion
3.97 (0.65)
3.73 (0.68)
3.77 (0.72)
.007
G>A,D
0.36
0.29
-0.06
Politeness
3.73 (0.64)
3.80 (0.54)
3.71 (0.60)
.435
-
-0.12
0.03
0.16
Conscientiousness
3.47 (0.51)
3.68 (0.45)
2.92 (0.47)
< .001
A>G>D
-0.44
1.12
1.65
Industriousness
3.51 (0.63)
3.54 (0.54)
2.94 (0.66)
< .001
G,A>D
-0.05
0.88
1.00
Orderliness
3.42 (0.54)
3.83 (0.58)
2.91 (0.57)
< .001
A>G>D
-0.73
0.92
1.60
Extraversion
3.48 (0.44)
3.21 (0.48)
3.11 (0.56)
< .001
G>A,D
0.59
0.73
0.19
Enthusiasm
3.54 (0.43)
3.13 (0.44)
3.11 (0.53)
< .001
G>A,D
0.94
0.89
0.04
Assertiveness
3.42 (0.62)
3.28 (0.67)
3.10 (0.76)
.001
G>D
0.22
0.46
0.25
Openness
3.79 (0.59)
3.73 (0.49)
3.79 (0.58)
.660
-
0.11
0.00
-0.11
Openness
3.78 (0.67)
3.52 (0.63)
3.80 (0.64)
.001
-
0.40
-0.03
-0.44
Intellect
3.81 (0.61)
3.95 (0.63)
3.77 (0.72)
.102
A>G,D
-0.23
0.06
0.27
N.B. p values are from F-tests comparing all 3 groups. Post-Hoc tests are Bonferroni corrected comparisons, where ‘>‘ indicates p < .05 and ‘,’ indicates p .05. d i 10 1371/j
l
0140867 t004 Table 4. Comparison of Groups in Sample 1 on Big Five Aspects and Traits. doi:10.1371/journal.pone.0140867.t004 type). The aversive/discerning types were highest on conscientiousness (overall, and orderli-
ness aspect) and lowest on openness (aspect of Openness trait). Finally, the deluded/speculative
types were higher than the greedy/faithful types (but not aversive/discerning types) on neuroti-
cism (withdrawal and volatility aspects, as well as neuroticism trait) and lowest on conscien-
tiousness (industriousness and orderliness aspects, as well as conscientiousness trait). Sample 2: Relationships to Approach/Avoidance, Attentional Functioning, Interpersonal
functioning, and decision making. Group differences were largely consistent with hypotheses
(see Table 5). The greedy/faithful types were highest on overall approach orientation (BAS), and
the drive and reward-responsiveness subscales of the BAS, the overall approach scale of the CRI,
and positive reappraisal, and seeking guidance subscales of the CRI, the LOT-R (optimism), and
the Trust Inventory. The aversive/discerning types were lowest on overall approach orientation
(BAS) and the fun-seeking subscale of the BAS, lowest on the overall avoidance scale of the CRI,
and significantly higher than the deluded/speculative types (but not the greedy/faithful types)
on the vigilance subscale of the MDMQ. The deluded/speculative types were highest on all
aspects of the CAARS (inattention and memory problems, hyperactivity and restlessness,
impulsivity and emotional lability, and self-concept problems) as well as on the inconsistency
index. They were also higher on cognitive avoidance, acceptance and resignation, and the emo-
tional discharge subscales of the CRI, along with the overall avoidance scale of the CRI, as well
as lowest on the overall approach scale of the CRI, and the logical analysis and problem-solving
subscales of the CRI. The deluded/speculative types were additionally lowest on the LOT-R and
highest on the Buck-Passing, Hypervigilance, and Procrastination subscales of the MDMQ. Construct Validation with Sample 2 Sample 1: Relationships to Big Five. After classification of each participant into a pri-
mary temperament type based on the BTQ, the three groups were compared on scales and sub-
scales of the BFAS. ANOVAs with Bonferroni corrected post-hoc tests were conducted on all
10 aspect scales and the Big Five scales. Group differences are reported in Table 4. The greedy/faithful types were (relatively) highest on compassion (aspect scale of agreeable-
ness) and extraversion (enthusiasm, and assertiveness, relative to the deluded/speculative 12 / 21 PLOS ONE | DOI:10.1371/journal.pone.0140867
November 4, 2015 Behavioral Tendencies Table 5. Comparison of Groups in Sample 2 on Behavioral Characteristics. Greedy
Aversive
Deluded
GvA
GvD
AvD
Scale
Subscale
M (SD)
M (SD)
M (SD)
Post-Hoc
Cohen’s d
BAS
40.29 (5.80)
35.78 (6.06)
37.13 (5.67)
G>D>A
0.76
0.55
-0.23
Drive
11.69 (2.78)
10.35 (2.63)
10.02 (2.38)
G>A,D
0.50
0.65
0.13
Reward Resp. 17.27 (2.30)
16.08 (2.41)
16.22 (2.56)
G>A,D
0.51
0.43
-0.06
Fun-Seeking
11.33 (2.37)
9.35 (2.60)
10.89 (2.43)
G,D>A
0.80
0.18
-0.61
BIS
19.23 (4.22)
20.06 (4.61)
21.70 (4.87)
D>G,A
-0.19
-0.54
-0.35
CAARS
Inconsistency
3.97 (2.52)
4.06 (2.18)
5.04 (2.31)
D>G,A
-0.04
-0.44
-0.44
Inattn/Memory
8.23 (3.08)
8.31 (2.64)
12.29 (3.29)
D>G,A
-0.03
-1.27
-1.33
Hyper Restless
9.87 (3.09)
9.48 (2.76)
11.46 (3.05)
D>G,A
0.13
-0.52
-0.68
Impulsive/Labile
7.91 (2.59)
7.77 (2.38)
10.12 (3.49)
D>G,A
0.06
-0.72
-0.79
Self-Concept
9.40 (3.76)
10.68 (4.11)
13.66 (4.32)
D>A>G
-0.32
-1.05
-0.71
CRI-Approach
74.20 (10.14)
71.52 (10.21)
66.66 (9.78)
G>A>D
0.26
0.76
0.49
Logical Analysis
18.73 (2.89)
19.13 (2.65)
17.90 (2.93)
G,A>D
-0.14
0.29
0.44
Pos. Reappraisal
18.95 (3.31)
17.34 (3.66)
16.36 (3.78)
G>A,D
0.46
0.73
0.26
Seek Guidance
16.95 (3.15)
15.64 (3.58)
14.96 (3.09)
G>A,D
0.39
0.64
0.20
Prob. Solving
19.57 (3.00)
19.41 (2.70)
18.95 (3.03)
G,A>D
0.06
0.21
0.16
CRI-Avoidance
57.44 (10.53)
54.40 (10.07)
61.33 (9.10)
D>G>A
0.30
-0.40
-0.72
Cog Avoidance
14.23 (3.81)
13.82 (4.11)
16.60 (3.87)
D>G,A
0.10
-0.62
-0.70
Accept Resign
13.86 (3.37)
13.55 (3.48)
15.54 (2.99)
D>G,A
0.09
-0.53
-0.61
Seek Alter Rew. 16.17 (3.55)
14.40 (3.20)
14.86 (3.20)
G>A,D
0.52
0.39
-0.14
Emot. Discharge
13.18 (3.22)
12.62 (2.82)
14.32 (2.72)
D>G,A
0.19
-0.38
-0.61
LOT-R
22.14 (5.40)
19.71 (6.11)
16.65 (6.06)
G>A>D
0.42
0.96
0.50
MDMQ
Buck Pass
9.66 (3.21)
9.94 (3.46)
12.00 (3.51)
D>G,A
-0.08
-0.70
-0.59
Hypervigilance
8.72 (2.49)
8.55 (2.50)
10.31 (2.84)
D>G,A
0.07
-0.60
-0.66
Procrastination
7.48 (2.48)
7.38 (2.50)
9.84 (3.00)
D>G,A
0.04
-0.86
-0.89
Vigilance
15.13 (2.41)
15.71 (2.26)
14.54 (2.72)
A>D
-0.25
0.23
0.47
Trust Inventory
61.68 (10.41)
55.90 (11.34)
54.82 (10.28)
G>A,D
0.53
0.66
0.10
N.B. All scales have been scored such that higher scores indicate higher levels of the primary construct. All F-tests were significant at p < .001, except LOT-R, where p = .001. Post-Hoc tests are Bonferroni corrected comparisons, where ‘>‘ indicates p < .05
and ‘,’ indicates p .05. Table 5. Comparison of Groups in Sample 2 on Behavioral Characteristics. doi:10.1371/journal.pone.0140867.t005 members of class 2), we selected the best discriminating blocks from the original 43 blocks (13
in total) to create a short version of the BTQ (BTQ-SF). The BTQ-SF is provided in S1 Appen-
dix. Conditional probabilities of preferring the first item in each pair are given in Table 6. Using conditional probabilities, the individual probabilities of belonging to each class may be
computed, and individuals classified in one of the three classes. We compared the estimated
class membership based on the full BTQ (43 blocks) and the short version (13 blocks). The
shortened version yielded very similar classification (see Table 7), with the coefficient of agree-
ment between the two classifications kappa = .798. Development of the BTQ short form Based on the odds ratios computed for all pairs in sample 1 (odds of preferring the first item in
the pair for members of class 1 divided by odds of preferring the first item in the pair for 13 / 21 PLOS ONE | DOI:10.1371/journal.pone.0140867
November 4, 2015 All F-tests were significant at p < .001, except LOT-R, where p = .001. Post-Hoc tests are Bonferroni corrected comparisons, where ‘>‘ indicates p < .05
and ‘ ’ indicates p 05 g
g
p
y
, except LOT-R, where p = .001. Post-Hoc tests are Bonferroni corrected comparisons, where ‘>‘ indicates p < .05 h that higher scores indicate higher levels of the primary construct. B. All scales have been scored such that higher scores indicate higher levels of the primary construct. N.B. All scales have been scored such that higher scores indicate higher levels of the primary construct.
All F-tests were significant at p < .001, except LOT-R, where p = .001. Post-Hoc tests are Bonferroni corrected comparisons, where ‘>‘ indicates p < .05
and ‘,’ indicates p .05. All F-tests were significant at p < .001, except LOT-R, where p = .001. Post-Hoc tests are Bonferroni corrected co
and ‘,’ indicates p .05. Behavioral Tendencies Table 6. Conditional probabilities of preferring first item in a pair for the BTQ-SF. block
pairs {G, A}
pairs {G, D}
pairs {A, D}
Greedy
Aversive
Greedy
Deluded
Aversive
Deluded
1
.47
.21
.62
.28
.77
.60
2
.60
.47
.72
.29
.76
.33
3
.57
.27
.77
.32
.89
.40
4
.52
.07
.58
.31
.79
.58
5
.55
.08
.55
.22
.87
.48
6
.37
.10
.49
.15
.78
.51
7
.52
.18
.79
.46
.92
.59
8
.47
.22
.59
.17
.75
.31
9
.54
.17
.57
.21
.75
.39
10
.39
.08
.51
.21
.80
.61
11
.55
.29
.58
.21
.59
.24
12
.42
.08
.81
.42
.96
.68
13
.61
.25
.79
.43
.87
.61
doi:10.1371/journal.pone.0140867.t006 Table 6. Conditional probabilities of preferring first item in a pair for the BTQ-SF. We purposefully retained the original forced-choice format, ranking 3 response options
with a single item stem, corresponding to each of the behavioral tendencies. Retaining this for-
mat was important, as the items were designed to represent categorical options broadly relating
to descriptions of what individuals of different types would do in different contexts [17, 19]. Latent Class Analysis of pairwise comparisons between items indicating Greedy and Aversive
types yielded two classes, which clearly corresponded to the hypothesized types. The same was
true for the contrasts between Greedy and Deluded, and between Aversive and Deluded types. Individual items were assessed for their ability to discriminate between the types; many items
had favorable properties, showing medium to large or large effect sizes. While we created an
item pool reflecting the many characteristics of the traditional temperaments (see Table 1), not
all item domains had acceptable psychometric properties. For example, items about food pref-
erences had poor discriminative ability. Overall, however, the items of the final scale (BTQ-SF,
see S1 Appendix) generally reflected the broad characteristics related to these historic tempera-
ment types. The class membership derived from the estimated conditional probabilities of
responses resulted in clear classification with one predominant behavioral tendency type, as
well as a secondary type. When examining the classification groups of the first sample in relation to the Big Five
Aspect Scales, the Greedy/Faithful types were compassionate and extraverted, the Aversive/
Discerning types were conscientious and closed-minded (opposite of openness to experience),
and the Deluded/Speculative were somewhat neurotic and careless (opposite of conscientious). Table 7. Discussion The goals of the current project were to establish a parsimonious temperament scale based on
historic observations and considerations of habitual behaviors [17] that had 1) good psycho-
metric properties, and 2) good nomothetic span (appropriate relationships to other self-report
measures). 14 / 21 PLOS ONE | DOI:10.1371/journal.pone.0140867
November 4, 2015 doi:10.1371/journal.pone.0140867.t006 Classification of participants from sample 1 based on the BTQ (43 blocks) and the BTQ-SF (13 blocks). BTQ-SF (13 blocks)
BTQ (43 blocks)
Greedy
Aversive
Deluded
Total
Greedy
143
6
13
162
Aversive
9
89
8
106
Deluded
12
4
110
126
Total
164
99
131
n = 394
Overlap (%)
87.2
89.9
84.0
doi:10.1371/journal.pone.0140867.t007
PLOS ONE | DOI:10.1371/journal.pone.0140867
November 4, 2015
15 / 21 Table 7. Classification of participants from sample 1 based on the BTQ (43 blocks) and the BTQ-SF (13 blocks). 7. Classification of participants from sample 1 based on the BTQ (43 blocks) and the BTQ-SF (13 blocks). sample 1 based on the BTQ (43 blocks) and the BTQ-SF (13 blocks). PLOS ONE | DOI:10.1371/journal.pone.0140867
November 4, 2015 15 / 21 Behavioral Tendencies The second sample completed measures of approach/avoidance (BIS/BAS), attentional prob-
lems (CAARS:S), coping styles (CRI), optimism, decision-making styles, and trust. As hypothe-
sized, the Greedy/Faithful types were the most Approach-oriented, with significantly higher
BAS scores than both other types on 2/3 approach-oriented subscales and the total BAS score. The Aversive/Discerning types had the lowest approach orientation overall (BAS), while the
Deluded/Speculative types had the highest behavioral inhibition (BIS) mean score. Given that
the BIS measures behavioral inhibition (more akin to inaction and/or fear) rather than avoid-
ance [10], it is perhaps not surprising that the Deluded/Speculative types were highest on this
measure. While the CAARS:S was designed to assess symptoms of ADHD, the nature of sub-
scales (inattention and memory problems, hyperactivity and restlessness, impulsivity and
emotional lability, and problems with self-concept) is consistent with the Deluded/Speculative
type, who exhibited the highest scale means for all these subscales. Of additional interest, the
Deluded/Speculative types exhibited the highest mean inconsistency scores, which provides
support for this typology as a behavioral indicator of equivocation and/or difficulty committing
to a given response (pattern). In terms of coping strategies, the Greedy/Faithful types were largely approach-oriented in
terms of problem-solving while the Deluded/Speculative types were largely avoidance-oriented. While we characterized the Aversive/Discerning types as generally avoidance-oriented, these
individuals are also generally logical and conscientious (see Table 1), thus it is reasonable to
conclude that they may not necessarily avoid problems while the indecisive nature of the
Deluded/Speculative types may lead them to avoid problems in a number of ways. In terms of optimism, the Greedy/Faithful types were highest and the Deluded/Speculative
types lowest. Limitations It is worth noting that the temperaments, as originally described, rely heavily on observable
aspects of behavior [17]. Historical and contemporary assignments of a particular tempera-
ment type were often done by experienced individuals who had detailed (longitudinal) knowl-
edge of the individuals they were categorizing [20]. The present study relied solely on self-
report of behavioral tendencies, which may pose unique problems for a typology historically
characterized by observable behaviors. Items which held up to psychometric analysis in the
present study may not represent aspects of the constructs that are better measured by observer
report, actual behavioral measure, etc. While it is hypothesis-consistent that the Greedy/Faithful types were highest, one
might have expected the Aversive/Discerning types to be lowest on optimism. However, one
possibility is that the general uncertainty and ambiguity with which the Deluded/Speculative
types view the world that leads them to be somewhat more pessimistic. Consistent with our
hypotheses, the Deluded/Speculative type had the highest levels of maladaptive decision-mak-
ing, exhibiting high levels of buck-passing, hypervigilance, and procrastination. Also consistent
with our hypotheses, the Greedy/Faithful types had the highest mean scores on the Trust
Inventory. Overall, it would seem that the types derived from the BTQ would seem to be largely
consistent with those identified by the early source text [17]. Future Directions Similarities to attachment types. The three insecure types of attachment [46] map rela-
tively well with the behavioral temperaments. The anxious attachment style is one of clinging
and neediness, as well as concern about involvement with others, similar to the Greedy temper-
ament. The avoidant attachment style is one of pushing others away, often with increased inter-
nally and externally directed anger, and efforts to be self-sustaining, similar to the Aversive 16 / 21 PLOS ONE | DOI:10.1371/journal.pone.0140867
November 4, 2015 Behavioral Tendencies temperament. Finally, the disorganized attachment style is one of volatility and distortion, lack-
ing a clear pattern or basis, similar to the Deluded temperament. Future studies might examine
whether different attachment styles (e.g., [47]) map onto the behavioral temperaments. Rela-
tionships between these two constructs would further establish construct validity and compati-
bility and might permit important elaboration of how an individual’s attachment style might
influence his/her behavioral predispositions. Similarities to theoretical neurobehavioral systems. Another important future project
may be to examine relationships between the behavioral temperaments and the approach/
avoidance systems as measured experimentally (e.g., [48]). The greedy/faithful type might be
described as very approach-oriented, while the aversive/discerning type might be described as
very avoidance oriented. The deluded/speculative type might be considered neither approach
nor avoidance oriented as this type is fundamentally confused about whether to pull towards
or push away. Accordingly, there may be interesting relationships between the behavioral tem-
peraments and related neurobehavioral functions of approach and avoidance systems [49]. Similarities to other measures of temperament. Other temperament scales have been
developed with the aim of characterizing human experience and proclivities. For example, the
Adult Temperament Questionnaire (ATQ) has shown similarities to the Big 5 in that it both
had the emergence of a 5 factor model and subsequently demonstrated a higher-order 2 factor
model [4, 50]. The Temperament and Character Inventory is another measure that has pro-
posed relationships to basic neurochemistry [5]. Given that the behavioral temperament ques-
tionnaire has potentially important relationships to other measures of temperament (especially
the ATQ; see e.g., [50]), future studies might examine these possible overlaps. Since the ATQ
has direct relationships to various attentional and self-regulatory systems, these findings could
potentially elucidate underlying neurocognitive components of the behavioral temperaments. PLOS ONE | DOI:10.1371/journal.pone.0140867
November 4, 2015 Future Directions Relationships of the BTQ to these scales might provide further insight into the construct valid-
ity of the BTQ, as well as helping to generate hypotheses about the neurophysiological basis of
basic habitual behaviors. Clinical and practical utility. Finally, measures like the Behavioral Tendency Question-
naire may provide a much-needed framework to tailor behavioral approaches to treatment. Indeed, the text from which we have drawn the description of these types presents the typology
primarily as a means to provide prescriptive practices in relation to each of the different tem-
peraments. This suggests that recent initiatives among medicine to individualize treatment
(e.g., [51]) can also be utilized in meditation training as well as other behavioral therapies. The
behavioral temperament scale presented here may be useful for indicating who might respond
best to what type of meditation techniques [19], body-based practices and lifestyle changes [18,
21]. We have noted, for instance, how the Visuddhimagga suggests that individuals who mani-
fest behavioral characteristic of the aversive type will do best meditating in visually pleasing,
beautiful settings, and should be given meditation practices such as developing loving kindness. In contrast, it is suggested that greedy types will find austere and even unpleasant conditions
more conducive to practice, presumably because these counteract the tendency to focus on
pleasant stimuli. Similarly, because of its function to cut off mind-wandering, mindfulness of
breathing is suggested as particularly suitable for those with deluded and speculative tendencies
[17] (p. 114). Notably, this characterization of temperament/personality is quite different from the most
commonly used approach (i.e., the Big 5). A great deal of psychopathology is associated with
Neuroticism. While perhaps unintended, the classification of one as predominantly neurotic
does not really permit much room for positive interpretation. Further, the proposed notion
that these personality traits are stable over time implies that the neurotic individual may always
be that way. In stark contrast, the behavioral temperament system promotes a positive and PLOS ONE | DOI:10.1371/journal.pone.0140867
November 4, 2015 17 / 21 Behavioral Tendencies negative aspect of each temperament that can be modified through training. The greedy indi-
vidual is also characterized as faithful, the aversive as discerning, and the deluded as specula-
tive. These reflect skillful and unskillful manifestations of the same types of behaviors. Thus an
individual characterized as one type can actively work to direct behaviors in the more skillful
direction. Supporting Information S1 Appendix. Behavioral Tendency Questionnaire. (DOC) S1 Appendix. Behavioral Tendency Questionnaire. (DOC) Acknowledgments The authors would like to thank Dave Perlman, Zach Schlosser, Jeremy Gray, and Colin
DeYoung for insightful comments and discussion that contributed to some of the ideas pre-
sented in this manuscript. Conclusions The present study aimed to develop and validate a behavioral tendencies questionnaire based
on habitual behavior that was true to the traditional Buddhist texts in which mindfulness prac-
tice is described, has present day validity and applicability, and that seems to have practical
utility in both scientific and clinical settings. The findings suggested good psychometric prop-
erties and construct consistent nomothetic span. Given the strong Buddhist influence on the
BTQ, the measure would seem to have potential implications for individualizing meditation
and mindfulness-based practices. Additional work is required to identify the extent to which
the BTQ is a useful predictor of observable behavior, task performance, and neurobehavioral
activity, but the present findings suggest that the scale has considerable potential. Future Directions Further, the nature of these simple classifications may serve to enhance an individu-
al’s awareness of his/her oft-overlooked behavioral tendencies and serve as a basis for discus-
sion. As the typology is relatively simplistic, recalling that one is predominantly of a particular
type could help one to place effort towards being more skillful, especially in the company of
others who may have known similar or dissimilar temperaments. These types of individualized approaches may be relatively easily adopted in modern medi-
cine, given the brevity and ease of use of screening tools such as the BTQ. Indeed, although the
Visuddhimagga’s discussion envisions a teacher having ample time to observe a student’s
behavioral habits in daily life, s/he recommends questioning the student as a primary means of
determining temperament [17] (p. 107). Moreover, given the origins of the scale, the BTQ may
offer high cultural acceptability and relevance to the increasing population of Buddhists in
Western societies. In the modern clinical context, individuals might fill out a screening BTQ
form before beginning practice with a teacher or taking a mindfulness-based clinical interven-
tion (e.g. Mindfulness-Based Stress Reduction; [52]), allowing the teacher to tailor instruction
accordingly. Using this empirically derived approach to tailoring treatments or practices to
individual temperaments may improve the efficacy of current treatments, though specific clini-
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https://openalex.org/W1551738228
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https://www.frontiersin.org/articles/10.3389/fpsyg.2015.00691/pdf
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English
| null |
Compensating for age limits through emotional crossmodal integration
|
Frontiers in psychology
| 2,015
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cc-by
| 12,816
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Compensating for age limits through
emotional crossmodal integration
Laurence Chaby 1,2*, Viviane Luherne-du Boullay 3, Mohamed Chetouani 2 and
Monique Plaza 2
1 Institut de Psychologie, Sorbonne Paris Cité, Université Paris Descartes, Boulogne-Billancourt, France, 2 Groupe Intégration
Multimodale, Interaction et Signal Social, Institut des Systèmes Intelligents et de Robotique, CNRS UMR 7222, Paris,
France, 3 Université Paris 8 Vincennes Saint Denis, Saint-Denis, France 1 Institut de Psychologie, Sorbonne Paris Cité, Université Paris Descartes, Boulogne-Billancourt, France, 2 Groupe Intégration
Multimodale, Interaction et Signal Social, Institut des Systèmes Intelligents et de Robotique, CNRS UMR 7222, Paris,
France, 3 Université Paris 8 Vincennes Saint Denis, Saint-Denis, France Social interactions in daily life necessitate the integration of social signals from different
sensory modalities. In the aging literature, it is well established that the recognition of
emotion in facial expressions declines with advancing age, and this also occurs with vocal
expressions. By contrast, crossmodal integration processing in healthy aging individuals
is less documented. Here, we investigated the age-related effects on emotion recognition
when faces and voices were presented alone or simultaneously, allowing for crossmodal
integration. In this study, 31 young adults (M = 25.8 years) and 31 older adults
(M = 67.2 years) were instructed to identify several basic emotions (happiness, sadness,
anger, fear, disgust) and a neutral expression, which were displayed as visual (facial
expressions), auditory (non-verbal affective vocalizations) or crossmodal (simultaneous,
congruent facial and vocal affective expressions) stimuli. The results showed that older
adults performed slower and worse than younger adults at recognizing negative emotions
from isolated faces and voices. In the crossmodal condition, although slower, older adults
were as accurate as younger except for anger. Importantly, additional analyses using the
“race model” demonstrate that older adults benefited to the same extent as younger
adults from the combination of facial and vocal emotional stimuli. These results help
explain some conflicting results in the literature and may clarify emotional abilities related
to daily life that are partially spared among older adults. Abbreviations: PASA, Posterior–Anterior Shift in Aging; fMRI, functional magnetic resonance imaging; ERP, event-
related potentials; RT, response times; STS, superior temporal sulcus, p-STC, posterior superior temporal cortex; BDI,
Beck Depression Inventory; MMSE, Mini Mental State Examination; CDFs, cumulative distribution functions. Edited by:
Andrea Hildebrandt,
Ernst-Moritz-Arndt Universität
Greifswald, Germany
Reviewed by:
Natalie Ebner,
University of Florida, USA
Cesar F. Lima,
University College London, UK
*Correspondence:
Laurence Chaby,
Groupe Intégration Multimodale,
Interaction et Signal Social,
Institut des Systèmes Intelligents
et de Robotique, CNRS UMR 7222,
4 Place Jussieu, Paris 75005, France
laurence.chaby@parisdescartes.fr Edited by:
Andrea Hildebrandt,
Ernst-Moritz-Arndt Universität
Greifswald, Germany Edited by:
Andrea Hildebrandt,
Ernst-Moritz-Arndt Universität
Greifswald, Germany
Reviewed by:
Natalie Ebner,
University of Florida, USA
Cesar F. Lima,
University College London, UK
*Correspondence:
Laurence Chaby,
Groupe Intégration Multimodale,
Interaction et Signal Social,
Institut des Systèmes Intelligents
et de Robotique, CNRS UMR 7222,
4 Place Jussieu, Paris 75005, France
laurence.chaby@parisdescartes.fr Reviewed by:
Natalie Ebner,
University of Florida, USA
Cesar F. Lima,
University College London, UK Correspondence:
Laurence Chaby,
Groupe Intégration Multimodale,
Interaction et Signal Social,
Institut des Systèmes Intelligents
et de Robotique, CNRS UMR 7222,
4 Place Jussieu, Paris 75005, France
laurence.chaby@parisdescartes.fr Keywords: aging, emotion, faces, voices, non-verbal vocalizations, multimodal integration, race model Introduction Specialty section:
This article was submitted to
Perception Science,
a section of the journal
Frontiers in Psychology Specialty section:
This article was submitted to
Perception Science,
a section of the journal
Frontiers in Psychology Specialty section:
This article was submitted to
Perception Science,
a section of the journal
Frontiers in Psychology
Received: 01 April 2015
Accepted: 10 May 2015
Published: 27 May 2015 Emotion recognition is a fundamental component of social cognition. The ability to discrim-
inate and interpret others’ emotional states from emotional cues plays a crucial role in social
functioning and behaviors (Carton et al., 1999; Adolphs, 2006; Corden et al., 2006; Frith and
Frith, 2012). From early and throughout lifespan, emotion recognition is an essential mediator
of successful social interactions and well-being (Izard, 2001; Engelberg and Sjöberg, 2004; Kryla-
Lighthall and Mather, 2009; Suri and Gross, 2012). Hence, impaired recognition of others’ emo-
tional states may result in severe social dysfunctions, including inappropriate social behaviors,
poor interpersonal communication and reduced quality of life (Feldman et al., 1991; Shimokawa
et al., 2001; Blair, 2005). Such difficulties have been observed not only in disorders characterized Received: 01 April 2015
Accepted: 10 May 2015
Published: 27 May 2015 Keywords: aging, emotion, faces, voices, non-verbal vocalizations, multimodal integration, race model ORIGINAL RESEARCH
published: 27 May 2015
doi: 10.3389/fpsyg.2015.00691 ORIGINAL RESEARCH
published: 27 May 2015
doi: 10.3389/fpsyg.2015.00691 Citation: Chaby L, Luherne-du Boullay V,
Chetouani M and Plaza M (2015)
Compensating for age limits through
emotional crossmodal integration. Front. Psychol. 6:691. Abbreviations: PASA, Posterior–Anterior Shift in Aging; fMRI, functional magnetic resonance imaging; ERP, event-
related potentials; RT, response times; STS, superior temporal sulcus, p-STC, posterior superior temporal cortex; BDI,
Beck Depression Inventory; MMSE, Mini Mental State Examination; CDFs, cumulative distribution functions. doi: 10.3389/fpsyg.2015.00691 May 2015 | Volume 6 | Article 691 1 Frontiers in Psychology | www.frontiersin.org Aging and multimodal emotional integration Chaby et al. by prominent social-behavioral deficits (i.e., autism spectrum dis-
orders, schizophrenia, neurodegenerative dementia; e.g., Chaby
et al., 2012; and see for review Kennedy and Adolphs, 2012;
Kumfor et al., 2014) but also in normal aging, which is frequently
associated with social withdrawal and loneliness (e.g., Szanto et al.,
2012; Steptoe et al., 2013). cortex (Hornak et al., 2003; Wildgruber et al., 2005; Tsuchida and
Fellows, 2012) and the temporal lobes, particularly the superior
temporal gyrus (Beer et al., 2006; Ethofer et al., 2006). The amyg-
dala is also involved in this processing (Iidaka et al., 2002; Fecteau
et al., 2007). Prefrontal cortex atrophy (in particular atrophy of
the orbitofrontal region; Resnick et al., 2003, 2007; Lamar and
Resnick, 2004) is a known marker of normal aging and could
explain the difficulties identifying some facial emotions, in par-
ticular anger. Moreover, although the amygdala does not decline
as rapidly as the frontal regions, some studies have reported a
linear reduction of its volume with age (Mu et al., 1999; Allen
et al., 2005). When comparing elderly people with young adults,
neuroimaging studies observed a less significant activation of this
structure among the elderly during the processing of emotional
faces, especially negative ones (Mather et al., 2004). This was
coupled with increased activity in the prefrontal cortex (Gunning-
Dixon et al., 2003; Urry et al., 2006; Ebner et al., 2012). Conversely,
other studies found a decrease in functional connectivity between
the amygdala and posterior structures, which may reflect a decline
in the perceptual process (Jacques et al., 2009). Overall, these
patterns of brain activity observed in neuroimaging studies during
a variety of emotional tasks (including recognition) are consistent
with the Posterior–Anterior Shift in Aging (PASA; for review, see
Dennis and Cabeza, 2008), which reflects the effect of aging on
brain activity. Citation: p
)
Although older adults report high levels of satisfaction and bet-
ter emotional stability with advancing age (Reed and Carstensen,
2012; Sims et al., 2015), they have difficulties processing some
types of emotional information, which is often marked by a
decline in emotion recognition (Ruffman et al., 2008; Isaacowitz
and Blanchard-Fields, 2012). Most past studies have identified
age-related difficulties in the visual channel, particularly when
participants were asked to recognize emotion from posed facial
expressions (see for review, Chaby and Narme, 2009; Isaacowitz
and Stanley, 2011). These posed expressions were created to con-
vey a single specific emotion, typically with exaggerated individual
features, without any distracting or irrelevant features. However,
emotions are not usually expressed solely by the face during
daily social interactions; typically, voice (including non-verbal
vocalizations) is also an important social signal, which needs to
be processed quickly and accurately to allow successful interper-
sonal interactions. The rare studies that have explored how the
ability to recognize vocal emotion changes with age have been
conducted on speech prosody using words or sentences spoken
with various emotional expressions. Theses studies concluded
that advancing age is associated with increasing difficulties in
recognizing emotion from prosodic cues (Kiss and Ennis, 2001;
Paulmann et al., 2008; Mitchell et al., 2011; Lambrecht et al., 2012;
Templier et al., 2015). However, vocal emotions could also be
experienced via non-verbal affect bursts (e.g., screams or laughter;
see Scherer, 1994) that typically accompany intense emotional
feelings and that might be considered as the vocal counterpart of
facial expressions. The processing of non-verbal vocal affects in
aging individuals has rarely been studied (see Hunter et al., 2010;
Lima et al., 2014); thus, this issue needs to be further investigated. Another explanation for older adults’ lower performance on
negative emotion recognition emerges within the framework of
the socio-emotional selectivity theory (Carstensen, 1992). With
advancing age, adults appear to concentrate on a few emotionally
rewarding relationships with their closest partners, report greater
emotional control, and reduce their cognitive focus on nega-
tive information. Based on these observations, it was suggested
that “paradoxically,” the recognition of negative emotion declines
(Carstensen et al., 2003; Charles and Carstensen, 2010; Mather,
2012; Huxhold et al., 2013). Losses in cognitive and sensory functions are also possible
explanations for age-related changes in emotion recognition. Frontiers in Psychology | www.frontiersin.org Citation: Increasing age is often associated with a decline in cognitive
abilities (e.g., Verhaeghen and Salthouse, 1997; for review, see
Salthouse, 2009), as well as with losses in visual and auditory
acuity (Caban et al., 2005; Humes et al., 2009), which could
hamper higher-level processes such as language and perception
(Sullivan and Ruffman, 2004). However, these sensory attributes
are shown to be poor predictors of the age-related decline in
visual or auditory emotional recognition (e.g., Orbelo et al., 2005;
Mitchell, 2007; Ryan et al., 2010; Lima et al., 2014). g
Altogether, the above studies showed evidence of age-related
decline of some basic emotions via unimodal visual or audi-
tory channels. These changes might start early, at approximately
40 years, for both facial (Williams et al., 2009) and prosodic emo-
tions (Paulmann et al., 2008; Mill et al., 2009; Lima and Castro,
2011), and decline may occur linearly with advancing age (see
Isaacowitz et al., 2007). In particular, compared to young adults,
older adults could experience difficulties recognizing fear, anger
and sadness from faces but experience no deficits recognizing
happy or neutral faces (see for review, Isaacowitz et al., 2007;
Ruffman et al., 2008). The recognition of disgust also seems highly
preserved in older adults (e.g., Calder et al., 2003). Data from
voices are less coherent, as difficulties have been found in older
adults only for anger and sadness (Ruffman et al., 2008) or for
almost all emotions (e.g., Paulmann et al., 2008). Previous research on age-related differences in the recogni-
tion of basic emotions has focused predominantly on a single
modality, and thus little is known about age-related differences
in crossmodal emotion recognition. However, in daily life, people
perceive emotions through multiple modalities, such as speech,
voices, faces and postures (e.g., Young and Bruce, 2011; Belin et al.,
2013). This indicates that our brain merges information from dif-
ferent senses to enhance perception and guide our behavior (Ernst
and Bülthoff, 2004; Ethofer et al., 2013). Evidence supporting this
idea includes studies of brain-damaged patients, such as traumatic
or vascular brain injuries and brain tumors. These studies found Different mechanisms have been proposed to explain these
age-related changes in emotion recognition. One preeminent
explanation concerns structural and functional brain changes
associated with age. Multiple interconnected brain regions are
implicated in visual and auditory emotional processing. Citation: These
regions include the frontal lobes, particularly the orbitofrontal May 2015 | Volume 6 | Article 691 Frontiers in Psychology | www.frontiersin.org 2 Aging and multimodal emotional integration Chaby et al. similar impairments in processing emotions from faces and voices
in a single modality, but found that brain-damaged patients expe-
rienced greater performance using both facial and vocal stimuli
(e.g., Hornak et al., 1996; Borod et al., 1998; Calder et al., 2001;
Kucharska-Pietura et al., 2003; du Boullay et al., 2013; Luherne-du
Boullay et al., 2014). processed in separate channels, which suggests an underlying
integrative mechanism (see Laurienti et al., 2006; Girard et al.,
2013; Charbonneau et al., 2013). To date, the processing mechanisms responsible for multisen-
sory enhancement in older compared to young adults remains
unclear, and crossmodal emotional integration in aging evaluated
by the race model has not been investigated. To characterize the
age-related effect on emotional processing, we used emotional
human stimuli (i.e., happy, angry, fear, sad and disgust) and a
neutral expression in the form of unimodal (facial or vocal) or
crossmodal (simultaneous congruent facial and vocal expressions)
cues. Isolated facial expression was studied using pictures of posed
facial expressions, and isolated vocal expression was studied using
non-verbal affect stimuli. Our primary focus concerned cross-
modal emotional processing in aging, and we aimed to explore
whether older adults benefit from congruent crossmodal integra-
tion and to better understand the nature of this benefit. According
to recent studies of multisensory integration mechanisms during
aging (e.g., Lambrecht et al., 2012; Freiherr et al., 2013; Mishra and
Gazzaley, 2013), we hypothesized that older adults benefit from
congruent crossmodal presentation when identifying emotions. To assess this hypothesis, we calculated redundancy gains for
scores and used the race model for RTs to determine the nature
of multisensory integration achieved by combining redundant
visuo-auditory information. Some studies in young adults have demonstrated that congru-
ent emotional information processed via multisensory channels
optimizes behavioral responses, which results in enhanced accu-
racy and faster response times (RT; De Gelder and Vroomen, 2000;
Kreifelts et al., 2007; Klasen et al., 2011). Citation: In older adults, audio-
visual performances have been shown to be equivalent or even
improved relative to younger adults (Laurienti et al., 2006; Peiffer
et al., 2007; Diederich et al., 2008; Hugenschmidt et al., 2009;
DeLoss et al., 2013), with more rare exceptions showing reduced
multisensory integration in older adults (Walden et al., 1993;
Sommers et al., 2005; Stephen et al., 2010). Some of these studies
have explored the effects of age on crossmodal emotional pro-
cessing and found evidence for preserved multisensory processing
in older adults when congruent auditory and visual emotional
information were presented simultaneously (Hunter et al., 2010;
Lambrecht et al., 2012). Multisensory integration refers to the process by which unisen-
sory inputs are combined to form a new integrated product (Stein
et al., 2010). This process has been studied in humans using
neuroimaging techniques, which show that different regions of
the human brain are implicated in the integration of multimodal
cues, including “convergence” areas such as the superior temporal
sulcus (STS; Laurienti et al., 2005; James and Stevenson, 2011;
Watson et al., 2014; see for review, Stein et al., 2014). Neuroimag-
ing techniques such as functional magnetic resonance imaging
(fMRI) generally show greater activity in response to bimodal
stimulation. More precisely, in a series of fMRI experiments
conducted by Kreifelts and collaborators (e.g., Kreifelts et al.,
2007; see for review, Brück et al., 2011) the posterior superior
temporal cortex (p-STC) emerges as a crucial structure for the
integration of facial and vocal cues. In event-related potential
(ERP) studies (e.g., Giard and Peronnet, 1999; Foxe et al., 2000;
Molholm et al., 2002), multisensory enhancement is measured
by comparing the ERP from the multisensory condition to the
sum of the ERPs from each unimodal condition. Multisensory
enhancement is also commonly measured in behavioral studies by
calculating a redundancy gain between the crossmodal stimulus
and the more informational unimodal stimulus. Another inter-
esting method performed in studies using RT, is to test whether
the redundant target effect (shorter RT under the crossmodal
condition) reflects an actual multisensory integrative process by
comparing the observed RT distribution with the distribution
predicted by the “race model” (proposed by Miller, 1982; see also
Colonius and Diederich, 2006). The “race model” assumes that
a crossmodal stimulus presentation produces parallel activation
(i.e., in a separate way) of the unimodal stimuli. Citation: According to this
model, the shortening of RT for crossmodal relative to unimodal
stimuli derives from the fact that either unimodal stimulus can
produce a response. Thus, any violation of the race model (i.e.,
if the observed RTs in crossmodal trials are shorter than those
predicted by the race model) indicates that the stimuli are not Frontiers in Psychology | www.frontiersin.org May 2015 | Volume 6 | Article 691 Participants p
The study participants consisted of 31 younger (20–35; M = 25.8,
SD = 6.4; 16 females) and 31 older adults (60–76; M = 67.2,
SD = 5.8; 17 females); see Table 1. The participants spoke French
and reported having normal or corrected-to-normal vision and
good hearing abilities at the time of testing. All participants were
living independently in the community and were in good gen-
eral physical health. None of the participants had any history of
psychiatric or neurological disorders, which might compromise
cognitive function. They also had a normal score on the Beck
Depression Inventory (Beck et al., 1996; BDI II, 21 item version; a
score of less than 17 was considered to be in the minimal range). All elderly adults completed the Mini Mental State Examination
(Folstein et al., 1975; MMSE), on which they scored above the cut-
off score (26/30) for risk of dementia. Grade level was calculated
with the Mill Hill Vocabulary Scale (French adaptation: Deltour,
1993), and this did not differ between groups (p = 0.55). TABLE 1 | Participant demographic characteristics. Younger adults
Older adults
(n = 31)
(n = 31)
Age (years)
25.8 ± 6.4
67.2 ± 5.8
Education (years)
14.18 ± 1.6
13.55 ± 2.8
Mill-hill
36.87 ± 3.0
37.48 ± 4.8
Sex ratio (M/F)
16/15
17/14
BDI-II (/63)
5.65 ± 6.5
5.25 ± 3.8
MMSE (/30)
–
29.33 ± 0.6 TABLE 1 | Participant demographic characteristics. TABLE 1 | Participant demographic characteristics. Frontiers in Psychology | www.frontiersin.org 3 Aging and multimodal emotional integration Chaby et al. faces obtained from the Karolinska Directed Emotional Faces
Database) and non-emotional voices (i.e., six pairs of neutral
voices obtained from the Montreal Affective Voices database). The
stimuli were different from those used in the main task. FIGURE 1 | Schematic representation of the stimuli. Examples of the
stimuli for the three different modalities, including visual (facial expressions),
auditory (non-verbal affective vocalizations) and crossmodal stimuli (congruent
facial and vocal emotions presented simultaneously). Then, after a short familiarization period, the experiment
began. The experiment consisted of three blocks (visual, auditory,
crossmodal) of 60 trials. Each trial started with the presentation of
a fixation cross for 300 ms and was followed by the target stimulus,
which was presented or repeated until the subject responded. Materials Examples of stimuli and the task design for each condition are
illustrated in Figure 1. Examples of stimuli and the task design for each condition are
illustrated in Figure 1. Visual stimuli. Visual stimuli consisted of pictures of human
facial expressions obtained from the Karolinska Directed Emo-
tional Faces database (Lundqvist et al., 1998). This database was
chosen because it provided good examples of universal emotion
categories with a high accuracy of labeling. The faces of 10 models
(5 females, 5 males) expressing facial expressions of happiness,
sadness, anger, fear, disgust or neutral constituted a set of 60
stimuli. All stimuli (presented on a black background) were 10 cm
in height and subtended a vertical visual angle of 8° at a viewing
distance of 70 cm. First, the data were entered into an overall analysis of variance
(ANOVA), with age (young adults, older adults) as a between-
subjects factor and with modality (visual, auditory, crossmodal)
and emotion (neutral, happiness, fear, anger, sadness, disgust)
as within-subjects factors. Effect sizes are reported as partial
eta-squared (η2
p). ANOVAs were adjusted with the Greenhouse-
Geisser non-sphericity correction for effects with more than one
degree of freedom. To provide clarity, uncorrected degrees of free-
dom, the Greenhouse-Geisser epsilon (ε) and adjusted p values
are reported. Planned comparisons or post hoc Bonferroni tests
were conducted to further explore the interactions between age,
modality and emotion. The alpha level was set to 0.05 (p values
were corrected for multiple comparisons). Auditory stimuli. Auditory stimuli (Figure 1) consisted of non-
verbal affective vocalizations (cry, laugh, etc.) obtained from
The Montreal Affective Voices database (Belin et al., 2008). This
database was chosen because it provided a standardized set of
emotional vocalizations corresponding to the universal emotion
categories without the potential confounds from linguistic con-
tent. The voices of 10 actors (5 females, 5 males) expressing
happiness, sadness, anger, fear, disgust or neutral, vocalization
constituted a set of 60 stimuli. Second, to examine whether both groups showed redundancy
gains, as reflected by the difference in the scores when the visual
and auditory stimuli were presented together (crossmodal condi-
tion) compared to each modality alone (unimodal condition), we
calculated a “redundancy gain” for each participant separately by
subtracting the higher of the scores under the unimodal condi-
tions from the score under the crossmodal condition [(crossmodal
score—best modality score) × 100] (see Calvert et al., 2004; Girard
et al., 2013). Statistical Analysis Participants’ accuracy (scores of correct responses) and corre-
sponding RT (in milliseconds, ms) was computed for each condi-
tion. To control for outliers, trials with RT below 200 ms or greater
than two standard deviations above the mean of each condition
(0.90% of the trials in young adults; 1.25% of the trials in older
adults) were excluded. Materials The significance of the difference in redundancy gain
(in percent) between younger and older participants was tested
using an independent samples t - test. Crossmodal stimuli. Each emotional face was combined with
an affective vocalization to construct 60 congruent expressions of
faces and voices. The gender of the face and the voice were always
congruent. Participants Participants were asked to select (by clicking with the computer
mouse) one label from a list of choices that best described the
emotion presented. The six labels were displayed at the bottom
of the computer screen and were visible throughout the test. There was an inter-trial interval of 700 ms. The order of the three
blocks was counterbalanced across participants, and the order
of trials was pseudo-randomized across each block. During the
session, resting pauses were provided after every 10 trials, and
the participants could take breaks if necessary between blocks. No
feedback was given to the participants. FIGURE 1 | Schematic representation of the stimuli. Examples of the
stimuli for the three different modalities, including visual (facial expressions),
auditory (non-verbal affective vocalizations) and crossmodal stimuli (congruent
facial and vocal emotions presented simultaneously). The study was approved by the ethics committee of Paris
Descartes University (Conseil d’Evaluation Ethique pour les
Recherches en Santé, CERES, n IRB 2015100001072) and all
participants gave informed consent. Frontiers in Psychology | www.frontiersin.org Procedure Participants were tested individually in a single session that lasted
approximately 45 min. The protocol was run using E-prime
presentation software (Psychology Software Tools). Prior to the
experiment, short facial-matching and vocal-matching tasks were
administered to control for basic visual and auditory abilities in
processing faces and voices. The subjects were asked to match
the identity of non-emotional faces (i.e., six pairs of neutral Finally, to further test the advantage of crossmodal over uni-
modal processing, we investigated whether the RTs obtained
under the crossmodal condition exceeded the statistical facil-
itation predicted by the race model (Miller, 1982). In mul-
tisensory research, the race model inequality has become a
standard tool to identify crossmodal integration using RT data May 2015 | Volume 6 | Article 691 Frontiers in Psychology | www.frontiersin.org 4 Aging and multimodal emotional integration Chaby et al. (Townsend and Honey, 2007). To analyze the race model inequal-
ity, we used RMItest software (http://psy.otago.ac.nz/miller),
which implements the algorithm described in Ulrich et al. (2007). The procedure requires four steps. First, participants’
RTs in each condition (i.e., visual, auditory and crossmodal)
are converted to cumulative distribution functions (CDFs). Sec-
ond, the race model distribution is calculated by summing the
CDFs of observed responses to the two unimodal conditions
(visual and auditory) to create a “predicted” multisensory dis-
tribution. Third, percentile points (i.e., in the present study:
5th, 15th, 25th, 35th, 45th, 55th, 65th, 75th, 85th, and 95th)
are determined for every distribution of RT. Finally, in each
group, the mean RT for the crossmodal condition and the “pre-
dicted” condition are compared for each percentile using a t-
test. If significant values are obtained in the crossmodal con-
dition relative to the predicted condition, we conclude that the
race model cannot account for the facilitation of the redun-
dant signal conditions, supporting the existence of an integrative
process. FIGURE 2 | Mean accuracy scores (%) and response times (ms) for
both age groups under the visual, auditory and crossmodal
conditions. Error bars indicate standard errors of the means. FIGURE 2 | Mean accuracy scores (%) and response times (ms) for
both age groups under the visual, auditory and crossmodal
conditions. Error bars indicate standard errors of the means. 2Note that a ceiling effect was observed for happiness in both groups and for
neutral in the younger group. Procedure than younger adults for the negative emotions only2 (i.e., sadness,
anger and disgust in the visual modality, p < 0.01; anger and fear
in the auditory modality, p < 0.01), and (b) in the crossmodal
condition, older adults perform more poorly than younger adults
for anger only (p < 0.001). Concerning RTs, in the unimodal and
crossmodal conditions, older adults were slower to identify all
emotions (all p < 0.001) except for happiness (p > 0.1). Results Age-related Difference in Emotion Recognition
Mean performance and RTs for all conditions are presented in
Table 21. For both younger and older groups, the mean perfor-
mance accuracy was greater than 80% for the visual, auditory
and crossmodal conditions. However, we found significant main
effects of age, indicating that older adults performed less accu-
rately and more slowly than younger adults (85.23 ± 1.24% vs. 92.58 ± 0.51%, F(1,60) = 30.17, p < 0.001, η2
p = 0.33 for scores;
3619 ± 145 ms vs. 1991 ± 68 ms, F(1,60) = 103.4, p < 0.001,
η2
p = 0.63 for RTs). Importantly, we found a significant effect of
modality on the scores [F(2,120) = 137.54, p < 0.001, ε = 0.92,
η2
p = 0.7] and the RTs [F(2,120) = 62.48, p < 0.001, ε = 0.88,
η2
p = 0.51], indicating that participants responded more effec-
tively under the crossmodal condition than under either uni-
modal condition (all p < 0.001). There was a significant effect
of emotion on the scores [F(5,300) = 92.11, p < 0.001, ε = 0.62,
η2
p = 0.60] and the RTs [F(5,300) = 36.91, p < 0.001, η2
p = 0.38]. Furthermore, main effects were accompanied by several two-way
interactions: between group and modality (see Figure 2) on the
scores [F(2,120) = 6.98, p = 0.002, ε = 0.92, η2
p = 0.10] and the
RTs [F(2,120) = 7.66, p = 0.001, ε = 0.88, η2
p = 0.11]; between
group and emotion on the scores [F(5,300) = 8.13, ε = 0.61,
p < 0.001, η2
p = 0.12] and the RTs [F(5,300) = 8.62, p < 0.001,
η2
p = 0.12], and between modality and emotion on the scores
[F(10,600) = 27.01, p < 0.001, ε = 0.55, η2
p = 0.31] and the RTs
[F(10,600) = 10.52, p < 0.001, ε = 0.56, η2
p = 0.15]. Importantly,
there was a significant effect of the three-way interaction between
group, modality and emotion on the scores [F(10,600) = 3.23,
p = 0.005, ε = 0.55, η2
p = 0.05] and the RTs [F(10,600) = 2.7,
p = 0.016, η2
p = 0.04]. This reveals the following (see Table 2): (a)
in the visual and auditory modality, older adults have lower scores Integration of Crossmodal Emotional Information
in Aging FIGURE 3 | Test for the violation of race model inequality. The figure the existence of a crossmodal integrative process. The temporal
window in which this benefit was significant was from 1019 to
1410 ms. Similar to the responses by the younger group, the older
group responses were shorter than those predicted by the race
model for the 5th, 15th, 25th, and 35th percentiles of the RT
distribution (all p < 0.01). The temporal window in which this
benefit was significant was from 1647 to 2300 ms. Although the
maximal enhancement occurred at different absolute RTs between
the two populations, this peak enhancement occurred at the exact
same percentile of the cumulative distribution curve. Integration of Crossmodal Emotional Information
in Aging To explore the ultimate crossmodal gain in the scores, we cal-
culated a “redundancy gain” (i.e., the difference between the
crossmodal condition and the unimodal condition with the higher
score) for each participant in the two groups (see Materials and
Methods section). For the scores, our analysis indicated that the redundancy gain
was greater for the older (8.82%) than for the younger adults
(5.86%, p = 0.007). In the older group, all but two subjects showed
a redundancy gain (29/31; one performed equally between the
auditory modality and the crossmodal condition, and the other
performed slightly better under the visual condition compared to
the crossmodal condition). Moreover, there was a significant dif-
ference between the unimodal and crossmodal conditions for all
emotions (all p < 0.003). In the younger group, all subjects except
for one (30/31; who performed equally between the auditory
condition and the crossmodal condition) showed a redundancy
gain. Our analysis showed a significant difference between the
unimodal and crossmodal conditions for negative emotions only
(fear, sadness, anger and disgust) (all p < 0.007); for the neutral
emotion and for happiness, performance ceilings may explain the
lack of significant effects (all p > 0.1). For RTs, we used the race model to explore crossmodal integra-
tion and to determine whether the observed crossmodal behav-
ioral enhancement (i.e., shorter RTs) was beyond that predicted
by statistical summation of the unimodal visual and auditory
conditions (Figure 3). In the younger group, we observed a vio-
lation of the race model prediction for the 5th, 15th, 25th, and
35th percentiles of the RT distribution (all p < 0.01, but not
for the slowest percentiles (all p > 0.1). These results support 1To control for potential gender differences, this variable was initially entered
as a between-subject factor in the analyses. However, gender failed to yield any
significant main effects (F < 1) or interactions (p > 0.1) so we collapsed across
gender in the reported analysis. May 2015 | Volume 6 | Article 691 Frontiers in Psychology | www.frontiersin.org 5 Aging and multimodal emotional integration Chaby et al. TABLE 2 | Mean accuracy scores (%) and response times (ms) by age group and emotion. Standard errors of the means are shown in parentheses. Integration of Crossmodal Emotional Information
in Aging Mean accuracy (%)
Neutral
Happy
Fear
Sadness
Anger
Disgust
Older
Visual
90.9 (2.7)
99.3 (0.4)
83.2 (2.3)
73.5 (3.4)
66.1 (4.7)
72.5 (2.5)
Auditory
92.6 (2.7)
95.2 (1.9)
73.2 (3.9)
94.5 (1.3)
47.7 (3.4)
86.4 (2.3)
Crossmodal
98.7 (0.6)
100 (0.0)
89.6 (2.1)
95.8 (1.8)
76.8 (4.2)
97.7 (0.8)
Younger
Visual
97.1 (1.1)
99.7 (0.6)
87.7 (2.2)
90.9 (2.6)
84.5 (2.4)
85.4 (2.5)
Auditory
96.7 (0.9)
98.0 (0.9)
86.7 (2.3)
94.2 (1.3)
62.9 (3.0)
94.8 (1.2)
Crossmodal
99.6 (0.3)
99.0 (0.7)
97.4 (0.9)
97.4 (0.8)
94.5 (1.5)
99.3 (0.4)
Response times (ms)
Neutral
Happiness
Fear
Sadness
Anger
Disgust
Older
Visual
3447 (227)
2502 (136)
4366 (225)
4805 (325)
4243 (280)
4173 (224)
Auditory
4355 (342)
3195 (213)
4370 (265)
3824 (238)
4920 (354)
3871 (250)
Crossmodal
2796 (158)
2188 (105)
3135 (138)
3115 (146)
3273 (190)
2567 (132)
Younger
Visual
1871 (107)
1555 (50)
2408 (162)
2464 (157)
2569 (177)
2538 (163)
Auditory
2148 (157)
1967 (117)
2345 (153)
2082 (74)
2281 (124)
2026 (80)
Crossmodal
1613 (49)
1370 (35)
1689 (54)
1722 (50)
1571 (45)
1624 (35)
FIGURE 3 | Test for the violation of race model inequality. The figure
illustrates the cumulative probability curves of the RT under the visual (blue
circles), auditory (green squares), and crossmodal conditions (red circles). The
summed probability for the visual and auditory responses is depicted by the
race model curve (marked by an asterisk). Note that the crossmodal
responses are faster than the race model prediction for the four fastest
percentiles, i.e., the 5th, 15th, 25th, and 35th percentiles (all p < 0.01). from emotional faces, fewer studies have examined vocal emo-
tion recognition, and hardly any studies have investigated the
recognition of emotion from emotional faces and voices pre-
sented simultaneously (Hunter et al., 2010; Lambrecht et al.,
2012). The purpose of this study was to compare unimodal
facial and vocal emotion processing in older and younger adults
and, in addition, to test whether older adults benefit from the
combination of congruent emotional information from different
channels, which reveals crossmodal integration. Our results first
confirm that older adults experience difficulties in emotion recog-
nition. They were less accurate and slower overall than younger
adults in processing emotion from facial or non-verbal vocal
expressions presented alone. Second, the participants similarly
recognized facial and vocal cues, and both groups benefitted
from the crossmodal condition. Effects of Age on Emotion Recognition Based on
Unimodal Stimuli Our findings indicated that emotion recognition based on uni-
modal stimuli changes with age. In the visual modality, our results
support previous findings showing age-related difficulties in the
ability to recognize emotion from facial cues (see for a meta-
analysis, Ruffman et al., 2008). However, most of these studies Integration of Crossmodal Emotional Information
in Aging Third, age-related differences
were modulated by emotion, as older adults were particularly
affected in term of accuracy with regards to processing negative
emotions under both the facial and vocal conditions. Finally,
our results provide compelling evidence for the multisensory TABLE 2 | Mean accuracy scores (%) and response times (ms) by age group and emotion. Standard errors of the means are shown in parentheses. nd response times (ms) by age group and emotion. Standard errors of the means are shown in parentheses. from emotional faces, fewer studies have examined vocal emo-
tion recognition, and hardly any studies have investigated the
recognition of emotion from emotional faces and voices pre-
sented simultaneously (Hunter et al., 2010; Lambrecht et al.,
2012). The purpose of this study was to compare unimodal
facial and vocal emotion processing in older and younger adults
and, in addition, to test whether older adults benefit from the
combination of congruent emotional information from different
channels, which reveals crossmodal integration. Our results first
confirm that older adults experience difficulties in emotion recog-
nition. They were less accurate and slower overall than younger
adults in processing emotion from facial or non-verbal vocal
expressions presented alone. Second, the participants similarly
recognized facial and vocal cues, and both groups benefitted
from the crossmodal condition. Third, age-related differences
were modulated by emotion, as older adults were particularly
affected in term of accuracy with regards to processing negative
emotions under both the facial and vocal conditions. Finally,
our results provide compelling evidence for the multisensory
nature of emotional processing in aging. The important finding
of this study was that older adults benefit to the same extent
as younger adults from the combination of information pre-
sented in the visual and auditory modalities. This suggests that
crossmodal processing represents a mechanism compensating for
deficits in the visual or auditory channels that often affect older
adults. FIGURE 3 | Test for the violation of race model inequality. The figure
illustrates the cumulative probability curves of the RT under the visual (blue
circles), auditory (green squares), and crossmodal conditions (red circles). The
summed probability for the visual and auditory responses is depicted by the
race model curve (marked by an asterisk). Note that the crossmodal
responses are faster than the race model prediction for the four fastest
percentiles, i.e., the 5th, 15th, 25th, and 35th percentiles (all p < 0.01). e times (ms) by age group and emotion. Standard errors of the means are shown in parentheses.
Mean accuracy (%)
Happy
Fear
Sadness
Anger
Disgust
99.3 (0.4)
83.2 (2.3)
73.5 (3.4)
66.1 (4.7)
72.5 (2.5)
95.2 (1.9)
73.2 (3.9)
94.5 (1.3)
47.7 (3.4)
86.4 (2.3)
100 (0.0)
89.6 (2.1)
95.8 (1.8)
76.8 (4.2)
97.7 (0.8)
99.7 (0.6)
87.7 (2.2)
90.9 (2.6)
84.5 (2.4)
85.4 (2.5)
98.0 (0.9)
86.7 (2.3)
94.2 (1.3)
62.9 (3.0)
94.8 (1.2)
99.0 (0.7)
97.4 (0.9)
97.4 (0.8)
94.5 (1.5)
99.3 (0.4)
Response times (ms)
Happiness
Fear
Sadness
Anger
Disgust
2502 (136)
4366 (225)
4805 (325)
4243 (280)
4173 (224)
3195 (213)
4370 (265)
3824 (238)
4920 (354)
3871 (250)
2188 (105)
3135 (138)
3115 (146)
3273 (190)
2567 (132)
1555 (50)
2408 (162)
2464 (157)
2569 (177)
2538 (163)
1967 (117)
2345 (153)
2082 (74)
2281 (124)
2026 (80)
1370 (35)
1689 (54)
1722 (50)
1571 (45)
1624 (35)
equality. The figure
under the visual (blue
ditions (red circles). The
ses is depicted by the
he crossmodal
or the four fastest
les (all p < 0.01).
rocess. The temporal
nt was from 1019 to
nger group, the older
redicted by the race
ercentiles of the RT
indow in which this
00 ms. Although the
absolute RTs between
from emotional faces, fewer studies have examined vocal e
tion recognition, and hardly any studies have investigated
recognition of emotion from emotional faces and voices
sented simultaneously (Hunter et al., 2010; Lambrecht et
2012). The purpose of this study was to compare unim
facial and vocal emotion processing in older and younger ad
and, in addition, to test whether older adults benefit from
combination of congruent emotional information from diffe
channels, which reveals crossmodal integration. Our results
confirm that older adults experience difficulties in emotion re
nition. They were less accurate and slower overall than youn
adults in processing emotion from facial or non-verbal v
expressions presented alone. Second, the participants simi
recognized facial and vocal cues, and both groups benefi
from the crossmodal condition. Third, age-related differe
were modulated by emotion, as older adults were particu
affected in term of accuracy with regards to processing nega
emotions under both the facial and vocal conditions. Fin
our results provide compelling evidence for the multisen
nature of emotional processing in aging. The important find
of this study was that older adults benefit to the same ex
as younger adults from the combination of information
sented in the visual and auditory modalities. This suggests
crossmodal processing represents a mechanism compensating
deficits in the visual or auditory channels that often affect o
adults.
Effects of Age on Emotion Recognition Based Discussion While a large body of evidence shows that older adults are less
accurate than younger adults in recognizing specific emotions May 2015 | Volume 6 | Article 691 Frontiers in Psychology | www.frontiersin.org 6 Aging and multimodal emotional integration Chaby et al. used the collection of posed black-and-white photographs of
human faces from the 1970s Ekman dataset (e.g., Orgeta and
Phillips, 2008; Hunter et al., 2010; Slessor et al., 2010) that has
been criticized for its lack of ecological validity, which leads to
questions about the generalizability of the results (Murphy and
Isaacowitz, 2010). The present study used emotional expressions
consisting of static color photographs of faces (see also, Ebner
et al., 2010; Eisenbarth and Alpers, 2011), and this study con-
firmed the robustness of age-related difficulties. The fact that
the same results were found using dynamic facial expressions
(Lambrecht et al., 2012) confirms that widespread difficulties in
recognizing emotion from facial cues are encountered by older
adults. In the auditory modality, the ability to recognize emotion
from non-verbal vocal cues also becomes less efficient with age. This result is in accordance with that of Hunter et al. (2010), who
used non-verbal affective vocalizations. It is also in line with some
recent studies using spoken words in a neutral context that showed
impairments in decoding emotional speech with advancing age
(Paulmann et al., 2008; Mitchell et al., 2011; Lambrecht et al.,
2012). As normal variations of prosodic emotion ability could
be associated with depression, relationship satisfaction or well-
being in younger populations (Noller and Feeney, 1994; Emerson
et al., 1999; Carton et al., 1999), the question remains whether
and how the age-related decline in emotional vocal processing
influences social interactions. However, it is important to note
that in our study, the performance of the older group reached
80%, suggesting a relatively mild deficit. This suggest that non-
verbal vocalizations, that are devoid of linguistic information,
are however, effective at communicating diverse emotions in
aging. sadness, disgust), whereas Lima et al. (2014) found that older
adults performed poorly for all emotions (positive and negative
ones). Note however, that for scores, interpretations about age-
related difference in responses to specific emotions are limited
because of the presence of ceiling effects for happy and neutral
expressions. Interestingly, for RTs the effects seem to be more
general since older adults were especially slow to respond to all
emotions. Discussion These divergent results across aging studies may be due to the
individual variability of the samples and the use of different types
of emotional stimuli with varying presentation times, which might
influence the identification of the given emotion. In the present
study, the stimuli were presented or repeated until the subject
provided a response. The observed slower RT for all negative
emotions contrasts with the findings of recent studies (Pell and
Kotz, 2011; Rigoulot et al., 2013) using verbal emotional stimuli,
which showed that listeners are generally faster at identifying
fear, anger, and sadness and slower at identifying happiness and
disgust. This suggests that non-verbal affective vocalizations are
processed at different rates. Interestingly, this time window is
consistent with a work by Pell (2005); when happy, sad, or neutral
pseudo-utterances spoken in English were cut from the onset of
the sentence to last 300, 600, or 1000 ms in duration, emotional
priming of a congruent static face was only observed when vocal
cues were presented for 600 or 1000 ms, but not for only 300 ms. Hence, vocal information enduring at least 600 ms maybe neces-
sary to presumably activate shared emotion knowledge responsi-
ble for multimodal integration. More importantly, our data show
that the participants did not find it easier to identify emotion from
isolated facial or non-verbal vocal cues. By contrast, Hunter et al. (2010), who used facial and vocal non-verbal emotions, found that
emotion recognition was easier in response to facial cues than
vocal cues. However, our experiment used not only negative but
also happy and neutral expressions, which potentially improved
the performance of older adults in both the visual and auditory
modalities. Age-related Difference in the Responses to
Different Sensory Modalities and Specific
Emotions However, the main effect of age was tempered by a set of interac-
tions, suggesting that age-related differences varied across modal-
ities and across specific emotions. Specifically, in response to the
visual and auditory stimuli, we found an age-related reduction
in accuracy for negative expressions (i.e., fear, sadness, anger
and disgust) and comparable performance for neutral and happy
expressions. For the visual modality, this result is in accordance
with individual studies using images of static faces showing dif-
ferent emotional expressions, which showed that certain discrete
emotions, notably negative ones, are more sensitive to age-related
variation (see for review, Ruffman et al., 2008). Studies regarding
the auditory channel are more inconsistent because they are based
on diverse paradigms. The results differ inasmuch as the studies
did not isolate specific emotions (e.g., Orbelo et al., 2005; Mitchell,
2007; Mitchell et al., 2011), they investigated negative emotions
only (e.g., Hunter et al., 2010), they explored a few contrasting
emotions (e.g., Lambrecht et al., 2012) or they included several
positive and negative emotions (e.g., Wong et al., 2005; Lima
et al., 2014). Wong et al. (2005) found that older adults poorly
recognized only sadness and happiness in speech; in contrast,
using non-verbal vocalizations, Hunter et al. (2010) found that
older adults poorly identified negative emotions (fear, anger, Overall, these results are consistent with the fact that age-
related emotional difficulties do not reflect general cognitive aging
(Orbelo et al., 2005) but rather a complex change affecting discrete
emotions; notably, the same authors also suggest that the age-
related decline in emotional processing is not explained by sex
effects or age-related visual or hearing loss. Nevertheless, assessing
hearing and seeing abilities objectively could have informed the
pattern of our findings and we can consider the lack of measuring
these covariates as a limitation of the study. For example, recent
findings (Ruggles et al., 2011; Bharadwaj et al., 2015) suggest that
despite normal or near-normal hearing thresholds, a significant
portion of listeners exhibit deficits in everyday communication
(i.e., in complex environments such as noisy restaurants or busy
streets). These results could also be interpreted in terms of the socio-
emotional selectivity theory, which states that aging increases
emotional control, diminishes the impact of negative emotions
and facilitates concentration on more positive social interac-
tions (e.g., Charles and Carstensen, 2010; Huxhold et al., 2013). Age-related Difference in the Responses to
Different Sensory Modalities and Specific
Emotions However, Frank and Stennett (2001) have noted that using only
a few basic emotion categories allows participants to choose May 2015 | Volume 6 | Article 691 Frontiers in Psychology | www.frontiersin.org 7 Aging and multimodal emotional integration Chaby et al. crossmodal RTs of older adults was longer than that of younger
adults for each emotion. their response based on discrimination and exclusion rules,
which is less likely to be the case in a real-life setting. In
particular, if happiness is the only positive emotion, partici-
pants can make the correct choice as soon as they recognize a
smile. Therefore, a ceiling effect can be an alternative explana-
tion to the socio-emotional selectivity theory. An alternative to
examine possible valence-specific effects is the use of a simi-
lar number of positive and negative emotions (see Lima et al.,
2014). Furthermore, a recent study using ERPs by Mishra and Gaz-
zaley (2013) among healthy older adults (60–90 years old)
suggested the existence of compensatory mechanisms suscepti-
ble to sustaining efficient crossmodal processing. The authors
showed evidence that distributed audio-visual attention results
in improved discrimination performance (faster RTs without any
differences in accuracy in congruent stimuli settings) compared
to focused visual attention. They noted that the benefits of dis-
tributed audio-visual attention in older adults matched those
of younger adults. Interestingly, ERPs recoding during the task
further revealed intact crossmodal integration in higher perform-
ing older adults, who had results similar to those of younger
adults. As suggested by Barulli et al. (2013), attention, execu-
tive function and verbal IQ may play a role in the generation
of a “cognitive reserve” that reduces the deleterious effects of
aging and, thus, buffers against a diminished adaptive strat-
egy (Hodzik and Lemaire, 2011). These results show the neces-
sity of taking into account individual cognitive differences in
aging. It is clear that significant cognitive decline is not an
inevitable consequence of advancing age and that each cogni-
tive domain is differentially affected. As aging can have diverse
effects on cognitive functions, it is therefore important to empha-
size the maintained functions rather than taking a customary
approach that only underlines the loss of capacities among the
elderly. Integration of Crossmodal Emotional Information
in Aging Individuals The principal goal of the current study was to explore whether
older adults benefit from congruent crossmodal integration and
to better understand the nature of this benefit. In daily life, the
combination of information from facial and vocal expressions
usually results in a more robust representation of the expressed
emotion (e.g., De Gelder and Vroomen, 2000; Dolan et al., 2001;
De Gelder and Bertelson, 2003), which thus results in a more
unified perception of the person (Young and Bruce, 2011). p
p
p
g
In our study, emotional faces and voices come from different
sensory modalities to build a unified and coherent representation
of the same percept (i.e., an emotion) as defined by crossmodal
integration mechanisms (Driver and Spence, 2000). We showed
that whereas older adults exhibited slower RTs under the cross-
modal condition, resulting in a different temporal window of
multisensory enhancement, a multisensory benefit occurred to
the same extent in the two groups. However, early studies of mul-
tisensory integration in aging individuals showed that compared
to younger adults, older adults did not benefit from multisensory
cues (Stine et al., 1990; Walden et al., 1993; Sommers et al.,
2005) and experienced a suppressed cortical multisensory integra-
tion response that was associated with poor cortical integration
(Stephen et al., 2010). By contrast, more recent studies point
toward an enhancement of multisensory integration effects in
older adults, notably reporting shorter RT in response to mul-
tisensory events (e.g., Mahoney et al., 2011, 2012; DeLoss et al.,
2013). It should be noted however, as a possible limitation of the
current study, that our stimuli are quite unnaturalistic since they
combine non-dynamic (photographs) and dynamic (sound) stim-
uli. Although our participants did not report any incongruent
perception of crossmodal stimuli, the use of emotional expres-
sions that contain truly multimodal expressions (video and audio
obtained from the same person), which are not posed, but enacted
using the Stanislawski technique (see the Geneva Multimodal
Emotion Portrayals, GEMEP; Bänziger et al., 2012) could be
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10.1016/j.neuroimage.2012.09.057 Calder, A. J., Keane, J., Manly, T., Sprengelmeyer, R., Scott, S., Nimmo-Smith, I.,
et al. (2003). Facial expression recognition across the adult life span. Neuropsy-
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multisensory enhancement with the time-window-of-integration model. Neu-
ropsychologia 46, 2556–2562. doi: 10.1016/j.neuropsychologia.2008.03.026 Calder, A. J., Lawrence, A. Conclusion Consistent with the latter works, the present study indicates
that in younger and older adults, emotional information derived
from facial and vocal cues is not reducible to the simple sum of
the unimodal inputs and suggests that multisensory integration is
maintained with increasing age and could play a compensatory
role in normal aging. This is in accordance with a magneto-
encephalography study (Diaconescu et al., 2013), which indi-
cated that sensory-specific regions showed increased activity after
visual-auditory stimulation in young and old participants but that
inferior parietal and medial prefrontal areas were preferentially
activated in older subjects. Activation of the latter areas was
related to faster detection of multisensory stimuli. The authors
proposed that the posterior parietal and medial prefrontal activity
sustains the integrated response in older adults. This hypothesis
is supported by the theory of PASA and that of cortical dediffer-
entiation, stating that healthy aging is accompanied by decreased
specificity of neurons in the prefrontal cortex (Park and Reuter-
Lorenz, 2009; Freiherr et al., 2013). This could explain why the In conclusion, our results suggest that despite a decline in facial
and vocal emotional processing with advancing age, older adults
integrate facial and vocal cues to yield a unified perception of the
person. Given the changes in facial and vocal modality exhib-
ited by older adults, it may be helpful for family members and
caregivers to use multiple sensory modalities to communicate
important affective information. Thus, supplementing facial cues
with vocal information may facilitate communication, prevent-
ing older individuals from withdrawing from the community
and reducing the development of affective disturbances such
as depression. Future research is required to further examine
whether crossmodal integration can benefit older adults who
exhibit cognitive impairments (e.g., Mild Cognitive Impairments,
Alzheimer’s Disease). Such studies would be of particular interest
in the context of recently developed assistive robotics platforms
that prolong the ability of persons who have lost their autonomy
to remain at home. For instance, serious games and socially aware May 2015 | Volume 6 | Article 691 Frontiers in Psychology | www.frontiersin.org 8 Aging and multimodal emotional integration Chaby et al. interpretation and the final version of the manuscript, which all
approved. assistive robots have actually been designed without consider-
ing the age-specific effects on social signal recognition. There-
fore, improving the efficiency and suitability of these interactive
systems clearly requires a better understanding of crossmodal
integration. Acknowledgments This work waspartially supported bythe Labex SMART(ANR-11-
LABX-65) under French state funds managed by the ANR within
the Investissements d’Avenir program under reference ANR-11-
IDEX-0004-02 and by the FUI PRAMAD2 project. We are also
grateful to all of the volunteers who generously gave their time to
participate in this study. Author Contributions Study concept and design was performed by LC, VL, MC, and
MP. Data acquisition was conducted by VL. Data analysis was
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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) 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. Watson, R., Latinus, M., Noguchi, T., Garrod, O., Crabbe, F., and Belin, P. (2014). Crossmodal adaptation in right posterior superior temporal sulcus
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